Articles on Medical Diseases and Conditions

Tag: Serum Proteins

  • Lipoprotein Phenotyping

    In 1965, Frederickson, Levy, and Lees published an article that caught the attention of the medical world. They divided lipoprotein disorders into five basic “phenotypes,” based primarily on electrophoresis of serum obtained after 10 hours of overnight fasting. The sixth phenotype was added later when type II was split into IIa and IIb. Lipoprotein phenotyping was originally proposed as a means of classifying congenital disorders of lipid metabolism according to the lipoprotein abnormality involved to provide more specific therapy. In this way abnormal serum levels of specific lipids such as cholesterol could be traced to abnormalities in specific lipoprotein groups, which, in turn, could suggest certain congenital or acquired etiologies. In time, however, the original intent and limitations of the system were sometimes forgotten, and treatment was sometimes begun—based on the phenotype suggested by the patient’s lipid profile or lipoprotein electrophoretic pattern—as though the patient had a congenital lipoprotein disease. Congenital disease cannot be treated directly, so therapy is directed against the abnormal lipids. Since congenital disease is present in only a small percentage of persons with abnormal serum lipid values, and since lipid disorders due to acquired conditions are best treated by therapy directed at the condition responsible for the lipid abnormality, some of these persons were not being managed appropriately. In other words, the symptoms (abnormal levels of lipids or lipoproteins) were being treated rather than the underlying etiology.

    Laboratory tests in lipoprotein disorders

    Screening tests for lipoprotein abnormality include determination of serum cholesterol and TG levels plus visual inspection of serum (or plasma) for presence of chylomicrons after the specimen has been kept overnight at refrigerator temperature. After this incubation, chylomicrons will rise to the surface as a creamlike surface layer. If the serum or plasma remains cloudy without formation of a definite surface layer, this represents VLDLs. The specimen should be obtained after a 10- to 12-hour fast. This triad of tests serves not only as a screening procedure but in the majority of cases is sufficient to establish the phenotype. Normal results on all three tests are reasonable evidence against serious lipoprotein disease. However, occasional patients with disease can be missed, due sometimes to laboratory variation, borderline abnormality, or the overlap of normal and abnormal persons in statistically established reference ranges referred to earlier. Lipoprotein electrophoresis and ultracentrifugation are useful in a minority of cases as confirmatory or diagnostic tests. Electrophoresis is helpful in differentiating Frederickson type I from type V disease, some cases of type IIa from IIb, and some cases of type II from type IV. Ultracentrifugation is useful mainly in diagnosis of type III disease. Electrophoresis is not needed in the majority of patients. Lipoprotein phenotyping is done preferably on outpatients rather than hospitalized patients due to the short-term effects of serious illness on lipid metabolism as well as the other factors mentioned in the previous discussion of serum cholesterol measurement. However, screening of hospital inpatients can detect possible abnormality that can be verified later under more basal conditions.

    A short summary of lipoprotein phenotype patterns shows that the presence of a creamlike layer of chylomicrons after overnight incubation of fasting serum in the refrigerator indicates type I or type V. If the serum below the chylomicron layer is clear, this suggests type I; if the serum below the chylomicron layer is cloudy or turbid, this suggests type V. Fasting serum obtained from patients with the other phenotypes does not contain chylomicrons. If fasting serum does not contain chylomicrons, elevation of serum cholesterol levels with normal TG levels suggests type II disease, whereas the reverse suggests type IV disease. If both cholesterol and TG levels are significantly abnormal, the disease may be type II or type III. Type II disease has recently been subdivided into IIa and IIb. Type IIa has increased cholesterol but normal TG. Type IIb has elevated cholesterol and TG. Type III is uncommon. It is similar to IIb in that both cholesterol and TG levels are elevated, but it frequently has a slightly different electrophoretic pattern (broad beta) and always has a peculiar “floating beta” component (a beta-mobility protein that floats at 1.006 density instead of 1.013), which can be demonstrated only by ultracentrifugation. Type IV phenotype has elevated TG and normal cholesterol levels.

    Although the majority of patients can be phenotyped using the triad of biochemical tests, some overlap occurs among the phenotypes because cholesterol is present to some extent in all of the major lipoprotein fractions and because triglyceride is found in both chylomicrons and the VLDL fractions. Also, the laboratory reference range may inadvertently influence a phenotype decision in some cases, depending on whether the upper limit of the reference range was derived from a sample of the local population, obtained from data published in the literature, or structured according to findings in populations with low risk of atherosclerosis.

    Phenotypes I, III, and V are uncommon. Phenotypes II and IV constitute the majority of hyperlipoproteinemias. Type IV is probably more common than type II. Most type IV patients have the acquired form. The majority of type II patients also have the acquired form (the most common etiology being a high-cholesterol diet), but type II is more frequently congenital than type IV.

    Some serum specimens from patients with types IIb, III, or IV disease may have a somewhat cloudy or faintly milky appearance. This must be differentiated from the thicker, creamlike precipitate characteristic of increased chylomicrons.

    Certain considerations affect interpretation of these laboratory results. Patients should be on a normal diet for several days before testing and must be fasting for at least 10 hours before a specimen is drawn. If a test cannot be done the same day, the serum must be refrigerated but not frozen; freezing alters prebeta and chylomicron fractions, although cholesterol and triglyceride determinations can be done. Various diseases may produce certain phenotype patterns or may falsely change one pattern into another. Changes in diet, medications, activity levels, stress, and other factors may alter a mild or borderline abnormality or produce mild abnormality. In addition, the laboratory results have ± 5%-10% built-in variability for technical reasons.

    On electrophoresis, occasional persons display increased prebeta but normal TG levels. If laboratory error is ruled out, these patients may have a congenital variant called “Lp system” or “sinking prebeta.” This is found in up to 10% of the U.S. population and is the same as the lipoprotein group now collectively called Lp(a). Lp(a) was discussed earlier as an independent risk factor for CHD.

    Plasma or serum may be used for lipoprotein analysis. Plasma collected with EDTA is preferred if the specimen cannot be tested the same day. Plasma values for TG are about 2%-4% lower than for serum.

    Two rare diseases display characteristic lipoprotein patterns on electrophoresis. Tangier disease has no alpha peak. Bassen-Kornzweig syndrome (associated with “pincushion” RBCs called acanthocytes, and neurologic abnormalities) lacks a beta peak.

  • Laboratory Tests Used to Assess Risk of Coronary Heart Disease (CHD)

    There has been much interest in the significance of the lipoproteins in atherosclerosis. Large numbers of studies have been carried out, different populations have been examined, various diets have been tried, and endless pages of statistics have been published. Several laboratory assays that have general, but sometimes not unanimous, acceptance as predictors of atherosclerotic risk have emerged from these data. Some of these risk factors include cigarette smoking, fibrinogen (which can be elevated in part due to cigarette smoking but is still a risk factor in nonsmokers), diabetes mellitus, hypertension, and various serum lipids. Discussion follows of laboratory tests currently used to assess the level of coronary heart disease (CHD) risk induced by various risk factors.

    Serum (total) cholesterol. Total serum cholesterol comprises all of the cholesterol found in various lipoproteins. Cholesterol is the major component of LDLs and a minority component of VLDLs and HDLs. Since LDL has consistently been associated with risk of atherosclerosis, and since LDL is difficult to measure, serum total cholesterol has been used for many years as a substitute. There is general agreement that a strong correlation exists between considerably elevated serum cholesterol levels and an increased tendency for atherosclerosis. Disadvantages include the following:

    1. There is considerable overlap between cholesterol values found in populations and individuals at normal risk for atherosclerosis and those at higher risk. This leads to controversy over what values should be established as “normal” for the serum cholesterol reference range. A related problem is a significant difference between the reference range values for cholesterol based on “ideal” populations (i.e., derived from populations with a low incidence of atherosclerosis) compared with reference ranges from populations with a higher incidence of atherosclerosis (e.g., unselected clinically asymptomatic persons in the United States). This has led to objections that data from many persons with significant but subclinical disease are being used to help derive the reference values for populations with higher risk of CHD.

    Whereas the upper limit of statistically derived U.S. values is about 275-300 mg/100 ml (7.2-7.6 mmol/L), some investigators favor 225 mg/100 ml (5.85 mmol/L) as the acceptable upper limit since that is the value representing average risk for CHD in the Framingham study. However, the average risk for CHD in the U.S. population of the Framingham study is higher than the average risk for a low-risk population. The National Institutes of Health (NIH) Consensus Conference on cholesterol in heart disease in 1984 proposed age-related limits based on degree of CHD risk. The NIH Conference guidelines were widely adopted. In some studies, serum cholesterol (as well as triglyceride) reference values are sex related as well as age related.

    To make matters more confusing, many investigators believe that 200 mg/100 ml (5.15 mmol/L) should be considered the acceptable upper limit because that is the approximate upper limit for low-risk populations. The Expert Panel of the National Cholesterol Education Program (NCEP, 1987) chose 200 mg/100 ml without regard to age or sex (Table 22-6). Although the NCEP advocated use of total cholesterol as the basic screening test for CHD risk, they recommended that therapy should be based on LDL cholesterol values.

    The NCEP Guidelines seem to be replacing the NIH Consensus Guidelines. One possible drawback is lack of consideration of HDL cholesterol effects (discussed later), which may be important since HDL is an independent risk factor.

    2. Serum cholesterol can have a within-day variation that averages about 8% (range, 4%-17%) above or below mean values during any 24-hour period (±8% variation represents about ±20 mg/100 ml if the mean value is 250 mg/100 ml).
    3. Day-to-day cholesterol values in the same individual can fluctuate by 10%-15% (literature range, 3%-44%).
    4. Serum cholesterol values may decrease as much as 10% (literature range 7%-15%) when a patient changes from the erect to the recumbent position, as would occur if blood were drawn from an outpatient and again from the same person as a hospital inpatient. Two studies have shown less than 5% average difference between serum cholesterol obtained from venipuncture and from fingerstick capillary specimens in the same patient.
    5. Various lipid fractions are considerably altered during major illnesses. For example, total cholesterol values often begin to decrease 24-48 hours after an acute myocardial infarction (MI). Values most often reach their nadir in 7-10 days with results as much as 30%-50% below previous levels. The effect may persist to some extent as long as 6-8 weeks. In one study not all patients experienced postinfarct cholesterol decrease. Although theoretically one could obtain valid cholesterol results within 24 hours after onset of acute MI, the true time of onset is often not known. Surgery has been shown to induce similar changes in total cholesterol to those following acute MI. HDL cholesterol also temporarily fell in some studies but not in others. Triglyceride levels were relatively unchanged in some studies and increased in others. In bacterial sepsis and in viral infections, total cholesterol levels tend to fall and triglyceride levels tend to increase. Besides effects of illness there are also effects of posture and diet change, stress, medications, and other factors that make hospital conditions different from outpatient basal condition. For example, thiazide diuretic therapy is reported to increase total cholesterol levels about 5%-8% (range, 0%-12%) and decrease HDL cholesterol to a similar degree. However, several studies reported return to baseline levels by 1 year. Certain medications can interfere with cholesterol assay. For example, high serum levels of ascorbic acid (vitamin C) can reduce cholesterol levels considerably using certain assay methods.
    6. Certain diseases are well-known causes of hypercholesterolemia; these include biliary cirrhosis, hypothyroidism, and the nephrotic syndrome. A high-cholesterol diet is another important factor that must be considered.
    7. Total cholesterol becomes somewhat increased during pregnancy. In our hospital, data from 100 consecutive patients admitted for delivery showed 16% with values less than 200 mg/100 ml (5.2 mmol/L); the lowest value was 169 mg/100 ml (4.4 mmol/L). Thirty-five percent were between 200-250 mg/1000 ml (5.2-6.5 mmol/L); 36% were between 250-300 (6.5-7.8 mmol/L); 10% were between 300-350 (7.8-9.1 mmol/L); and 3% were between 350-400 (9.1-10.4 mmol/L), with the highest being 371 (9.6 mmol/L). On retesting several patients 3-4 months after delivery, all had values considerably less than previously, although the degree of decrease varied considerably.
    All of the major lipoprotein fractions, including chylomicrons, contain some cholesterol. Therefore, an increase in any of these fractions rather than in LDL alone potentially can elevate serum total cholesterol values. Of course, for lipoproteins with low cholesterol content the degree of elevation must be relatively great before the total cholesterol value becomes elevated above reference range.

    In summary, according to the NCEP, 200 mg/100 ml (5.15 mmol/L) is the upper acceptable limit for any age. Lipid values obtained during hospitalization may be misleading, and borderline or mildly elevated values obtained on a reasonably healthy outpatient may have to be repeated over a period of time to obtain a more accurate baseline. Changes between one specimen and the next up to 20-30 mg/100 ml (0.52-0.78 mmol/L), or even more—may be due to physiologic variation rather than alterations from disease or therapy.

    Although one would expect cholesterol in food to raise postprandial serum cholesterol values, actually serum cholesterol levels are very little affected by food intake from any single meal. Cholesterol specimens are traditionally collected fasting in the early morning because serial cholesterol specimens should all be drawn at the same time of day after the patient has been in the same body position (upright or recumbent) and because triglyceride (which is greatly affected by food intake) or HDL cholesterol assay are frequently performed on the same specimen.

    Cholesterol assay on plasma using EDTA anticoagulant is reported to be 3.0-4.7 mg/100 ml (0.078-0.12 mmol/L) lower than assay on serum (depending on the concentration of EDTA).

    Low-density lipoprotein cholesterol. The LDL (beta electrophoretic) fraction has been shown in various studies to have a better correlation with risk of atherosclerosis than total serum cholesterol alone, although the degree of improvement is not marked. As noted previously, the NCEP bases its therapy recommendations on LDL values. The major disadvantage of this approach is difficulty in isolating and measuring LDL. The most reliable method is ultracentrifugation. Since ultracentrifugation is available only in a relatively few laboratories and is expensive, it has been standard procedure to estimate LDLs as LDL cholesterol by means of the Friedewald formula. This formula estimates LDL cholesterol from results of triglyceride, total cholesterol, and HDL cholesterol.

    One report suggests that modifying the formula by dividing triglyceride by 6 rather than 5 produces a more accurate estimate of LDL levels. A disadvantage of the Friedewald formula is dependence on results of three different tests. Inaccuracy in one or more of the test results can significantly affect the formula calculations. In addition, the formula cannot be used if the triglyceride level is greater than 400 mg/100 ml (4.52 mmol/L).

    High-density lipoprotein cholesterol. Several large-scale studies have suggested that HDL levels (measured as HDL cholesterol) have a strong inverse correlation with risk of atherosclerotic CHD (the higher the HDL level, the less the risk). HDL seems to be a risk factor that is independent of LDL or total cholesterol. Some believe that HDL cholesterol assay has as good or better correlation with risk of CHD than total or LDL cholesterol. In general, the Framingham study suggested that every 20 mg/100 ml (5.2 mmol/L) reduction of HDL cholesterol corresponds to approximately a doubling of CHD risk. Disadvantages include certain technical problems that affect HDL assay, although methodology is becoming more simple and reliable. These problems include different methods that produce different results and need for two procedure steps (separation or extraction of HDL from other lipoproteins and then measurement of the cholesterol component), all of which produce rather poor correlation of results among laboratories. Ascorbic acid (vitamin C) may interfere (5%-15% decrease) with some test methods but not others. Reliability of risk prediction is heavily dependent on accurate HDL assay, since a relatively small change in assay values produces a relatively large change in predicted risk. HDL values are age and sex dependent. HDL values tend to decrease temporarily after acute MI, as do total serum cholesterol values. Hypothyroidism elevates HDL values and hyperthyroidism decreases them; therefore, in thyroid disease HDL values are not reliable in estimating risk of CHD. The possible effects of other illnesses are not as well known. Certain antihypertensive medications (thiazides, beta-blockers without intrinsic sympathomimetic activity, sympathicolytic agents) decrease HDL by a small but significant degree.

    Since serum total cholesterol and HDL are independent risk factors, some patients may have values for one that suggest abnormality but values for the other that remain within reference limits. As independent risk factors, a favorable value for one does not entirely cancel the unfavorable effect of the other.

    Serum cholesterol/high-density lipoprotein cholesterol ratio. Some investigators use the serum cholesterol/HDL cholesterol ratio as a convenient way to visualize the joint contribution of risk from these important risk factors. The ratio for normal risk is 5, for double risk is 10, and for triple risk is 20. Some believe that the ratio is the best single currently available predictor of CHD risk. Others believe that the ratio does not adequately demonstrate the independent contributions of the two factors and may be misleading in cases in which one or both factors may be abnormal, but the ratio does not suggest the actual degree of abnormality.

    It should be mentioned that some uncertainty exists whether mortality data involving total cholesterol and HDL cholesterol is still valid in persons over age 60, and if so, to what degree.

    Apolipoproteins. Apolipoprotein A (apo A) is uniquely associated with HDL, and measurement of apolipoprotein A1 (apo A1) has been proposed as a better index of atherogenic risk than assay of HDL cholesterol. Apolipoprotein B (apo B) comprises most of the protein component of LDL, which is composed of a core of cholesterol esters covered by a thin layer of phospholipids and free cholesterol around which is wrapped a chainlike molecule of the principal subgroup of apo B known as apo B100. Apo B100 is also the major B apolipoprotein component of VLDL. The apo B subgroup known as apo B48 (produced by the intestine) is a major structural protein in chylomicrons. Some research suggests that apo B may have a role of its own in cholesterol synthesis and that apo B measurement may provide a better indication of atherosclerotic risk than LDL cholesterol measurement. The apo A1/apo B ratio has been reported by some to be the best single predictor of CHD. However, there is some controversy over the role of apoprotein assay in current management of CHD. In my experience the total cholesterol/HDL ratio and the Apo A1/Apo B ratio, done simultaneously, gave approximately the same CHD risk assessment in the great majority of patients. The apoproteins have been quantitated mostly by immunoassay. Apo E4 gene has been proposed as a risk factor for Alzheimer’s disease. Apoprotein assay is still not widely available or widely used, and quality control surveys have shown problems in accuracy between laboratories with one international survey finding within-lab coefficients of variation (CVs) of 5%-10% and between-lab CVs of 15%-30%.

    Triglyceride (TG). Triglyceride (TG) is found primarily in chylomicrons and in VLDLs. In fasting plasma, chylomicrons are usually absent, so TG provides a reasonably good estimate of VLDL. The usefulness of VLDL or TG as an indicator of risk for CHD has been very controversial. The majority opinion in the early 1980s was that TG levels do not of themselves have a strong predictive value for CHD. The majority opinion in the early 1990s cautiously suggests that such an independent role is possible but is not yet unequivocally proven. Several large studies reported a strong correlation between increased TG and increased CHD values. However, when the effect of other risk factors was considered, there was thought to be less evidence of an independent TG role. There is a roughly inverse relationship between TG and HDL levels, so that elevated TG levels tend to be associated with low HDL levels (which are known to be associated with increased risk for CHD). Currently, the major use of TG assay still is to calculate LDL using the Friedewald formula, to help screen for hyperlipidemia, and to help establish lipoprotein phenotypes.

    Other factors that influence TG levels are frequently present. Nonfasting specimens are a frequent source of elevated TG levels. Postprandial TG levels increase about 2 hours (range, 2-10 hours) after food intake with average maximal effect at 4-6 hours. Therefore, a 12- to 16-hour fast is recommended before obtaining a specimen. Within-day variation for triglyceride averages about ± 40% (range, 26%-64%), with between-day average variation about ± 25%-50% (range, 18%-100%). Obesity, severe acute stress (trauma, sepsis, burns, acute MI) pregnancy, estrogen therapy, alcohol intake, glucocorticoid therapy, high-fat diet, and a considerable number of diseases (e.g., diabetes, acute pancreatitis, nephrotic syndrome, gout, and uremia) increase TG levels. Levels more than 1,000 mg/100 ml (11.29 mmol/L) interfere with many laboratory tests, and predispose for acute pancreatitis. There are also certain laboratory technical problems that may falsely decrease or increase TG values. High alkaline phosphatase levels increase TG levels to some degree in all TG methods. All TG methods actually measure glycerol rather than triglyceride, so that glycerol that is not part of TG (from a variety of etiologies) can falsely increase the result unless a “blank” is prepared and subtracted. Increased bilirubin, uric acid, or vitamin C levels interfere with some TG methods.

    Plasma TG fasting values of 250 mg/100 (2.82 mmol/L) were considered to be the upper limit of normal in adults by an NIH Consensus Conference on hypertriglyceridemia in 1993. Fasting values more than 500 mg/100 ml (5.65 mmol/L) were considered definitely abnormal. Most laboratories perform TG assays on serum rather than plasma and apply the NIH cutoff values to the results, although serum values are about 2%-4% less than results obtained from plasma.

    Lipoprotein (a) [Lp(a)] Lipoprotein (a) [Lp(a)] is a lipoprotein particle produced in the liver and composed of two components: one closely resembling LDL in structure which, like LDL, is partially wrapped by a chainlike apo B100 molecule, and an apolipoprotein (a) glycoprotein molecule covalently linked to apo B100 by a single disulfide bond. Apo (a) has a structure rather similar to plasminogen, which is the precursor molecule of the anticoagulant enzyme plasmin. The apo (a) gene is located on the long arm of chromosome 6 next to the gene for plasminogen. However, there are at least 6 alleles (isoforms) of apo (a), so that small variations in the structure and size of apo (a)—and therefore of Lp (a)—may occur. The apo (a) isoforms are inherited in a codominant fashion and Lp(a) is inherited as a autosomal dominant. In Europeans, Lp(a) distribution is considerably skewed toward the lower side of value distribution; while in African Americans there is a gaussian bell-shaped value distribution that is relative to Europeans results in a greater number of elevated values. Familial hypercholesterolemia, chronic renal failure requiring dialysis, the nephrotic syndrome, and postmenopausal decreased estrogen levels (in females) are associated with higher Lp(a) levels. Chronic alcoholism may decrease Lp(a) levels.

    There now are a number of studies indicating that Lp(a) elevation is a very significant independent risk factor for atherosclerosis, especially for CHD and probably for stroke and abdominal aneurisms. About 10% of the general population have elevated levels of Lp(a). Lp(a) values over 30 mg/100 ml increase CHD risk two to threefold. When high levels of LDL and Lp(a) coexist, this raises the relative CHD risk up to fivefold. However, a few studies deny that Lp(a) is an important independent risk factor.

    Lp(a) can be quantitated by a variety of immunoassay methods. Concentration has been reported as total Lp(a) mass; this includes both the lipid (HDL) and protein (apo[A]) components of Lp(a). The majority of the population has values less than 20 mg/dl (0.2 g/L). Elevation above 30 mg/dL (0.3 g/L) is associated with a twofold or more increase in CHD risk. Concentration has also been reported as apo(a) protein mass. Elevation above 0.5-0.7 g/L increases risk for CHD. However, these cutoff points were established in predominately European populations and may not be exactly applicable to other racial populations. There are problems with assay standardization (since currently there is no international standard material) and significant variations between laboratories and various assays. There is also a potential problem because apo(a) and plasminogen have considerable structural similarities, and therefore antibodies against either molecule may have some degree of cross-reaction. Postprandial specimens are reported to be 11%-13% lower than fasting specimens.

    Summary. The most widely used current procedure to estimate risk of coronary heart disease is to obtain serum or plasma total cholesterol levels (as a substitute for LDL assay) and HDL cholesterol levels. If desired, the total cholesterol/HDL ratio can be calculated, and LDL cholesterol levels can be derived from the same data plus TG assay by means of the Friedewald formula. These studies are best performed when the patient is in a basal state. It is important to note that many investigators caution that such studies may be misleading when performed on hospitalized patients, due to the effects of disease and the hospital environment. Accuracy of total cholesterol, HDL, TG, and apolipoprotein measurements is increased if two or preferably three specimens are obtained, each specimen at least 1 week apart (some prefer 1 month apart), each obtained fasting at the same time of the day to establish an average value to compensate for physiologic and laboratory-induced fluctuations in lipoprotein measurements.

  • Lipoprotein Metabolism

    Triglycerides (TG) enter the blood from exogenous (food) and endogenous (liver) sources. Food provides neutral fat, which is primarily triglyceride and which is hydrolyzed by pancreatic lipase into free fatty acids (FFA) and monoglycerides. These enter small intestine mucosal cells. The lipid fractions are recombined into TG within the mucosal cells and are incorporated into chylomicrons. Chylomicrons have a thin outer shell of phospholipid and unesterified cholesterol with protein that is mostly in the form of apoproteins B-48, C-II, and E (the outer shell components collectively are known as “polar lipids”). There is a central core of TG plus some cholesterol esters (“non-polar lipids”). Chylomicrons give postprandial plasma its characteristic milky appearance.

    Chylomicrons enter the capillaries of adipose tissue, heart muscle, and skeletal muscle where an enzyme called lipoprotein lipase that is activated by apo C-II splits off much of the TG and hydrolyzes it to FFA and glycerol. FFA are used for energy in heart and skeletal muscle. Some is transported to the liver bound to albumin and some is converted back to TG by reesterification with glycerol in fat cells and stored there for future use. The remainder of the chylomicrons (now called “chylomicron remnants,” composed mostly of apo B, apo E, and cholesterol) is taken to the liver and metabolized there.

    The liver synthesizes triglyceride from FFA and glycerol derived partly from chylomicron origin and partially from hepatic synthesis by the glucose metabolism pathway. The liver also synthesizes cholesterol, cholesterol esters, apo B-100 and C-II and combines these with TG to form very low-density lipoprotein (VLDL), which is structurally rather similar to chylomicrons. In body tissues, lipoprotein lipase (activated by apo C-II) hydrolyzes TG to release FFA, leaving a “VLDL remnant.” This is taken to the liver, where about half is converted to low density lipoprotein (LDL) by addition of cholesterol esters derived from action on free cholesterol by an enzyme called lecithin-cholesterol acyltransferase (LCAT). LCAT is partially located in the liver and partially in high density lipoprotein (HDL).

    Body cholesterol is derived from exogenous dietary sources and from endogenous synthesis by the liver from acetate. The majority is produced by the liver. The liver excretes some cholesterol as a component of bile.

    LDL differs from VLDL in that LDL has lower triglyceride content, higher cholesterol content, and no C or E apoproteins. LDL molecules are taken into tissue cells from specific receptor sites in the cell membrane. Inside the cell, the LDL molecules are metabolized into their component parts. Some of the cholesterol is used by the cell, and some can leave the cell under proper conditions. Thus, LDLs are thought to have a major role in providing body cells with cholesterol and thereby are an important part of the atherogenic process.

    HDLs have two contrasting roles in lipid metabolism. On one hand, they help create LDL from VLDL and thus enhance the possibility of atherogenesis. On the other hand, they are thought to help transport cholesterol out of peripheral tissues to the liver (although the mechanism for this is still not completely understood) and thus help “protect” against atherosclerosis.

  • Serum Complement

    Serum complement is an important immunologic enzyme system that comprises about 10% of the serum globulins. Complement has many activities, some of which are undoubtedly still unknown. Most attention has been focused on its role in the immunologic system, where effects have been demonstrated on vascular permeability, chemoaxis, phagocytosis, immune adherence, and immune cell lysis. The best-known laboratory procedure directly involving complement is the complement fixation (CF) test method. There are nine major components of complement, ranging from alpha to gamma in electrophoretic mobility. There are also inhibitors of some of these components. Nomenclature for this system has been confusing because numbers assigned to the components do not correspond to the sequence in which the components are activated and also because subcomponents exist in some of the components. Total complement is abbreviated C (some use the symbol ў instead of C). Total complement is sometimes referred to as total hemolytic complement, or CH50. The major complement components are numbered C1 through C9. Component C1 has three subcomponents: C1q, C1r, and C1s. Component C3 has also been called beta-1C.

    The actual order of complement component activation in the classic complement pathway is C1, C4, C2, C3, C5, C6, C7, C8, and C9. Classic complement activation usually begins by IgM or IgG type of antibody that binds to C1q. A chain reaction then successively involves C1r and C1s, resulting in activation of the complete C1 molecule (when activated, C1 is often called C1 esterase). C1 esterase then activates C4 to begin the complement activation sequence. There is an alternate pathway involving properdin, which activates C3 directly and bypasses C1, C4, and C2.

    Complement has been assayed in two ways: methods that test complement overall functional activity and methods that depend on immunologic quantitative measurement of individual components of the complement system. Functional assessment is usually done through a hemolytic system using antibody-coated sheep RBCs in which complement is necessary for RBC lysis. The assay is dependent on proper function of the entire complement pathway. The end point is lysis of 50% of a standard antibody-coated RBC suspension with the results being reported in complement hemolytic (CH50) units per milliliter of test specimen (this refers to the dilution of patient serum required to produce the end point). The total hemolytic complement (CH50) test assesses overall function of the entire complement pathway. If CH50 is decreased, then direct quantitation of C3 and C4 component are measured. The amount of the component is assumed to be directly related to its functional activity. A decreased C4 level suggests some defect in the classic pathway. A decreased C3 level with normal C4 level suggests abnormality in the alternate pathway.

    The most common congenital disease associated with complement is hereditary angioedema. This is due to absence of C1 inhibitor, and the diagnosis is established by assay of C1 (C1 esterase) inhibitor. In 10%-20% of cases C1 inhibitor is present but nonfunctional. If immunologic methods are used for assay, this would lead to apparent normal results. Since C1, when activated, will split C2 and C4, lack of C1 inhibitor leads to decrease of CH50, C2, and C4 levels during an acute attack. During remission the C4 level usually remains decreased, but CH50 and C2 levels may return to their reference ranges. These assays permit the diagnosis of functional decrease in the C1 esterase inhibitor level even if immunologic C1 esterase inhibitor values are within reference range.

    Acquired complement abnormalities are much more common than congenital ones. Total complement is temporarily elevated following onset of various acute or chronic inflammatory diseases or acute tissue damage, although hepatitis virus type B infection is associated with decreased complement. Most of the clinical conditions in which complement measurement is useful are associated with decreased levels, either because of decreased production secondary to severe liver disease or to increased consumption secondary to glomerulonephritis or because of activation by circulating immune complexes. Total complement (C or CH50), C3, and C4 levels are all usually reduced in active systemic lupus erythematosis (SLE) nephritis. They may also be decreased in serum sickness, infectious endocarditis (subacute bacterial endocarditis), immune complex disease, and renal transplant rejection. They are normal in most patients with rheumatoid arthritis but may be reduced in a subgroup with severe disease accompanied by vasculitis. Total complement and C3 levels are usually reduced (but the C4 level is often normal) in poststreptococcal acute glomerulonephritis and in membranoproliferative nephritis.

    Complement C3 function is unstable and may decrease significantly in blood or serum left standing at room temperature for more than 1-2 hours. This results in low CH50 levels with normal C3 and C4 levels. Serum or plasma that must be preserved should be frozen immediately.

    Complement assays are not available in most laboratories and must be done by university hospitals or large reference laboratories. Problems with maintaining complement levels in specimens and the length of time before results are available have limited the popularity of complement assay. The assays currently are used mainly to diagnose angioedema, evaluate some patients with poststreptococcal or SLE nephritis, and to monitor therapy in some patients with SLE nephritis or membranoproliferative nephritis.

  • Secondary Monoclonal Gammopathy

    Secondary monoclonal gammopathy may be further subdivided into diseases associated with neoplasia and those associated with nonneoplastic disorders. In the neoplasm group, monoclonal proteins are most often found with malignant lymphoma and chronic lymphocytic leukemia. Among carcinomas, those of the rectosigmoid are most frequent, followed by carcinomas of the prostate, breast, and lung. The incidence in one large cancer hospital ranged from 0.2% in the fourth decade of life to 5.7% in the ninth. In three large series of patients with monoclonal gammopathy, 6%-8% of cases were associated with lymphoma or lymphocytic leukemia and 4%-8% with other types of neoplasms. Nonneoplastic diseases that have been associated with monoclonal proteins are many and varied, but the greatest number appear in the rheumatoid-collagen-autoimmune group, cirrhosis, chronic infection (particularly tuberculosis and chronic infection of the biliary tract, urinary tract, and lung), Gaucher’s disease, osteitis deformans (Paget’s disease of bone), and sarcoidosis. One study detected monoclonal or oligoclonal serum proteins in 9% of patients who had HIV-1 infection without the criteria for acquired immunodeficiency syndrome (AIDS). There is also increased incidence in AIDS. Most of these nonneoplastic conditions are ordinarily associated with polyclonal hyperglobulinemia rather than monoclonal gammopathy. The incidence of nonneoplastic monoclonal protein in the three series mentioned varied from 4%-10%. Monoclonal protein type in the secondary paraproteinemias may be IgG, IgA, or IgM. Occasionally patients in either the neoplastic or nonneoplastic group excrete Bence Jones protein in the urine, usually in small amounts. In some cases the heat test gives false positive results due to an increase in normal light chains associated with polyclonal gammopathy.

    Diagnostic techniques

    In many instances the diagnosis of myeloma or Waldenstrцm’s macroglobulinemia can be made by serum protein electrophoresis, followed by bone marrow aspiration. In patients without a monoclonal-type serum peak, urine electrophoresis on a concentrated specimen is essential to detect cases in which the only protein abnormality is urinary excretion of Bence Jones protein. In problem cases, it is necessary to resort to serum and urine immunoelectrophoresis. Many authorities advocate immunoelectrophoresis in all patients since this is the only way to classify monoclonal immunoglobulin disorders with certainty (even subgrouping of IgG and IgA is now possible.) Although subclassification of myeloma into immunoglobulin categories at present has more academic than practical application from the standpoint of therapy, such classification may become important in the future, and in any event, it provides additional confirmation of the diagnosis. About 1%-2% of myeloma patients fail to secrete abnormal proteins that are detectable in either serum or urine by immunoelectrophoresis (“non-secretory” myeloma).

    Immunoelectrophoresis consists of three steps. First, the unknown (patient’s) serum is subjected to ordinary electrophoresis in a substance such as agar gel; this separates the immunoglobulins from one another to some extent. Second, antiserum against a specific type of human globulin (or a polyvalent antiserum against several types) is added to a trench cut nearby and parallel to the electrophoretic separation bands. Then the immunoglobulins and antiimmunoglobulin antibodies diffuse toward each other. Finally, the reaction between the patient’s immunoglobulin fractions and any specific antibodies against one or more of those immunoglobulin fractions forms visual precipitin lines. The combination of electrophoresis and agar diffusion antigen-antibody reaction produces better separation of the immunoglobulin components and demonstrates abnormal quantities of any type present. Immunofixation is a modification of the immunoelectrophoresis technique that takes a little longer to perform but is easier to interpret and may be a little more sensitive. The test sample (usually diluted serum or concentrated urine) is spotted into each of 6 side-by-side slots on a cellulose acetate or agar gel plate at the same end of each slot. The proteins are then separated by electrophoresis. After that, antiserum against IgG, IgA, IgM, kappa light chain, and lambda light chain are placed into different slots. One slot does not receive antiserum. Incubation permits antigen-antibody reaction, if any. A protein stain is applied to visualize any antigen-antibody reaction. Monoclonal proteins are seen as a sharp narrow band; polyclonal proteins appear to be a wider, more diffuse band. Immunoelectrophoresis and immunofixation are most useful to demonstrate monoclonal proteins, differentiate monoclonal from polyclonal proteins, and to identify any monoclonal or polyclonal proteins. Therefore, these methods differentiate between macroglobulinemia (IgM) and other categories of monoclonal gammopathy. It should be noted that antisera against IgD and IgE are ordinarily not used, because these monoclonal gammopathies are uncommon, so that no reaction with the antisera used does not exclude the possibility of IgD or IgE. To exclude them would require repeating the procedure with these antisera. The same is true for immunoelectrophoresis.

    If a monoclonal peak is shown to be IgM, this is evidence against myeloma, since only a few cases of IgM myeloma have been reported. On the other hand, if the peak is not IgM, this rules out Waldenstrцm’s macroglobulinemia. The idiopathic or secondary paraproteinemias can be of the IgG, IgA, or IgM class.

    There are two types of immunoglobulin light chains: kappa and lambda. Normally about twice as much kappa is produced as lambda. Immunoelectrophoresis can detect these light chains, differentiate kappa from lambda, demonstrate whether one is increased or decreased in relation to the other, and afford a rough visual estimate whether either one is increased or decreased in quantity. Malignant monoclonal gammopathies, such as myeloma or Waldenstrцm’s macroglobulinemia, usually have an abnormal predominance of either kappa or lambda, with the other markedly decreased or absent. Unfortunately, commercial companies have had problems in producing consistently good antisera to kappa and lambda light chains. Controls must be run with every lot of antiserum to guard against false results.

    Differentiation of benign and malignant monoclonal gammopathies

    Patients with monoclonal gammopathies are usually detected in one of two ways. Either clinical symptoms suggest myeloma and electrophoretic studies are performed, or the studies are ordered for some other reason and monoclonal abnormality is discovered by accident. In 80%-90% of patients with myeloma, bone marrow aspiration findings make the correct diagnosis relatively easy. In those patients with monoclonal gammopathy but normal or equivocal results of bone marrow aspirate, diagnosis becomes a problem. As listed in Table 22-1, nonmyelomatous monoclonal gammopathies include Waldenstrцm’s macroglobulinemia, leukemia and lymphoma (usually the lymphocytic B-cell types), secondary monoclonal gammopathies (carcinomas and inflammatory disease), and idiopathic cases without known associated disease. Some investigators divide the monoclonal gammopathies into two categories, malignant (myeloma, Waldenstrцm’s macroglobulinemia, and lymphoproliferative disorders) and benign (secondary and idiopathic types). The majority of diagnostic problems involve the secondary and idiopathic monoclonal gammopathies, and the usual difficulty consists of excluding a plasma cell or lymphocytic malignancy when bone marrow results are normal and other studies are negative.

    In general, if the monoclonal serum protein is greater in quantity than 3.0 gm/100 ml (30 g/L), if the bone marrow contains more than 20% plasma cells, or if the patient excretes more than 60 mg/L of Bence Jones protein, the disorder is probably malignant. On the other hand, if serum monoclonal protein does not exceed 2.0 gm/100 ml, marrow plasma cells do not exceed 5%, and Bence Jones protein does not exceed 60 mg/L, the condition is more likely to be benign. However, up to 5% of those with benign monoclonal gammopathies are reported to exceed at least one of the three criteria for malignancy, although usually not more than one criterion. About 15% of myeloma patients are reported to have less than 10% plasma cells in their initial bone marrow aspirate, with about 5% of myeloma patients having less than 5%. About 30% of myeloma patients have monoclonal protein in serum less than 3.0 gm/100 ml, and about 20% (literature range, 17%-22%) have less than 2.0 gm/100 ml. About 20%-30% have no Bence Jones protein in the urine, and of those who do, about 10% excrete less than 60 mg/L. Thus, the criteria for malignancy are more reliable than those for benign monoclonal etiology. In addition, in one study about 10% of patients thought to have benign monoclonal gammopathy developed myeloma or macroglobulinemia within 5 years.

    Some patients require quantitation of one or more immunoglobulin groups. The most frequent reasons include serial quantitation of monoclonal protein to monitor the effects of therapy and immunoglobulin quantitation to detect deficiency of one or more specific immunoglobulin groups. The current standard method to quantitate immunoglobulins is some type of immunologic technique (radial immunodiffusion, immunonephelometry, immunoassay, and others). It must be emphasized that immunoelectrophoresis is not suitable for immunoglobulin quantitation. Immunoelectrophoresis detects and classifies immunoglobulins, but the quantity of any detectable immunoglobulin can only be very roughly estimated as normal, increased, or decreased rather than measured. Serum protein electrophoresis is sometimes used to monitor myeloma protein response to therapy. However, protein electrophoresis is not ideal for this purpose, since standard methods depend on measuring total protein, finding the percentage that each protein fraction contributes to total protein, and then multiplying the total protein numerical value by the percentage representing each protein fraction to derive the numerical quantity of each protein fraction. This means that abnormality in one fraction (e.g., albumin) can change total protein and secondarily change the percentage of all the protein fractions relative to total protein. This can produce some degree of artifactual change in quantity of protein fractions calculated from percent of total protein. Finally, if serial determinations of a specific protein fraction are necessary, the same laboratory should perform the assays, since different techniques can yield somewhat different results on the same specimen.

    Cryoglobulins

    Cryoglobulins are immunoglobulins that precipitate reversibly in serum or at least partially gel at cold temperature. The most common symptoms are purpura (60%-100% of cases), arthralgias (60%-90%), or Raynaud’s phenomenon (about 50% of cases). The symptoms are usually referable to cryoglobulin precipitation in blood vessels. Cryoglobulins can be primary (“idiopathic,” “essential”) or secondary (associated with some disease). In either case the cryoglobulins can be monoclonal or mixed. In type I cryoglobulins (about 25% of cryoglobulinemia), there is monoclonal IgM or IgG (rarely IgA) only; in type II, there is monoclonal IgM rheumatoid factor (RF) plus polyclonal IgG; and in type III, there is polyclonal IgM RF and IgG.

    Cryoglobulins most often do not appear as discrete peaks in serum protein electrophoresis but are incorporated into areas occupied by other globulins. Although the classic cryoglobulin test takes place at 4°C, some cryoglobulins agglutinate to some degree at higher temperatures, with reports even as high as 35°C. Cryofibrinogens exist as well as cryoglobulins. The most common conditions associated with cryoglobulins are vasculitis, rheumatoid-collagen diseases, leukemias and lymphomas, myeloma and Waldenstrцm’s macroglobulinemia, infections, and liver diseases.

    Diagnosis consists of drawing a blood specimen and maintaining it at 37°C until clotting is completed. After that, the serum is incubated at 4°C. There is disagreement over the maximum time needed to terminate the test if no agglutination or gelation occurs. Some use a cutoff point of 3 days and others propose 7 days. If cryoglobulins become visible, they should be analyzed to determine which immunoglobulins are present. For this, it would often be necessary to send the specimen to a reference laboratory. Other applicable tests are a screening test for RF and for antinuclear antibodies (ANA). To detect cryofibrinogenemia, a plasma specimen collected in ethylenediamine tetraacetic acid (EDTA) or citrate plus a serum specimen obtained at the same time are incubated together at 4°C. A precipitate in the plasma but not serum would indicate cryofibrinogens.

  • Multiple Myeloma

    Multiple myeloma is a malignancy of plasma cells that, in turn, are derived from B-type lymphocytes. The neoplastic plasma cells involve bone marrow and frequently produce multisystem disease. Patients are usually age 40 or older, with a peak incidence occurring in age group 60-65 years. About two thirds of the patients are male (literature range, 54%-72%). Clinically, the most common symptom is bone pain, reported in about 70%. Pathologic fractures are common. Infections are more common in myeloma (10%-52% of cases) than in the general population. The classic infecting organisms are encapsulated bacteria such as pneumoccoci and, much less frequently, Haemophilus influenzae; some reports indicate that gram-negative infections are becoming more frequent. Recurrent infections are relatively frequent. Weight loss, weakness, and various GI symptoms are found in 35%-65% of cases. Neurologic symptoms are present in 30%-50% of cases. Some are due to nerve root or spinal cord compression by myelomatous infiltrates; some to amyloid deposition, and some to peripheral neuropathy. Hepatomegaly is present in about 20% of cases (literature range, 10%-38%) and splenomegaly in about 10% (range, 5%-15%). Acute or chronic renal failure occurs in over 50% of patients. When patients over age 40 are seen because of bone pain, recurrent infection or unexplained nonpulmonary infection or fracture, myeloma should be considered.

    Laboratory abnormalities

    Standard laboratory test findings. Anemia is found in 60%-80% of patients. It is generally moderate in degree (although sometimes severe) and is usually of the normocytic-normochromic type. RBC rouleaux formation (RBCs adhering together like a stack of coins due to the presence of abnormal protein) in peripheral blood smears is a clue to possible myeloma and can be found in 60%-85% of cases. Frequency of rouleaux detection depends to some extent on the degree of abnormality and also on the experience and interest of the examiner. Total white blood cell (WBC) count is usually normal except during episodes of infection, with leukopenia reported in 15%-20% of cases. Although myeloma plasma cells infiltrate the bone marrow at some time during the course of the disease, ordinarily this does not result in peripheral blood plasmacytosis. In 1%-2% of patients there are more than 5% plasma cells in the peripheral blood, and some of these patients have been considered to have plasma cell leukemia. Some require as many as 20% of the peripheral blood WBCs to be plasma cells in order to make that diagnosis. Occasionally patients display some degree of myeloid cell immaturity. Thrombocytopenia is found in approximately 10% of patients.

    Among other laboratory test results that may be abnormal, the ESR is usually moderately to markedly elevated (90% of patients in a large Mayo Clinic series). Hypercalcemia occurs in about 30% (literature range, 20%-50%). Azotemia is reported in 40%-55% of patients, and proteinuria is detected in 60%-90%. Hyperglobulinemia is present in approximately 60% of patients (literature range, 50%-80%) and hypogammaglobulinemia in about 10%. Serum albumin is decreased in about 50% of patients. The alkaline phosphatase level is most often normal unless a fracture is present, but reports of alkaline phosphatase abnormality in the literature range from 0%-48%. The total lactic dehydrogenase value (LDH) is elevated (according to several reports) in 24%-60% of cases; LDH isoenzyme fractionation most often shows elevated LDH-3 values. Amyloid is found in 5%-10% of cases and cryoglobulins in approximately 5%. Uric acid levels are increased in about 40% of patients.

    X-ray findings. X-ray examination displays abnormality in 80%-85% of patients. The most typical finding is the “punched-out” osteolytic lesion, most commonly in the skull, vertebral spine, and pelvis. Between 6% and 25% of patients demonstrate diffuse osteoporosis as the only abnormality; the rest have various combinations of focal osteolytic lesions, osteoporosis, and pathologic fractures. Results of radionuclide bone scan are not as frequently abnormal in myeloma as in metastatic tumor involving bone.

    Bone marrow diagnosis. Diagnosis of myeloma is made through bone marrow aspiration. The bone marrow of myeloma patients usually contains more than 20% plasma cells (however, criteria listed in the literature range from 5%-30%, sometimes based on presence or absence of other abnormalities such as bone lesions). If the plasma cell percentage is less than 20%, a considerable number of the plasma cells must be immature to make the diagnosis, because other diseases may be associated with an increase in mature marrow plasma cells. Ordinarily, a benign plasmacytic marrow reaction produces a plasma cell percentage less than 10%, but in some cases this figure may reach 20% and, rarely, even higher (e.g., in a few patients with human immunodeficiency virus [HIV] infection). Bone marrow aspiration is usually diagnostic by the time symptoms appear. However, about 15% of patients have less than 20% plasma cells in the marrow aspirate, and about 8% have less than 20% plus no evidence of plasma cell immaturity. Since marrow infiltration may have an irregular distribution in some patients, a repeat aspiration may be necessary. In some cases a cytologic diagnosis cannot be made until a later date. In occasional patients a bone marrow biopsy specimen may show changes suggestive of myeloma when the marrow smear is not diagnostic.

    Abnormal proteins (monoclonal immunoglobulins). About 75%-80% of myeloma patients have plasma cell secretion of abnormal monoclonal serum protein with a molecular weight (160,000 daltons or 7S) typical of a normal complete immunoglobulin molecule. This has a highly concentrated appearance or spikelike densitometer pattern on electrophoresis; usually located in the gamma-globulin area, occasionally in the beta-globulin area, and rarely in the alpha-2 globulin area. Of all patients with monoclonal protein, about two thirds have myeloma. Of those myeloma patients with normal weight serum monoclonal protein, roughly 70% have monoclonal protein categorized as IgG, about 25% have IgA, and fewer than 2% have IgD or IgE.

    In addition to normal weight serum monoclonal protein, many patients excrete an abnormal, incomplete, low weight protein known as Bence Jones protein. Bence Jones protein is composed only of immunoglobulin light chains and therefore has a low molecular weight (40,000 daltons, or 3.5S). Unlike normal weight monoclonal proteins, it is able to pass the glomerular filter into the urine. In most cases it is cleared rapidly from plasma; therefore, even when Bence Jones proteinuria is marked, this substance usually is not demonstrable in serum by ordinary electrophoretic techniques and frequently not even by immunoelectrophoresis. About 70%-80% of myeloma patients show Bence Jones protein on urine electrophoresis, whereas urine Bence Jones protein can be detected only in about 50% of patients using the old heat test. About 50%-60% have a normal weight serum monoclonal protein in addition to Bence Jones protein in the urine, and about 20% (literature range, 10%-26%) have Bence Jones protein in the urine as the only protein abnormality (“light chain” myeloma). Light chain myeloma is frequently associated with hypogammaglobulinemia on standard serum protein electrophoresis. Clinically, there tends to be a somewhat greater incidence of azotemia, hypercalcemia, and lytic bone lesions than in ordinary myeloma.

    The classic method of detecting Bence Jones protein is by a carefully done heat coagulability test, in which the protein appears on heating to 60° C and disappears on boiling, only to reappear if the urine is cooled. As mentioned in Chapter 12, with a few exceptions the sulfosalicylic acid test result for urine protein is positive with Bence Jones protein, but dipsticks frequently give negative results. Since various technical and human factors make the heat method unreliable, urine electrophoresis is the best method for demonstrating Bence Jones protein. In a large Mayo Clinic series, 49% of myeloma patients revealed urine Bence Jones protein by heat test, whereas 75% had a Bence Jones peak in nonconcentrated urine on electrophoresis. On urine electrophoresis, Bence Jones protein appears as a single homogeneous spike similar to that of monoclonal protein in serum. The normal weight monoclonal serum proteins of myeloma usually do not appear in the urine. In many cases it is necessary to concentrate the urine (5-100 times) to detect small quantities of Bence Jones protein. Urine immunoelectrophoresis is about 5% more sensitive than standard urine protein electrophoresis.

    Bence Jones protein is excreted in urine by approximately 70% (literature range, 54%-80%) of myeloma patients, about 30% (0%-78%) of patients with Waldenstrцm’s macroglobulinemia, about 20% (15%-62%) of patients with monoclonal gammapathy associated with lymphoproliferative malignancy, and about 10% (0%-24%) of patients with so-called benign (secondary and idiopathic) monoclonal gammopathy. In general, patients with benign monoclonal gammopathy and Bence Jones protein excrete only small amounts of Bence Jones protein (Ј60 mg/L). Only 2%-3% (0%-6%) of these patients excrete greater quantities. Patients with malignant gammopathies and Bence Jones proteinuria tend to excrete quantities greater than 60 mg/L. However, about 10% of myeloma patients with Bence Jones proteinuria excrete less than 60 mg/L.

    About 2% (range, 1%-5%) of myeloma patients do not show detectable serum or urine monoclonal proteins or free light chains (“nonsecretory” myeloma).

    Immunoglobulin D myeloma

    IgD myeloma has some unusual features that are worth mentioning. This entity is rare, seen in about 1% of myeloma patients. In IgD myeloma, the light chain of the abnormal IgD molecule is lambda type in about 90% of cases, whereas in other types of myeloma about 66% are kappa and about 33% are lambda. Also, Bence Jones proteinuria occurs in more than 90% of IgD cases, compared with an incidence of about 75% in IgG and IgA myeloma (literature range, 60%-80%). It is claimed that IgD myeloma is more likely to be associated with extraosseus myeloma spread, although about 50%-75% of myeloma patients display microscopic extramedullary (nonbone) foci (predominantly in liver, spleen, and lymph nodes), which usually are not evident clinically.

    Amyloidosis

    A word should be said about amyloidosis. There are several categories of amyloidosis, including primary, secondary, familial, localized, and senile. The amyloid of primary amyloidosis is derived from the variable region of immunoglobulin light chains (most often lambda), and myeloma is associated with 20%-30% of these patients. Standard electrophoresis is said to detect a monoclonal peak in serum and urine in about 50% of cases, whereas immunoelectrophoresis of serum and urine is abnormal in 90% of cases. Diagnosis is made through tissue biopsy and special stains with or without polarized light. Biopsy can be obtained from clinically affected tissue (e.g., the carpal ligament in those patients with carpal tunnel syndrome) or from certain other tissues such as subcutaneous fat aspirates (in two reports, positive in 75% or more cases). However, the yield from subcutaneous fat aspiration is only about 17% if the patient has amyloid-induced carpal tunnel syndrome without any other clinical evidence of amyloidosis, and is only 0%-40% if the patient has amyloidosis but is on renal dialysis.

    Solitary plasmacytoma

    About 3% (literature range, 2%-5%) of patients with plasma cell dyscrasias have a single plasma cell localized tumor. There is considerable confusion in the literature as to the nomenclature of these lesions. In general, those within bone tend to be regarded as solitary myeloma, whereas those in soft tissue are usually called “extramedullary plasmacytomas.” The most common location for extramedullary plasmacytoma is the upper respiratory tract, whereas solitary bone lesions are found most often in the spine. About 60% of patients with solitary (bone) myeloma lesions when first seen have disseminated myeloma by 10 years after diagnosis. Extramedullary plasmacytomas are usually regarded as having a better prognosis than myeloma, with some being cured by therapy, some recurring, and some metastasizing or developing into myeloma in spite of treatment. In one review of the literature, only about 20% of patients with localized plasma cell tumors in bone were found to have monoclonal proteins in serum on standard electrophoresis and about 25% on immunoelectrophoresis.

    Heavy chain disease

    Whereas light chain myeloma involves selective production of the light chain fragment of immunoglobulin molecules, there is a rare condition known as “Franklin’s disease” (heavy chain disease) that is characterized by selective production of the heavy chain fragment. The clinical picture most often resembles that of malignant lymphoma. Bone marrow aspiration findings are variable, and lymph node biopsy may suggest lymphoma or contain a mixed cell infiltrate.

    Waldenstrцm’s (primary) macroglobulinemia

    Waldenstrцm’s macroglobulinemia is a lymphoproliferative disorder characterized by monoclonal IgM (molecular weight 1,000,000 daltons or 19S) production, with classic clinical features of lymphadenopathy, hepatosplenomegaly, anemia, hyperglobulinemia with rouleaux formation, and the hyperviscosity syndrome. From 10%-30% of patients with Waldenstrцm’s macroglobulinemia (literature range, 0%-60%) excrete Bence Jones protein in the urine. Although typical biopsy findings are a mixture of mature lymphocytes and plasmacytoid lymphocytes, in some cases the histologic picture is suggestive of a diffuse type of lymphocytic lymphoma. Bone marrow aspiration may yield either normal findings, nonspecific lymphoid infiltration, or lymphoma-like infiltrate. On skeletal x-ray film, punched-out osteolytic lesions of the type seen in myeloma are usually absent. The hyperviscosity syndrome consists of shortness of breath, various neurologic abnormalities, and visual difficulty with sausage-shaped segmentation of retinal veins. Serum viscosity (as measured by the Ostwald viscosimeter or other method) is increased. In some patients the disease could be interpreted as malignant lymphoma or lymphocytic leukemia with IgM production. In a few instances, plasma cells predominate, and osteolytic bone lesions compatible with an IgM myeloma are present. Since plasma cells are derived from lymphocytes, many are inclined to view these disorders as a spectrum. However, since clinicians usually insist on a specific diagnosis, the “intermediate” forms create a problem.

    Idiopathic monoclonal gammopathy

    Idiopathic monoclonal gammopathy is defined as a monoclonal protein detected in a person without any monoclonal protein-associated disease. There is confusion with the term “monoclonal gammopathy of uncertain significance” (MGUS), that usually includes both idiopathic monoclonal gammopathy and noneoplastic secondary gammopathies (page 252). The incidence of nonneoplastic monoclonal gammopathy apparently is increased with advanced age. Some investigators found an incidence of 0.3%-3.0% in populations tested, with the higher rates in the elderly. Using high-sensitivity detection methods, monoclonal peaks (usually small) have been reported by a few investigators in 10% or even up to 20% of persons age 75-90. In a Mayo Clinic series of 241 patients with monoclonal gammopathy of uncertain significance, 19% developed myeloma, Waldenstrцm’s, amyloidosis, or lymphoma in 10 years of follow-up, and about 25% after 20-35 years.

    IgM monoclonal proteins. In one series of 430 patients who had monoclonal IgM detected, 56% had idiopathic IgM at the time of diagnosis, 17% had Waldenstrцm’s macroglobulinemia, 7% had malignant lymphoma, 5% had chronic lymphocytic leukemia, 14% had other malignant leukemias or lymphomas, and 17% had amyloidosis. About 20% of those who had idiopathic IgM eventually developed lymphocytic leukemia or lymphoma.

  • 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 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.

  • Serum Proteins

    Serum albumin levels decrease to variable degrees in many severe acute and chronic disorders. Albumin is synthesized in the liver, so most acute or chronic destructive liver diseases of at least moderate severity also result in decreased serum albumin levels. In addition, there may be other serum protein changes. In cirrhosis of moderate to severe degree, there is a decreased albumin level and usually a “diffuse” (“polyclonal”) gamma-globulin elevation, sometimes fairly marked. About 50% of patients with well-established cirrhosis have a characteristic serum protein electrophoretic pattern with gamma-globulin elevation that incorporates the beta area (so-called beta-gamma bridging). However, about 35% of cirrhotic patients show only various degrees of gamma elevation without any beta bridging, and about 10% have normal gamma levels. Hepatitis may also be associated with moderate elevation of the gamma globulins. Biliary obstruction eventually causes elevated beta-globulin levels, since beta globulins carry cholesterol.