Category: Clinical Laboratory Medicine

The clinical laboratory has a major role in modern medicine. A bewildering array of laboratory procedures is available, each of which has its special usefulness and its intrinsic problems, its advantages and its drawbacks. Advances in biochemistry and radioisotopes, to name only two conspicuous examples, are continually adding new tests or modifying older methods toward new usefulness. It seems strange, therefore, that medical education has too often failed to grant laboratory medicine the same prominence and concern that are allotted to other subjects

  • Immunoglobulins

    Gamma globulins (called immunoglobulins, or Igs, in current immunologic terminology) are not a homogeneous group. There are three main subdivisions: IgG, which migrates in the gamma region on electrophoresis; IgA, which migrates in the pregamma region or in the area between gamma and beta; and IgM, which migrates in the prebeta or beta region (Fig. 22-3). There are two additional groups called IgD and IgE. IgG comprises about 75% of serum immunoglobulins and has a normal 7S molecular weight. IgG constitutes the majority of the antibodies, especially the warm-temperature incomplete type. IgM accounts for 5%-7% of total immunoglobulins and is a macroglobulin (19S group). The IgM class includes the earliest antibodies produced against infectious organisms (later followed by IgG antibodies), cold agglutinins, ABO blood group isoagglutinins, and rheumatoid factor. IgA constitutes about 15% of immunoglobulins. Although most are 7S, some molecules are larger. It is found primarily in secretions, such as saliva, tears, gastrointestinal secretions from stomach and accessory organs, and secretions from the respiratory tract. Selective deficiency of IgA (the other immunoglobulins being normal) is the most common primary immunodeficiency, and is associated with frequent upper respiratory and GI infections. There is also increased frequency of autoimmune disease. Phenytoin (Dilantin) is reported to decrease IgA levels to some extent in 20%-85% of patients on long-term therapy. In one report, about 15% of patients had IgA levels below reference range, and about 4% had very low levels. IgD is a normal 7S molecular weight molecule that makes up less than 1% of the immunoglobulins; its function is not known. IgE also has a 7S weight and occurs with less than 1% frequency. It is elevated in certain allergic conditions, especially atopic disorders, and it is associated with reaginic antibody.

    A normal Ig molecule is composed of two heavy chains (each chain of 50,000-dalton molecular weight) and two light chains (kappa and lambda, normally in 2:1 K/L ratio, each of 20,000-dalton molecular weight) connected by disulfide bridges. IgM is a pentomeric arrangement of five complete Ig units.

  • Typical Electrophoretic Patterns

    Several typical electrophoretic patterns are presented with diseases in which they are most commonly found (Fig. 22-2). It must be strongly emphasized that no pattern is pathognomonic of any single disease and that there is considerable variation in the shape and height of the electrophoretic peaks in individual patients that may obscure a pattern. In addition, the patterns will not always appear when they should be expected and in some cases may be altered when two diseases are present together (e.g., acute infection in a patient with cirrhosis).

    One electrophoretic configuration is called the acute reaction pattern. It consists of decreased albumin level and elevated alpha-2 globulin level and is found in acute infections in the early stages; some cases of myocardial infarct and tissue necrosis; some cases of severe burns, surgery, and other stress conditions; and in some of the rheumatoid diseases with acute onset. A second pattern consists of a slightly or moderately decreased albumin level, a slightly or moderately elevated gamma-globulin, and a slightly elevated or normal alpha-2 globulin level. This is the chronic inflammatory pattern and is found in chronic infections of various types, the granulomatous diseases, cirrhosis, and rheumatoid-collagen diseases. There may, of course, be various stages of transition between this chronic pattern and previously described acute one. A third pattern is typically found in the nephrotic syndrome. There is greatly decreased albumin level and a considerably increased alpha-2 level with or without an increase in the beta-globulin level. This differs from the acute stress pattern in that the alpha-2 elevation of the nephrotic syndrome is usually either slightly or moderately greater than that seen in acute reaction, whereas the albumin fraction in the nephrotic syndrome has a definitely greater decrease (sometimes to extremely low levels) than the albumin fraction of acute reaction.

    A fourth pattern represents changes suggestive of far-advanced cirrhosis. It consists of a decreased albumin level with a moderately or considerably increased gamma-globulin level and variable degrees of incorporation of the beta peak into the gamma. The more pronounced the beta-gamma bridging becomes, the more suggestive the pattern is for cirrhosis. However, complete incorporation of the beta peak into the gamma region is actually uncommon, since it is found in only about 20% of well-established cases of cirrhosis. About 10% of cirrhotic patients have no gamma elevation at all, about 25% have mild to moderate gamma elevation without any beta-gamma bridging, about 8% have marked gamma elevation without any bridging, and about 33% have mild to moderate gamma elevation with mild to moderate beta-gamma bridging but without complete loss of the beta peak. Surprisingly, the electrophoretic pattern correlates poorly with degree of liver function abnormality. Correlation is not consistent even with microscopic findings at autopsy, although there is a tendency for more pronounced electrophoretic changes to be associated with more advanced microscopic abnormality.

    A fifth pattern consists of a polyclonal gamma-globulin elevation, that is, a greatly increased gamma-globulin level that involves the entire gamma zone rather than a focal area and does not have a thin spikelike appearance. There may or may not be some degree of beta-gamma bridging, but the beta peak does not totally disappear. This pattern is most often seen in some cases of cirrhosis, in patients with chronic infection, in granulomatous diseases such as sarcoidosis or far-advanced pulmonary tuberculosis, in subacute bacterial endocarditis, and certain rheumatoid-collagen diseases such as rheumatoidarthritis systemic lupus or polyarteritis nodosa.

    A sixth pattern consists of hypogammaglobulinemia, defined electrophoretically as decreased gamma-globulin level, usually without very marked changes in other globulin zones. This configuration is suggestive of the light chain variant of multiple myeloma (about 20% of myeloma cases) in which Bence Jones protein is excreted into the urine without a serum myeloma protein being evident with ordinary electrophoretic techniques. Patients with a substantial degree of hypogammaglobulinemia from other causes have the same electrophoretic picture.

    Finally, there is the so-called monoclonal gammopathy spike (M protein and paraprotein are synonyms). This is located in the gamma area (much less frequently in the beta and rarely in the alpha-2) and consists of a high, relatively thin spike configuration that is more homogeneous and needle shaped than the other gamma or beta elevations discussed earlier. The majority of persons with the monoclonal spike on serum electrophoresis have myeloma. However, a sizable minority do not, and these cases are divided among Waldenstrцm’s macroglobulinemia, secondary monoclonal gammopathies, and idiopathic monoclonal gammopathy (discussed later).

    Certain conditions may produce globulin peaks that simulate a small- or medium-sized monoclonal peak rather closely. A configuration of this sort in the alpha-2 area may occur in the nephrotic syndrome or in conditions that produce the acute reaction pattern (the alpha-2 elevation in both cases is due to elevated haptoglobin levels). A small monoclonal-type peak in the beta area may be seen in the third trimester of normal pregnancy or in some patients with chronic iron deficiency anemia (both due to increased transferrin levels). Similar beta region peaks may be caused by the presence of fibrinogen from incompletely clotted blood, by serum free hemoglobin, or by beta-2 microglobulin elevation. In the gamma area, somewhat similar peaks (but usually not as slender and needle like) may be found in rare patients with chronic disease, most often of the granulomatous type.

  • Serum Protein Electrophoresis

    Electrophoresis is still the most commonly used screening test for serum protein abnormalities except for measurement of albumin only. Before electrophoresis was readily available, the albumin/globulin ratio was widely used; this was determined with a chemical method and should no longer be ordered, since electrophoresis not only provides the same information but also pinpoints areas where globulin abnormalities may be found. Various electrophoretic methods produce some differences within the same basic framework of results due to differences in technical factors and the type of electrophoretic migrating field material used. Some of the standard materials include filter paper, cellulose acetate film, agarose gel, and polyacrylamide gel. Each method has advantages and disadvantages in terms of laboratory ease of performance, cost, or sensitivity to certain protein fractions. However, all are capable of identifying the areas where major serum protein shifts take place, which sometimes have as much diagnostic importance as the shifts themselves.

    Serum protein electrophoresis ordinarily displays bands corresponding to albumin, alpha-1 and alpha-2 globulins, beta globulins, and gamma globulins. A special instrument (densitometer) translates the quantity of protein or density of these bands into a linear pattern, which is a rough visual approximation of the amount of substance present. The main problem with this method is the difficulty in separating some of the serum components. It has been found that by using potato starch in a gel-like state, the separation of some of these fractions can be sharpened, especially the separation of some of the abnormal hemoglobins. Polyacrylamide gel has also been used. However, these procedures are technically somewhat more difficult than the other techniques, so that filter paper, cellulose acetate, and agarose gel remain the routine clinical laboratory methods of choice. On filter paper, cellulose acetate, or agarose, the alpha-2 fraction migrates faster toward the anode than the beta fraction. On starch or polyacrylamide gel, the reverse occurs.

    Proteins visualized by serum protein electrophoresis: acute reaction proteins. Certain reasonably predictable changes take place in plasma protein levels in response to acute illness, including acute inflammation, trauma, necrosis, infarction, burns, and chemical injury. The same changes may occur in focal episodes associated with malignant tumors (possibly due to infarction of a portion of the tumor). The acute reaction protein pattern has also been called “acute inflammatory response pattern,” “acute stress pattern,” and “acute-phase protein pattern.” The protein changes involved are increases in fibrinogen, alpha-1 antitrypsin, haptoglobin, ceruloplasmin, C-reactive protein (CRP), C3 portion of complement, and alpha-1 acid glycoprotein (orosomucoid) levels. There frequently is an associated decrease in albumin and transferrin levels. Of those proteins that are increased, the greatest effect is produced by haptoglobin, alpha-1 antitrypsin, CRP, and fibrinogen. The CRP level increase begins approximately 4-6 hours after onset of the acute episode, with alterations of the other proteins occurring 12-36 hours after onset. CRP is located in the beta-gamma interface area on electrophoresis, and a distinct peak is not usually seen. Fibrinogen is normally not present in serum (unless the blood has not completely clotted), so ordinarily it is not detected on serum electrophoresis. Haptoglobin migrates in the alpha-2 region, with the result that alpha-2 elevation is the most common abnormality associated with acute reaction. An alpha-1 elevation due to alpha-1 antitrypsin increase is seen less frequently but can be present. Albumin is often decreased under acute reaction conditions, presumably due to decreased liver synthesis, for which there often is no good explanation. Albumin is not always decreased; due to the wide reference range, a substantial reduction could occur in a person whose normal level is toward the upper end of the reference range, with the final level remaining within the lower reference range despite the reduction. A decrease in transferrin levels is usually not seen on electrophoresis but is manifested by a decrease in the total iron-binding capacity. In summary, the typical electrophoretic change of acute reaction is increased alpha-2 globulin, frequently associated with decreased albumin and sometimes with increased alpha-1 globulin. Acute reaction frequently is accompanied by a polymorphonuclear leukocytosis and usually by an increase in CRP test values (especially when using immunoassay methods) and an increased erythrocyte sedimentation rate (ESR).

    Serum albumin. Elevation of the serum albumin level is very unusual other than in dehydration. Most changes involve diminution, although the normal range is somewhat large, and small decreases are thus hidden unless the individual patient’s normal levels are known. In pregnancy, albumin levels decrease progressively until delivery and do not return to normal until about 8 weeks post partum. In infants, adult levels are reached at about age 1 year. Thereafter, serum protein levels are relatively stable except for a gradual decrease after age 70. Malnutrition leads to decreased albumin levels, presumably from lack of essential amino acids, but also possibly from impaired liver manufacture and unknown causes Impaired synthesis may itself be a cause of decreased albumin levels, since it is found in most forms of clinical liver disease, especially cirrhosis. In chronic cachectic or wasting diseases, such as tuberculosis or carcinoma, the albumin level is often decreased, but it is not clear whether this is due to impaired synthesis or to other factors. Chronic infections seem to have much the same effect as the cachectic diseases.

    Serum albumin may be directly lost from the bloodstream by hemorrhage, burns, exudates, or leakage into the gastrointestinal (GI) tract in various protein-losing enteropathies. In the nephrotic syndrome there is a marked decrease in the serum albumin level from direct loss into the urine. Albumin frequently decreases rather quickly in many severe acute illnesses or injuries, beginning at about 12-36 hours, with the average maximal albumin decrease being reached in about 5 days. This is part of the acute reaction pattern described earlier and seems to have a different mechanism from hypoalbuminemia due to protein loss or to malnutrition. Finally, there is a rare genetic cause for low serum albumin levels known as “familial idiopathic dysproteinemia,” in which the albumin level is greatly decreased while all the globulin fractions are elevated and seem to take over most of the functions of albumin.

    Alpha-1 globulins. The alpha-1 globulin area on electrophoresis is almost 90% alpha-1 antitrypsin. The other 10% includes alpha-1 acid glycoprotein, alpha-fetoprotein, and certain carrier proteins such as cortisol-binding protein (transcortin) and thyroxine-binding globulin (which is actually located more toward the area between alpha-1 and alpha-2). Alpha-1 globulin is increased to some extent (usually not great) in pregnancy and by estrogen administration and in some patients with the acute reaction protein pattern. Alpha-1 globulin is absent or nearly so in alpha-1 antitrypsin deficiency, a hereditary disorder that predisposes to development of emphysema. Serum protein electrophoresis detects homozygous alpha-1 antitrypsin deficiency but frequently displays a normal alpha-1 peak in those who are heterozygous. Immunoassay is needed to detect heterozygotes or to confirm electrophoretic findings.

    Alpha-2 globulins. Alpha-2 globulins include haptoglobin, alpha-2 macroglobulin, and ceruloplasmin. This electrophoretic area is seldom depressed; diminution of one component is usually masked by the other components within the reference range. Haptoglobin values are decreased in severe liver disease, in patients on estrogen therapy, in megaloblastic anemia, and also whenever free hemoglobin appears in the blood, as occurs in red blood cell (RBC) hemolysis or even from resorption of a large hematoma located outside the vascular system. Haptoglobin levels are increased by adrenocorticosteroid therapy. Ceruloplasmin levels are decreased in Wilson’s disease, malnutrition, nephrotic syndrome, and protein-losing enteropathy. Ceruloplasmin levels are increased by estrogen therapy. Both haptoglobin and ceruloplasmin levels are increased, resulting in alpha-2 elevation, in the many disorders that produce the acute reaction pattern.

    In subacute and severe chronic illnesses the acute reaction pattern may exist in a lesser degree or may disappear. The alpha-2 increase usually diminishes and may return to normal. Albumin levels may return to normal, but an albumin decrease frequently persists or may even become more pronounced. Gamma-globulin levels may begin to increase.

    There are certain diseases other than acute injury that often produce alpha-2 increase. In the nephrotic syndrome there is classically a marked alpha-2 peak, which may sometimes be accompanied by a beta-globulin elevation. In addition there is greatly decreased albumin. In hyperthyroidism, far-advanced diabetes, and adrenal insufficiency, there is reportedly a slightly to moderately elevated alpha-2 globulin level in some cases.

    Beta globulins. The beta-globulin zone contains transferrin, beta-lipoprotein, and several components of complement.

    A decrease in the beta-globulin level is not very common. Transferrin is frequently decreased in protein malnutrition. Beta globulin increase may occur in many conditions. Probably the most common source is a nonfasting specimen. An increase in transferrin levels produced by chronic iron deficiency anemia, pregnancy in the third trimester, and free hemoglobin in serum, or fibrinogen from incompletely clotted blood may each produce a spikelike peak in the beta region that simulates a monoclonal peak. In conditions in which serum cholesterol levels are elevated, the beta-globulin levels are also likely to be increased; these include hypothyroidism, biliary cirrhosis, nephrosis, and some cases of diabetes. In liver disease, there is some variability. The beta-globulin level is usually, but not always, elevated in obstructive jaundice. It may be elevated to some extent in many cases of hepatitis but not as often as in obstructive jaundice. It is often elevated in cirrhosis; when so, it is often partially incorporated into the gamma globulin and occasionally does not even appear as a separate peak. This will be discussed later. Finally, beta elevation may occasionally be seen in certain other diseases, including malignant hypertension, Cushing’s disease, polyarteritis nodosa, and sometimes carcinoma. These changes are probably due to increase in complement. A double peak in the beta area is a frequent and normal finding when the cellulose acetate method is used. Electrophoresis on polyacrylamide gel produces reversal of the alpha-2 and beta area positions compared to paper or cellulose acetate electrophoresis (i.e., the beta area on paper becomes alpha-2 on polyacrylamide gel).

    Gamma globulins. The gamma region is predominantly composed of antibodies of the IgG type. Immunoglobulin A, IgM, IgD, and IgE antibodies underlie the beta-gamma junction area. The gamma-globulin zone is decreased in hypogammagobulinemia and agammaglobulinemia, which may be either primary or secondary. The secondary type sometimes may be found in patients on long-term steroid treatment, in the nephrotic syndrome, in occasional patients with overwhelming infection, and in a moderate number of patients with chronic lymphocytic leukemia, lymphocytic lymphoma, or multiple myeloma of the light chain type.

    Many diseases produce an increase in the gamma-globulin level. Many types of infections are followed by an increased gamma-globulin level that reflects antibody production, although the increase is often not sufficient to demonstrate a clear-cut elevation above reference range, especially if the infection is mild or acute. Chronic infections typically produce antibody responses that are prolonged and substantial enough to increase gamma-globulin values above reference limits. Granulomatous diseases such as tuberculosis, sarcoidosis, lymphogranuloma venereum, and tertiary syphilis are also chronic diseases and frequently result in marked gamma increase by the time they become well established. Rheumatoid-collagen diseases, notably rheumatoid arthritis and lupus erythematosis, have electrophoretic gamma values that range from normal to considerably increased. Gamma levels are usually elevated while the disease is active if activity has persisted for several months. Gamma-globulin levels may be increased in some patients with Hodgkin’s disease, malignant lymphoma, and chronic lymphocytic leukemia, although in other patients they may be decreased or normal. Multiple myeloma and Waldenstrцm’s macroglobulinemia characteristically demonstrate a homogeneous spikelike peak in a focal region of the gamma area, which may or may not result in the value for the total gamma area being increased. Liver diseases form a substantial group of etiologies for gamma elevation. In hepatitis there is classically a relatively mild separate increase in both beta- and gamma-globulin levels with a decrease in albumin level, but this does not always occur. About 90% of patients with well-established cirrhosis show some degree of gamma elevation. The gamma elevation is of considerable degree in about 30% of patients and of slight to moderate degree in about 60%. There is no electrophoretic gamma elevation in about 10% of patients with histologically well-established cirrhosis. The most suggestive pattern is a broad-based gamma-globulin elevation plus a fusion of beta and gamma globulin without the usual separation of the two peaks (“beta-gamma bridging”). However, only about 20% of cirrhotics display complete beta-gamma fusion with about 33% more showing partial fusion. In obstructive jaundice, alpha-2, beta, and gamma levels may all become elevated to some degree.

  • Laboratory Problems in Serum Protein Assay Methods

    The various techniques just mentioned are based on different properties of protein molecules (e.g., chemical, dye-binding, electrical charge, antibody binding sites). Therefore, the different techniques may not produce identical values for all protein fractions or for individual proteins. Added to this are alterations in proteins from disease or genetic abnormalities, different interfering substances or medications, and technical problems unique or important for each technique or method. For example, the serum albumin value by electrophoresis may be about 0.5 gm/100 ml (5 g/L) less than the value determined by the usual chemical method. This is fortunately not often very important as long as the same laboratory performs the tests on the same patient and provides its own reference range. The differences in technique and methodology are magnified when the quantity of protein being assayed is small, such as in urine or spinal fluid. Since even modifications of the same method can produce slightly but significantly different results, reference values should be obtained by the individual laboratory for its own particular procedure.

  • Serum Protein Assay Methods

    There are many widely used techniques for fractionating the serum proteins. “Salting out” by differential chemical solubility yields rough separation into albumin and globulin. Cohn devised a more complicated chemical fractionation method by which certain parts of the protein spectrum may be separated from one another in a large-scale industrial-type procedure. Albumin is most often assayed by a chemical method (biuret) that reacts with nitrogen atoms, or with a dye (such as bromcresol green or bromcresol purple) that preferentially binds to albumin. The ultracentrifuge has been used to study some of the subgroups of the globulins. This is possible because the sedimentation rate at high speeds depends on the molecular size and shape, the type of solvent used to suspend the protein, and the force of centrifugation. The velocity of any particular class of globulins under standard conditions depends primarily on molecular size and is known as the “Svedberg number”; the most common classes of globulins are designated as 7S, 19S, and 22S. Electrophoresis separates molecules by the electrical charge of certain structural atomic configurations and is able to subdivide the globulins, but only into groups rather than into individual proteins. Serum protein nomenclature derived from electrophoresis subgroups the serum globulins into alpha, beta, and gamma, corresponding to electrophoretic mobility. Using antibodies against antigens on the protein molecule, immunoassay (including radial immunodiffusion, Laurell “rocket” electroimmunodiffusion, immunonephelometry, immunofluorometry, and radioimmunoassay, among others) is another technique for measuring serum proteins that is assuming great importance. Immunoassay techniques quantitate individual proteins rather than protein groups and in general produce reliable results with excellent sensitivity and specificity. Immunoelectrophoresis or similar techniques such as immunofixation goes one step beyond immunoassay and separates some of the individual globulin molecules into structural components or into subclasses. Immunoelectrophoresis also can detect abnormal proteins that either differ structurally from normal proteins or are produced with different proportions of structural components.

  • Serum Proteins

    Serum protein is composed of albumin and globulin, either nonbound or acting as carrier proteins. The word “globulin” is an old chemical fractionation term that refers to the non-albumin portion of serum protein; it was subsequently found that this portion includes a heterologous group of proteins such as glycoproteins, lipoproteins, and immunoglobulins. Most globulin molecules are considerably larger than albumin, although the total quantity of albumin is normally two to three times the level of globulin. Albumin seems most active in maintaining the serum oncotic pressure, where it normally has about 4 times as much importance as globulin, accounting for about 80% of the plasma oncotic pressure. Albumin also acts as a transport protein for some drugs and a few other substances. The globulins have more varied assignments than albumin and form the main transport system for various substances as well as constituting the antibody system, the clotting proteins, complement, and certain special-duty substances such as the “acute reaction” proteins. Most serum albumin is produced by the liver. Some of the globulins are produced by the liver, some by the reticuloendothelial system, and some by other tissues or by poorly understood mechanisms.
    Plasma contains fibrinogen in addition to the ordinary serum proteins.

  • Creatine Kinase (CK)

    Creatine kinase (CK) is found in heart muscle, skeletal muscle, and brain. It is elevated at some time in about 90%-93% (literature range, 65%-100%) of patients with acute MI. In acute MI, CK behaves similarly to AST. In addition, elevations have also been reported in myocarditis and also in some patients with tachyarrhythmias (mostly ventricular) for unknown reasons. Acute liver cell damage, which frequently causes an abnormal AST value, has no effect on CK. This is an advantage, since the situation often arises in which an elevated AST (or LDH) level might be due to severe hepatic passive congestion from heart failure rather than from acute MI.

    Use of CK measurements in diagnosing primary diseases of skeletal muscle is discussed elsewhere. A considerable number of conditions associated with acute muscle injury or severe muscle exertion affect CK values. Thus, CK values are usually elevated in muscle trauma, myositis, muscular dystrophy, after surgery, postpartum, after moderately severe exercise (e.g., long-distance running), and in delirium tremens or convulsions. Increased serum values have been reported in about 80% of patients with hypothyroidism (literature range, 20%-100%) and in patients with severe hypokalemia, due to changes induced in skeletal muscle. CK elevation can be due to effects of alcohol on muscle. For example, one study found that CK levels became abnormal after 24-48 hours in the majority of persons following heavy drinking episodes as well as in most patients with delirium tremens. Levels of CK are said to be normal in chronic alcoholics without heavy intake.

    CK levels are frequently elevated after intramuscular injection. Since therapeutic injections are common, this probably constitutes the most frequent cause of CK elevation. Specimens must be drawn before injection or at least within 1 hour after injection. Trauma to muscle makes the CK level unreliable for a few days postoperatively.

    Although CK is present in brain tissue as well as muscle, reports differ to some extent as to the effect of central nervous system (CNS) disease on serum CK levels. According to one report, CK levels may be elevated in a wide variety of conditions that affect the brain, including bacterial meningitis, encephalitis, cerebrovascular accident, hepatic coma, uremic coma, and grand mal epileptic attacks. Elevation is not always present, and when it is present, the degree of elevation varies considerably. Elevations in some patients in acute phases of certain psychiatric diseases, notably schizophrenia have been reported; the cause is not known. According to one report, CK is elevated in 19%-47% of patients with uremia.

    Since the major source for body CK is skeletal muscle, individuals with relatively small muscle mass will tend to have lower normal CK levels than the average person; those with increased muscle mass will tend to have relatively higher normal values. Normal CK values for African-American males are double those for European males; values for African-American and European females are nearly equal in most (but not all) reports.

    The major drawbacks of total CK are (1) the relatively short time period after onset of infarction during which the CK value is elevated and (2) false positive elevations due to skeletal muscle injury (especially intramuscular injections).

    Creatine kinase isoenzyme measurement. Total CK can be separated into 3 major fractions (isoenzymes): CK-BB (CK-1), found predominantly in brain and lung; CK-MM (CK-3), found in skeletal muscle; and the hybrid CK-MB (CK-2), found predominantly in cardiac muscle. CK isoenzyme assays are now available in most hospital laboratories. Isoenzymes offer a way to detect myocardial damage that minimizes skeletal muscle contribution to CK values.

    Creatine kinase MM fraction. CK-MM comprises well over 95% of skeletal muscle CK and about 70%-75% of myocardial CK. Since the total amount of body skeletal muscle is so much greater than myocardium, elevation of the MM fraction is usually due to skeletal muscle injury or hypoxia, including moderately severe or severe exercise, convulsions, inflammation, trauma, intramuscular injection, or muscular dystrophy. Some conditions producing less obvious effects on muscle, such as hypothyroidism and hypokalemia, may also produce CK-MM increase.

    Creatine kinase MB fraction. CK-MB can be reported in two ways: percentage of total CK (MB/total CK) or in mass units (either by multiplying total CK by the percentage of MB value or by assaying the MB fraction directly using immunoassay). The most recommended method is to screen with mass unit values, because a low normal MB value divided by a relatively low total CK value can give a misleading, rather high percentage of MB. Skeletal muscle contains mostly CK-MM isoenzyme, but there is also about 3%-5% MB present (the amount depends on the particular muscle assayed). Therefore, serum MB can be increased over baseline to some degree by sufficient skeletal muscle injury as well as by myocardial muscle injury. When acute skeletal muscle hypoxia or other injury of sufficient degree elevates CK-MB levels above the upper limit of the reference range in terms of CK-MB units, CK-MM levels are usually increased at the same time and to a much greater degree. Because of the concurrent CK-MM increase, the CK-MB level (although it may be increased) usually remains less than a small MB/total CK cutoff value (which ranges in the literature from 2.5%-5% in different laboratories). Therefore, when the CK-MB value is increased, it is very helpful to know the total CK value in order to calculate the percentage of MB relative to total CK (the “relative index”). If the MB value in terms of units is not increased, the percentage of MB is not useful and may be misleading (e.g., if normal CK-MB levels are 0-10 units and normal total CK units are 0-40 units, a CK-MB level of 2 units is 20% of a total CK value of 10 units, even though both values are well within reference range). Each laboratory should determine the MB “relative index” experimentally because of considerable differences in MB and total CK methodology and analysis conditions.

    The CK-MB level begins to rise 3-6 hours after onset of acute MI, reaches a peak in 12-24 hours, and returns to normal in 24-48 hours (sometimes earlier). There is a rough correlation between the size of the infarct and the degree of elevation. Since small infarcts may not elevate the MB fraction dramatically, it is important to time the collection of specimens so as not to miss the peak values. Some recommend five specimens: one immediately, then at 6, 12, 18, and 24 hours afterward. Others use 4 specimens: one immediately, then at 8, 16, and 24 hours or at 6, 12, and 18 hours. Some use 3 specimens: immediately, 12 hours, and 24 hours.

    Some laboratories do not perform CK-MB assay unless the total CK value is elevated. However, reports indicate that about 10% of acute MI patients (literature range 0%-16%) demonstrate elevated CK-MB levels with the total CK value remaining within reference range limits. This is especially common in persons with relatively small muscle mass, whose preinfarct normal total CK value is apt to be in the lower part of the population reference range.

    Since acute myocardial infarct may occur with CK-MB values elevated to levels diagnostic of MI but with total CK remaining within its reference range, the question arises whether there is a cutoff point within the total CK reference range below which CK-MB could be omitted. Zero percent to 16% of patients with acute MI are said to have elevated MB with concurrent normal total CK. Unfortunately, there is controversy surrounding this question and little data on which to base an answer. Much of the older literature is invalid because the MB methods were not specific for MB; other reports are hindered because MB results were reported only as a percentage of total CK (e.g., an increase of 10 MB units of activity or mass units is a much smaller percentage of total CK when total CK is high in its reference range than when it is low in its reference range). In addition, in some cases there was not adequate confirmation that acute MI had actually occurred (e.g., typical MB rise and fall pattern or LDH-1 isoenzyme fraction becoming elevated and greater than LDH-2). Frequently the location of the total CK values in its reference range when MB was elevated was not disclosed. It would probably be safe to say that MB would be unlikely to suggest acute MI if the total CK value were in the lower half of its reference range (one of 50 consecutive cases of documented acute MI at my hospital).

    There is some dispute as to whether ischemia without actual infarct will elevate either total CK or CK-MB levels. The controversy revolves around the fact that currently there is no way to rule out the possibility that a small infarct may have occurred in a patient who clinically is thought to have only ischemia.

    In equivocal cases it is helpful to obtain LDH isoenzyme determinations as well as CK-MB, both to enhance diagnostic specificity and because specimens for CK-MB may have been obtained too late to detect elevation. If a CK isoenzyme sample is obtained immediately, at 12 hours, and at 24 hours; and LDH isoenzyme determinations are performed at 24 and 48 hours, 95% or more acute MIs detectible by these tests will be documented. Of course, if one of the CK-MB determinations is strongly positive, it might not be necessary to obtain further samples.

    The question sometimes arises whether both CK-MB and LDH isoenzyme fractionation should be done. At our hospital, CK-MB assay is performed immediately and then 12 and 24 hours later; and LDH isoenzyme study is performed at 24 and 48 hours (both using electrophoresis). In 50 consecutive patients with either the CK-MB level elevated or LDH-1 level elevated accompanied by the LDH-1 level becoming greater than the LDH-2 level, CK level was elevated in the first sample in 28% of cases, initially elevated in the 12-hour sample in 50%, initially elevated in the 24-hour specimen in 4%, and not elevated in 18%. The LDH-1/LDH-2 ratio was initially reversed in the 24-hour specimen in 70%, in the 48-hour specimen in 18%, and in neither specimen in 12%. Therefore, with the commonly used protocol, 12% of patients would have been missed relying on LDH isoenzyme studies alone and 18% with CK-MB alone.

    CK-MB fraction and skeletal muscle

    Although CK-MB is found mainly in cardiac muscle (25%-30%; range, 14%-42% of total myocardial CK), skeletal muscle CK contains a relatively small amount of CK-MB (3%-5% as noted previously). Skeletal muscle contains two types of muscle fibers. Type 1 predominates in muscles such as the gastrocnemius; total CK of type 1 fibers only contains about 1% CK-MB (range, 0%-4%) in addition to CK-MM. Type 2 fibers predominate in other muscles such as the intercostal and soleus and comprise 40% of the fibers in other muscles such as the quadriceps. The total CK of type 2 fibers contains 2%-10% CK-MB in addition to CK-MM. In Duchenne’s muscular dystrophy, especially in the earlier stages, serum levels of CK-MB may be increased, presumably due to skeletal muscle type 2 involvement, with CK-MM levels increased to a much greater degree. An increase in CK-MB value may also occur in some patients with gangrene or severe ischemia of the extremities. In addition, patients with acute myositis of various types, long-distance runners, idiopathic myoglobinemia, Reye’s syndrome, and Rocky Mountain spotted fever frequently display some degree of CK-MB elevation. One study found that intestinal infarct raises CK-MB (both quantitative and %MB) without reversal of the LDH-1/LDH-2 ratio.

    CK-MB fraction in defibrillation, coronary angiography, and coronary angioplasty

    In patients undergoing defibrillation or cardiac resuscitation, the CK-MB value is usually normal unless actual myocardial injury (e.g., contusion) takes place. At least one report indicates that the CK-MB value is normal in most patients with myocarditis, pericarditis, and subacute bacterial endocarditis, even when LDH isoenzymes display a “cardiac” pattern. However, other investigators have reported elevated MB values in some patients with active myocarditis. Coronary angiography and even coronary angioplasty are likewise reported to be associated with normal MB values unless some degree of myocardial infarction is occurring. In open heart surgery, such as coronary artery revascularization, a CK-MB isoenzyme elevation raises the question of MI, even though operative manipulation of the heart takes place. Nearly 15% of such patients (ranging from 1%-37%) have displayed CK-MB elevation just before operation. Others have CK-MB increase during institution of cardiopulmonary bypass or during anesthesia induction before actual cardiac surgery begins.

    Creatine kinase MB fraction; some technical problems and pseudo-MB substances. CK isoenzyme fractionation can be performed in several ways. The most commonly used techniques are column chromatography, electrophoresis, and immunoinhibition. The accuracy of column chromatography depends to some extent on the method used. The major disadvantage is the tendency in some kits for some MM isoenzyme to carry over into the MB fraction if the value of the MM fraction is greatly increased. In addition, some patients have a variant of BB isoenzyme called “macro-CK” (or “macro-CK type 1”) in which BB isoenzyme becomes complexed to gamma globulin, with the resulting complex migrating electrophoretically between MM and MB instead of in the usual BB location on the opposite (anodal) side of MB from MM. Macro-CK is carried into the MB elution fraction in ordinary CK isoenzyme column methods and, if it is present, can falsely increase apparent MB values. Macro-CK is reported present in about 1% of all CK-MB isoenzyme studies (literature range, 0.2%-1.8%), and in one report it accounted for about 8% of elevated CK-MB results. Another CK-MB method is electrophoresis. Electrophoresis is reasonably sensitive, although there is some variation among different methods. Electrophoresis is more specific than column chromatography (and immunoinhibition methods, discussed later) since the various isoenzyme fractions can be inspected visually, and therefore atypically migrating CK variants such as macro-CK can be detected. However, macro-CK may be falsely included with CK-MB even with electrophoresis when automatic electrophoretic fraction quantitation devices are used or the technologist does not inspect the pattern carefully before manually quantitating the major isoenzyme fractions. Immunoinhibition methods are becoming widely used. Most kits currently available are based on elimination of the M subunit and measurement of the B subunit. This measures both MB and BB together. The rationale for this is the infrequency of elevated BB values. However, macro-CK is detected and produces falsely elevated MB results. Several immunoassay methods that are not affected by macro-CK are now available, and it is helpful to ask the laboratory whether their method will be affected.

    Other variant CK isoenzymes besides macro-CK have been reported. The best characterized one is a macromolecular CK isoenzyme variant derived from mitochondria (mitochondrial CK, or macro-CK type 2), which electrophoretically migrates cathodal to MM (on the other side of MM from MB). In the few reports in which data are provided, 0.8%-2.6% of patients who had CK-MB studies done had mitochondrial CK present. It was present more often in patients with cancer than in those with other conditions and tended to be associated with a poor prognosis. In addition to CK isoenzyme variants, there is an enzyme called “adenylate kinase,” present in RBCs as well as certain other tissues, which electrophoretically migrates in the same area as mitochondrial CK. Specimens with visible degrees of RBC hemolysis may contain increased adenylate kinase, which, in turn, may falsely increase (apparent) CK-MB results unless there is some way to detect the problem. Hemolysis, whether artifactual or due to hemolytic or megaloblastic anemia; renal cortex infarct; and some germ cell tumors may also elevate LDH-1 values, which adds to the potential for misdiagnosis of acute MI.

    Immunoassay methods specific for MB using monoclonal antibodies are now commercially available; these methods do not detect macro-CK or other atypical CK variants. Some laboratories use nonspecific MB methods only; some screen for elevated MB using a nonspecific method (which is usually less expensive than a specific method) and use a specific method to confirm any elevated specimen result; some use only a specific method. It is important to know whether any laboratory is using only a nonspecific method. In fact, even some of the MB-specific kits may produce false positive results in uncommon cases where the anti-MB antibody cross-reacts with a heterophilic or HAMA antibody against the animal tissue from which the monoclonal anti-MB antibody originated. However, if this happens all MB specimens would likely be elevated at approximately the same degree without the “rise and fall” pattern expected in acute MI.

    Creatine kinase BB Fraction. Elevation of the CK-BB value is not common. It is occasionally elevated after pulmonary embolization, and one report indicated that it may occasionally be increased in some patients who undergo cardiopulmonary resuscitation or in some patients in shock. It has also been reported in a few patients with some types of cancer (more often widespread and most frequently in prostate and lung small cell carcinoma) but not sufficiently often to be useful as a screening test. In brain disorders, where one theoretically would expect release of CK-BB, the actual isoenzyme detected in serum is usually CK-MM. In some patients (especially those with chronic renal disease) an unusually accentuated albumin may be mistaken for CK-BB on electrophoresis, since albumin migrates between MB and BB.

    CK-MM and CK-MB isoforms in acute myocardial infarction. There are three major CK isoenzymes, but each of the three has now been shown to contain subforms (isoforms). The isoform of CK-MM in myocardium is known as MM3; and when MM3 is released into blood during myocardial injury, carboxypeptidase-N enzyme in serum converts MM3 to MM2 isoform; later, MM2 is further converted to MM1. An MM3/MM1 ratio of more than 1.0 suggests myocardial or skeletal muscle injury. The rapid release of the tissue isoform MM3 from damaged myocardium creates a significant excess of MM3 over MM1 considerably sooner than the serum peak values of either MM3 or MM1 are reached. Therefore, most investigators used the MM3/MM1 ratio instead of one of the individual isoforms. The ratio is said to become abnormal 2-4 hours (or even earlier) after onset of acute MI and peaks before either its individual isoforms or CK-MB. In one study the MM3/MM1 ratio was elevated in 90% of acute MI patients by 6-9 hours, whereas 79% of patients had an elevated CK-MB at the same time interval. Thus, the isoform ratio measurement is similar to the myoglobin measurement regarding early abnormality. Unfortunately, like myoglobin, skeletal muscle also contains MM isoforms; so that cardiac MM isoform assay, while a little more specific than myoglobin, is frequently elevated in various types of skeletal muscle injury and in severe or prolonged physical activity.

    CK-MB has only two isoforms: the tissue-specific (unaltered tissue form) MB2 and the serum derivative form MB1, created by action of serum carboxypeptidase-N on MB2 released into blood. Similar to CK-MM isoform measurement, the ratio of MB2/MB1 becomes elevated consistently before elevation of either isoform alone or elevation of CK-MB. A MB2/MB1 ratio greater than 1.0 suggests acute myocardial injury. In several studies, the ratio was elevated in nearly 60%-67% of acute MI patients 2-4 hours after onset of symptoms and in 75%-92% of patients at 4-6 hours (versus 15%-50% of patients when using CK-MB). Also, specificity of the MB isoforms for myocardium is much greater than MM isoforms or myoglobin. However, as in CK-MB itself, the MB isoforms or their ratio may become elevated due to moderately severe or severe skeletal muscle injury. One report suggests that the ratio begins to decline after approximately 12 hours.

    The MM3/MM1 ratio is reported to peak at 4.0-5.5 hours after successful reperfusion, with reported sensitivity of about 85%. The MB2/MB1 ratio peaks at about 1.5 hours (range, 0.8-2.3 hours) after successful reperfusion.

    Troponin-T

    Troponin-T is a regulatory protein located in skeletal and cardiac muscle fibers. Skeletal muscle and myocardium have different forms of this protein, so that the antibody that detects cardiac troponin-T in serum is a specific test for myocardial fiber injury. After onset of acute MI, the troponin-T level begins to increase in about 4-6 hours, peaks at about 11 hours (range, 10-24 hours), and returns to reference range in 10 days or more. Therefore, Troponin-T behaves like CK-MB but remains elevated much longer.

    Troponin-I

    Troponin-I is a regulatory protein in the troponin cardiac muscle complex. This protein, like troponin-T, is specific for myocardium. The troponin-I level becomes elevated after onset of acute MI in about 4-6 hours (range, 3-19 hours), peaks at about 11 hours (range, 10-24 hours), and returns to reference range in about 4 days (range, 3.5-6 days). Therefore, troponin-I behaves much like troponin-T, except that return to reference range is faster. Response to acute MI is also somewhat similar to CK-MB. However, troponin-I may become elevated a little sooner than CK-MB in the first 4 hours after onset of acute MI (in one study, troponin-I was elevated at or before the fourth hour in 44% of MI patients, whereas when using CK-MB only 17% had elevated levels).

    Myoglobin in diagnosis of myocardial infarct

    Myoglobin is found in cardiac and skeletal muscle and is currently measured by immunoassay. The studies so far available indicate that serum myoglobin levels after onset of acute MI become elevated in about 3 hours (range, 1.5-6.5 hours), reach a peak in about 9 hours (range, 4-16 hours), and return to normal range in about 30 hours (range, 12-54 hours). By 4 hours serum myoglobin values are elevated in about 50%-60% of patients (range, 26%-100%). By 6-8 hours, about 85%-95% of patients have elevated values (range, 54%-100%). By 12-24 hours, after admission, levels remain elevated in fewer than 50% of patients. The serum myoglobin “false positive” rate in patients with chest pain but without strong evidence of acute MI has been reported to range from 0% to 50%. The major attraction of myoglobin in acute MI is elevation in a substantially greater percentage of patients with acute MI than seen with CK-MB before the desirable cutoff point of 6 hours after onset of MI, during which thrombolytic therapy has greatest benefit. However, the substantially greater false positive rate compared to CK-MB, mostly due to skeletal muscle etiology, has counterbalanced this advantage.

    Myoglobin is excreted in urine by renal glomerular filtration. There is evidence that myoglobinuria may appear as early as 3 hours after onset of infarct symptoms. In several patients, urine values returned to upper normal limits by 30 hours but remained elevated longer in most cases. Another study recorded 90% sensitivity 24 and 48 hours after hospital admission, diminishing to 76% by 72 hours.

    Besides cardiac muscle injury, skeletal muscle injury of various etiologies and other conditions may produce myoglobinemia. Since myoglobin is excreted through the kidneys, poor renal function can elevate serum levels. On the other hand, a sufficient degree of myoglobinuria (or hemoglobinuria) may cause renal failure. To screen for myoglobinuria, the simplest method is a dipstick urine test for blood (hemoglobin); this also reacts with urine myoglobin. Some recommend diluting the urine specimen 1:20 or even 1:40 to avoid detecting minor or clinically unimportant degrees of myoglobinuria. If positive, there are several chemical tests for myoglobin, none of which are completely satisfactory. Serum LDH isoenzyme fractionation by electrophoresis may be helpful; hemolysis elevates LDH-1, whereas myoglobin elevates LDH-4 and 5. An immunologic assay specific for myoglobin (which may have to be performed on serum) is the best diagnostic procedure, if available.

    Myosin light chains (MLCs)

    Myosin light chains (MLCs) are structural components of myosin, which with actin comprise the contractile proteins of muscle (including cardiac muscle). Each myosin molecule is composed of two heavy chains and two pairs of light chains (MLC-I and MLC-II). MLCs are released about 3-8 hours after onset of acute MI and the MLC value peaks at about 24-36 hours, remaining elevated for 10 days or more. Therefore, MLC assay behaves in the early phase like CK-MB and overall like troponin-T. MLCs are not cardiac-specific, and the MLC value becomes elevated in skeletal muscle injury.

    Causes of myoglobinemia and myoglobinuria
    Trauma and ischemic disease
    Acute myocardial infarct
    Muscle arterial ischemia
    Surgical procedures
    Muscle crush (trauma; pressure while immobile)
    Burns; electric shock
    Intramuscular injections (conflicting reports)
    Metabolic disorders
    Alcoholic myopathy
    Potassium depletion
    Hypothermia
    Myxedema
    Muscle exertion
    Convulsions
    Delirium tremens
    Severe exercise
    Heat cramps
    Muscle noninfectious inflammation/degeneration
    Systemic lupus
    Muscular dystrophies
    Dermatomyositis/myositis
    Infections/fever
    Viral influenza and various viral and bacterial infections
    Tetanus
    Gas gangrene
    Toxicity
    Carbon monoxide
    Certain drugs
    Miscellaneous
    Renal failure (myoglobinemia due to poor excretion)

    Enzymatic estimation of myocardial infarct size

    Several investigators reported that there was a reasonably good correlation between infarct size and the peak value of either total CK or CK-MB. Most investigators favor CK-MB because of its greater specificity for myocardium (especially in view of the effect of intramuscular injections on total CK). However, some reports indicate that infarct location is as important as size, and other reports did not find consistent clear-cut differences in prognosis between a considerable number of infarcts with moderate differences in CK-MB values. In addition to total CK and CK-MB, most other tests used to detect acute MI have also been used to estimate infarct size, but none to date has proved completely satisfactory.

    Detection of reperfusion after thrombolytic agents. After interventional therapy for acute MI, not all patients obtain reopening of the affected coronary artery. This information would be useful to assess the need for additional therapy or perhaps for more invasive measures such as balloon angioplasty or coronary artery bypass. Several investigators have found that enzymes released after myocardial fiber damage reach their peak earlier than would be the case if reperfusion did not take place. This is explained on the basis of release of accumulated enzyme after reperfusion occurs (“washout phenomenon”). The reperfusion peak for CK-MB was reached at an average of about 13 hours, whereas the peak for nonreperfused patients was reached at an average of about 18-22 hours. There is disagreement in the reports whether peak enzyme values are greater in the reperfused patients. In addition, although there is clear separation between the mean time duration before the peak in the two groups, in most reports there is overlap between the two groups regarding the time duration of individual patients, with some nonreperfused patients reaching a peak in the same time range as that of some reperfused patients. However, some of the overlap may be due to spontaneous reopening of coronary occlusion or arterial spasm in some nontreated patients. At least partially because of the overlap between patient values, early CK-MB peaking in one study showed a sensitivity of only 69% in detection of reperfusion. Since most of the reports to date have included only relatively small groups of patients, more definitive studies are needed. That includes the CK-MB isoform MB2/MB1 ratio, which is said to peak 1.5 hours after reperfusion (range, 0.8-2.3 hours), substantially earlier than CK-MB.

    Radionuclide heart scan

    Heart scanning can now be performed in two ways. Scan for acute MI is done with technetium pyrophosphate or certain other radiopharmaceuticals. These agents localize in acutely damaged myocardium, producing a focal area of increased radioactivity. The scan is not reliable less than 24 hours after onset of infarct and returns to normal by 6-7 days after infarct. Best results are obtained with transmural infarcts and with locations in the anterior and lateral walls of the left ventricle. Subendocardial infarcts, especially small ones, are much more likely to be missed. Another consideration is the necessity of transporting the patient to the scanner, unless the institution is one of the few that have a portable unit. Combined use of CPK and LDH isoenzyme determinations has lessened scan necessity in diagnosis. Heart scanning may, however, assist in the diagnosis of acute MI that occurs during or after cardiac surgery, when enzyme or isoenzyme diagnosis is not reliable. Some additional areas of difficulty in myocardial scanning include the fact that size of the scan abnormality cannot at present be reliably correlated with infarct size. Ventricular aneurysms may concentrate the phosphate radiopharmaceuticals in a manner suggesting infarct, and this unexplained capability may persist for years.

    Heart scanning can also be done by using radioactive elements, such as thallium, that localize in viable myocardium. Scars and infarcts are seen as areas without uptake. Areas of ischemia (if sufficiently large) also may be detected by scanning before and after exercise. The optimal scan time for acute MI is within 6 hours after onset of chest pain. After 24 hours, reports indicate that as many as 25% of patients may not demonstrate a lesion. Therefore, optimal scan times for thallium and for pyrophosphate are quite different.

    Ultrasound imaging of the heart (echocardiography) in two-dimensional B-mode is now widely used to visualize cardiac ventricle wall motion. A focal motion defect is suggestive of myocardial damage, although acute injury and scar cannot always be differentiated. This imaging technique is attractive because it is relatively easy to do with present-day equipment; is relatively inexpensive; and has good sensitivity compared to other scanning modalities. In addition, left ventricular ejection fraction (used as a parameter of left ventricular function) can be estimated. However, like ultrasound in general, much depends on operator scanning technique and interpretation. M-mode ultrasound can also be used to evaluate small ventricular wall areas.

  • Hydroxybutyric Acid Dehydrogenase (HBD)

    HBD has been used as a substitute for LDH-1 (heart) isoenzyme measurement. Actually, HBD is total LDH that is forced to act on a a-ketobutyric acid substrate instead of pyruvic or lactic acid. Under these conditions, LDH-1 and LDH-2 show relatively greater activity than LDH-5, so that HBD therefore indirectly measures LDH-1 (heart) activity. However, if the LDH-5 (liver) value is elevated sufficiently, it will also produce measurable HBD effect. Therefore, HBD is not as specific as electrophoresis or heat fractionation in separating heart from liver isoenzymes. Nevertheless, since HBD assay is easier to perform (and therefore cheaper) than LDH isoenzyme assay, some follow the practice of using a more specific isoenzyme method if in doubt about LDH heart versus liver contribution. Once there is proof that the heart fraction is elevated, they follow subsequent activity levels with HBD.

    Causes of lactic dehydrogenase fraction 1 elevation
    Acute MI
    Cardiac muscle hypoxia without definite acute MI
    Blood specimen hemolysis
    Hemolytic anemia
    Megaloblastic anemia
    Renal cortex infarct
    Germ cell tumors (some patients)
    LDH isomorphic isoenzyme pattern
    Multiorgan hypoxia
    Neoplasia
    Other conditions (less common; see Fig. 21-1, F)

  • Lactic Dehydrogenase

    Lactic dehydrogenase (LDH) values refer to total serum LDH. Total LDH levels are elevated at some time in 92%-95% (literature range, 82%-100%) of patients with acute MI. Statistics for sensitivity in acute MI refer to multiple sequential LDH specimens and are therefore not valid for any single determination. In acute MI, LDH becomes elevated 24-48 hours after MI, reaches a peak 48-72 hours after MI, and slowly falls to normal in 5-10 days. Thus, LDH values tend to parallel AST values at about double the time interval. Total LDH is slightly more sensitive than AST in acute MI and is reported to be elevated even in small infarcts that show no AST abnormality.

    LDH is found in many organs and tissues. In acute liver cell damage, the total LDH value is not as sensitive as the AST value. In mild acute or chronic passive congestion of the liver, the LDH level is frequently normal or only minimally increased. In moderate or severe congestion, LDH values range from mild to substantial degrees of elevation.

    Since LDH fraction 1 is contained in red blood cells (RBCs) as well as cardiac muscle, LDH is greatly influenced by accidental hemolysis in serum and thus must be collected and transported with care. Heart valve prostheses may produce enough low-grade hemolysis to affect LDH, and LDH levels are also abnormal in many patients with megaloblastic and moderate or severe hemolytic anemias. Skeletal muscle contains LDH, so total LDH (or even hydroxybutyric acid dehydrogenase [HBD]) values are not reliable in the first week after extensive surgery. LDH levels may be elevated in 60%-80% of patients with pulmonary embolism (reports vary from 30%-100%), possibly due to pulmonary tissue damage or to hemorrhage.

    Finally, LDH becomes elevated in some patients with malignant neoplasms and leukemia, and in some patients with uremia.

    The major drawback of total LDH, similar to AST, is the many conditions that can elevate LDH values.

    LDH sites of origin
    Heart
    Liver
    Skeletal muscle
    RBCs
    Kidney
    Neoplasia
    Lung
    Lymphocytes

    Lactic dehydrogenase isoenzymes. Total LDH is actually a group of enzymes. The individual enzymes (isoenzymes) that make up total LDH have different concentrations in different tissues. Therefore, the tissue responsible for an elevated total LDH value may often be identified by fractionation (separation) and measurement of individual isoenzymes. In addition, since the population normal range for total LDH is rather wide, abnormal elevation of one isoenzyme may occur without lifting total LDH out of the total LDH normal range.

    Five main fractions (isoenzymes) of LDH are measured. With use of the standard international nomenclature (early U.S. investigators used opposite terminology), fraction 1 is found mainly in RBCs and in heart and kidney, fraction 3 comes from lung, and fraction 5 is located predominantly in liver and to a lesser extent in skeletal muscle. Skeletal muscle contains some percentage of all the fractions, although fraction 5 predominates. Various methods of isoenzyme separation are available. The two most commonly used are heat and electrophoresis. Heating to 60°C for 30 minutes destroys most activity except that of fractions 1 and 2, the heat-stable fractions. With electrophoresis, the fast-moving fractions are 1 and 2 (heart), whereas the slowest-migrating fraction is 5 (liver). Electrophoresis has the advantage that one can see the relative contribution of all five fractions. Immunologic methods to detect LDH-1 are also available.

    The relative specificity of LDH isoenzymes is very useful because of the large number of diseases that affect standard heart enzyme tests. For example, one study of patients in hemorrhagic shock with no evidence of heart disease found an elevated AST level in 70%, an elevated total LDH level in 52%, and an elevated alanine aminotransferase (ALT); (formerly serum glutamate pyruvate transaminase) level in 37%. LDH enzyme fractionation offers a way to diagnose MI when liver damage is suspected of contributing to total LDH increase. In liver damage without MI, fraction 1 is usually normal, and most of the increase is due to fraction 5.

    Several characteristic LDH isoenzyme patterns are illustrated in Fig. 21-1. However, not all patients with the diseases listed necessarily have the “appropriate” isoenzyme configuration; the frequency with which the pattern occurs depends on the particular disease and the circumstances. Multiorgan disease can be a problem since it may produce combinations of the various patterns. For example, in acute MI the typical pattern is elevation of LDH-1 values with LDH-1 values greater than LDH-2. However, acute MI can lead to pulmonary congestion or hypoxia, with elevation of LDH-2 and LDH-3 values, and may also produce liver congestion or hypoxia, with elevation of LDH-4 and LDH-5 values. Acute MI can also produce multiorgan hypoxia or shock. In shock all LDH fractions tend to be elevated, and in severe cases the various fractions tend to move toward equal height. In malignancy, there may be midzone elevation only, elevation of only fraction 4 and 5, or elevation of all fractions. In my experience, the most common pattern in malignancy is elevation of all fractions with normal relationships preserved between the fractions.

    Fig. 21-1 Representative LDH isoenzyme patterns with most frequent etiologies. A, normal. B, Fraction 1 increased with fraction 1 greater than fraction 2: acute MI; artifactual hemolysis; hemolytic or megaloblastic anemia (cellulose acetate method, not agarose gel); renal cortex infarct; germ cell tumors. C, Fraction 5 increased: acute hepatocellular injury (hepatitis, passive congestion, active cirrhosis, etc); acute skeletal muscle injury. D, Fractions 2 and 3 elevated: pulmonary hypoxia (pulmonary embolization, cardiac failure, extensive pneumonia, etc); pulmonary congestion, lymphoproliferative disorders, myeloma, viral pulmonary infection. E, Fractions 2 through 5 elevated: lung plus liver abnormality (pulmonary hypoxia and/or congestion plus liver congestion, infectious mononucleosis or cytomegalovirus infection, lymphoproliferative disorders). F, All fractions elevated, relatively normal relationships preserved between the fractions (fraction 5 sometimes elevated disproportionately): multiorgan hypoxia and/or congestion (with or without acute MI); malignancy; occasionally in other disorders (trauma, infection/inflammation, active cirrhosis, chronic obstructive pulmonary disease, uremia, etc.)

    The LDH isoenzymes may be of help in evaluating postsurgical chest pain. Skeletal muscle mostly contains fraction 5 but also some fraction 1, so total LDH, HBD, or fraction 1 elevations are not reliable during the first week after extensive surgery. However, a normal LDH-1 value in samples obtained both at 24 and 48 hours after onset of symptoms is considerable evidence against acute MI, and an elevation of the LDH-1 value with the LDH-1 value greater than LDH-2 is evidence for acute MI. However, there have been reports that some athletes engaged in unusually strenuous (e.g., distance running) activity had reversal of the LDH-1/LDH-2 ratio after completing a race, and one report found that almost half of a group of highly trained star college basketball players had reversed LDH-1/LDH-2 ratios at the beginning of team practice for the basketball season. CK isoenzyme levels, if available, are of greater assistance than LDH isoenzyme levels in the first 24 hours.

    Immunoassay methods are now available for measurement of LDH-1 alone. Measurement of LDH-1 has been claimed to be superior to the LDH-1/LDH-2 ratio in diagnosis of acute MI. The typical reversal of the LDH-1/LDH-2 ratio is found in only 80%-85% of patients (literature range, 61%-95%) with acute MI. In some cases, reversal of the ratio is prevented (masked) by an increase of LDH-2 values due to pulmonary hypoxia occurring concurrently with the increase of LDH-1 values due to MI. An elevated LDH-1 fraction as demonstrated on immunoassay is more sensitive (detection rate about 95%; literature range, 86%-100%) than reversal of the LDH-1/LDH-2 ratio in acute MI. However, in my experience and that of others, LDH-1 values can be increased in myocardial hypoxia without any definite evidence of acute MI (e.g., in hypovolemic shock). In addition, the LDH-1 value by immunoassay is increased by all the noncardiac conditions that reverse the LDH-1/LDH-2 ratio (hemolytic or megaloblastic anemia, renal cortex infarct). Thus, the increase in sensitivity gained by LDH-1 immunoassay in acute MI must be weighed against the possible loss of specificity(increase in LDH-1 values not due to acute MI, see the box on this page). LDH isoenzyme fractionation by electrophoresis also demonstrates increased LDH-1 values even when the LDH-1/LDH-2 ratio is not reversed.

    Most LDH fractions are stable for several days at refrigerator temperature. If the specimen is frozen, LDH-5 rapidly decreases.

  • Aspartate Aminotransferase (AST)

    Certain enzymes are present in cardiac muscle that are released when tissue necrosis occurs. Serum aspartate aminotransferase (AST, formerly oxaloacetic transaminase, or SGOT) is elevated at some time in 90%-95% of acute MI patients (literature range, 87%-97%). These statistics for sensitivity of the AST in acute MI are based on multiple sequential AST determinations and are therefore not valid for any single determination. Aspartate transaminase levels become elevated 8-12 hours after infarction, reach a peak 24-48 hours after infarction, and fall to normal within 3-8 days. The AST blood levels correlate very roughly with the extent of infarct and may be only transiently and minimally abnormal.

    Besides myocardial injury, the AST level may become elevated because of acute damage to parenchymal cells of the liver, skeletal muscle, kidney, and pancreas. Abnormality due to liver cell injury is especially common (e.g., liver congestion, active cirrhosis, and acute or chronic hepatitis), but an increased AST level will occur with sufficient degrees of skeletal muscle injury (including trauma or extensive surgical damage) and is also found fairly frequently in acute pancreatitis. In some of these situations, myocardial infarct may have to be considered in the differential diagnosis of the patient’s symptoms. Chronic hypokalemia may elevate both AST and also creatine kinase (CK) levels; morphine and meperidine (Demerol) may temporarily raise AST levels, and AST elevations have been reported in occasional patients receiving warfarin sodium (Coumadin) anticoagulant therapy and in some patients taking large doses of salicylates.

    The major drawbacks of the AST in diagnosis of acute MI are the many conditions (especially liver congestion) that can produce AST elevation.