Laboratory Diagnosis in Neurology, 1 Ed.

General Serum Analysis

M. Zorn

This chapter discusses parameters that do not in themselves unambiguously indicate a diagnosis. However, when taken in combination with others, they can help to rule out possible differential diagnoses, and can also furnish information about concomitant diseases and the general condition of the patient. Unfortunately, many of these parameters have not been standardized, and for this reason the reference ranges depend on the measuring method used.

Inflammation Markers

Inflammation is characterized by the classic clinical symptoms of redness, swelling, warmth, dysfunction, and pain; along with these, a very complex immunological process takes place which is largely controlled by cytokines. These cytokines result in leukocytosis and the increased production of acute-phase proteins.

The concentration of cytokines increases very rapidly in the serum, thus giving an early indication of an inflammation. Particularly in intensive care medicine, therefore, they have advantages over classic inflammatory markers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR).

Interleukin 6 (IL-6)

Indication. Irrespective of liver function, IL-6 is produced within 2–4 hours, markedly more rapidly than CRP (see below). It is therefore well suited to the early diagnosis of acute inflammatory diseases.

Preanalytical requirements. The blood needs to be centrifuged and the serum removed within 2 hours of collection.

Analysis. IL-6 is determined by immunoassay (ELISA or chemiluminescence assay).

Reference range for IL-6 in plasma/serum:

image < 11.3 ng/L.

image Concentrations of > 400 ng/L strongly suggest sepsis.

Interpretation. The sensitivity of the test is almost 100%; the specificity depends on the underlying disease. Elevated levels are found in autoimmune diseases, psoriasis, glomerulonephritis, and malignant tumors. Increased concentrations are found during the first few days after surgery, but these are clinically unimportant. An increased concentration only allows the inference that an inflammation process is present; it is not usually possible to say more than that.

Test results from different providers are not always comparable. Some tests have been calibrated using a WHO standard and therefore agree better.

Interleukin 8 (IL-8)

Indication. IL-8 has much the same significance as IL-6; it is a prognostic factor for sepsis or trauma.

Preanalytical requirements. The plasma should be separated from the cells within 2 hours of collection.

Analysis. Like IL-6, IL-8 is determined by immunoassay (ELISA or chemiluminescence assay).

Reference range for IL-8 in plasma/serum:

image < 10 pg/mL.

Interpretation. Like IL-6, IL-8 is produced by immune cells, such as monocytes, and also by nonimmune cells:

• Similarly increased levels of both IL-8 and TNF-α are suggestive of overstimulation of monocytes. This occurs, for example, in systemic inflammatory response syndrome (SIRS).

• An increase only in IL-8 indicates activation of nonimmune cells, e. g., by binding of bacteria or bacterial lipopolysaccharides to endothelial cells.

Tissue hypoxia and trauma lead to a massive increase in IL-8. In general, follow-up tests for IL-8 are more important than single determinations—the exception being neonatal sepsis.

Tumor Necrosis Factor (TNF-α)

Indication. TNF-α is a marker for systemic inflammatory response syndrome (SIRS), which is associated with sepsis, trauma, and heart insufficiency.

Preanalytical requirements. Preanalytical conditions are very important in the determination of TNF-α. The cells must be separated from the plasma within 30 minutes of blood collection.

Analysis. Like IL-6 and IL-8, TNF-α is determined by immunoassay (ELISA). Tests detecting the bioactive TNF-α (a trimeric structure) should be distinguished from those that also detecting the inactive monomers and breakdown products (total TNF-α).

Reference range for TNF-α in plasma/serum:

image Total: < 20 pg/mL.

image Bioactive form: < 5 pg/mL.

Interpretation. TNF-α is secreted only for a short time (less than 6 hours) after stimulation, and it has a short half-life of less than 5 minutes. If elevated levels of bioactive TNF-α are measured, this indicates a systemic inflammation that is still ongoing or has just ended. Monomers and breakdown products, however, can be detected in the plasma for up to 24 hours. Determination of total TNF-α cannot be used to establish whether or not inflammation is still active.

TNF-α Pitfalls

Antibody therapy with anti-thymocyte globulin (ATG) and anti-CD 3 mAb (OKT3) may lead to false-positive results.

Soluble Interleukin-2 Receptor (sCD 25)

Indication. The soluble IL-2 receptor is an activity marker for a cell-mediated immune response—primarily in lymphoma patients, but also in patients with sarcoidosis (often regarded as a better activity marker than ACE) or after organ transplantation (indicating an infection or a rejection reaction).

Preanalytical requirements. This test does not have special preanalytical requirements.

Analysis. Determination of sCD 25 is by immunoassay; the various tests all yield comparable results.

Reference range for sCD 25 in plasma/serum:

image < 900 IU/mL.

Interpretation. sCD 25 measurements are good for monitoring the course of immune cell activation. Single tests are less informative.

sCD 25 Pitfalls

Antibody therapies yield falsely high results.

Procalcitonin (PCT)

Indication. Procalcitonin is the prohormone of calcitonin. It is normally produced in the C cells of the thyroid, but during infection probably comes from the liver. Within 2 hours after the secretion of TNF-α and IL-6 in bacterial, parasitic, and mycotic diseases, PCT concentrations rise, peaking after 6–8 hours.

Preanalytical requirements. There are no preanalytical requirements for PCT: the tests are highly automated, and results are rapidly available, even in an emergency.

Analysis. PCT is determined by immunoradiometric assay (IRMA) or luminescence immunoassay. There are also rapid tests that allow semiquantitative evaluation.

Reference range for PCT in serum/plasma:

image < 0.05 μg/L.

image Lower detection limit of the rapid test: 0.5 μg/L.

image Lower detection limit of the highly sensitive luminescence immunoassay: 0.05 μg/L.

Interpretation. In viral and local bacterial inflammations and chronic diseases, the PCT concentration increases not at all or only very slightly (< 1.5 μg/L). Systemic bacterial and parasitic infections, by contrast, cause a very large rise (up to 100 μg/L). Measuring the PCT concentration is also helpful in the differential diagnosis of meningitis:

• Bacterial meningitis is accompanied by a rise in PCT concentration to values above 5 μg/L.

• By contrast, in viral meningitis no increase is found, or only a very slight one (up to 2 μg/L).

Follow-up PCT measurements would be a good indicator of treatment success (e. g., after antibiotic treatment), but for cost reasons it is better to use c-reactive protein (CRP) measurements for this purpose instead.

C-Reactive Protein (CRP)

Pathophysiology. C-reactive protein is the classic acutephase protein. It got its name from its capacity to bind to the C-polysaccharide of the cell wall of Streptococcus pneumoniae. CRP consists of five identical subunits and belongs to the group of pentraxins. After stimulation by IL-6, it is rapidly synthesized in the liver. During maximum response to the acute phase, the synthesis of CRP constitutes almost 20% of the total protein output of the liver. In the presence of calcium, CRP binds to cell debris (e. g., phosphorylcholine from the cell walls of bacteria, fungi, and parasites), and to the cells themselves once the lipid bilayer of the cell membrane is destroyed. This opsonization leads to faster removal of substances from the blood, since CRP activates the complement system in the classic pathway and thus brings about elimination of the complex from the blood.

Preanalytical requirements. There are no preanalytical requirements for CRP: the tests are highly automated, and results are rapidly available, even in an emergency.

Analysis. Determination is usually by immunoturbidimetry or immunonephelometry, and the procedure is usually highly automated. However, there are also rapid immunological tests that are evaluated semiquantitatively or even quantitatively using a reflectometer. The method of latex agglutination, which allows only semiquantitative evaluation, is rarely used today.

Reference range for CRP in serum/plasma:

image < 5.0 mg/L.

image Lower detection limit: 2.0 mg/L.

Interpretation. CRP is an important marker of systemic inflammatory reactions, but does not show certain autoimmune diseases such as systemic lupus erythematosus (SLE) or ulcerative colitis. In viral meningitis, CRP levels are below 20 mg/L; in bacterial meningitis they rise to over 100 mg/L.

Comparing the relative merits of PCT and CRP with those of cytokines, cytokines certainly have the advantage of indicating acute inflammatory processes faster and, because of their short half-life, closer to real time. However, preanalytical conditions plays a major role with all cytokines, and there is no standardization of the tests. CRP concentrations have been standardized and are always comparable. There are many studies that describe in detail the value of CRP in particular clinical situations.

Erythrocyte Sedimentation Rate (ESR)

Indication. The ESR is one of the oldest laboratory markers of inflammation. Although it is very nonspecific, it is still widely used because it is simple and inexpensive to measure.

Pathophysiology. Dysproteinemia, such as in acute inflammatory diseases, and also hyperimmunoglobulinemia change the surface charge and aggregation tendency of erythrocytes (zeta potential, ξ-potential) and thus also their sedimentation rate.

Preanalytical requirements. The blood collection tubes required for this test contain sodium citrate. It is important to fill the tubes completely to ensure a blood/citrate ratio of 4 + 1.

Analysis. Shortly before measuring, the tubes are well mixed and placed vertically. One hour later the depth of the erythrocyte sediment is read off in millimeters.

Reference range for ESR:

image Men: up to 15 mm after 1 hour.

image Women: up to 20 mm after 1 hour.

Higher levels are found during pregnancy and in patients of advanced age.

Interpretation. The ESR indicates with great sensitivity any increase in immunoglobulins (rapid sedimentation, 80–100 mm in 1 hour) as found in, e. g., chronic inflammatory diseases, monoclonal gammopathy, and autoimmune diseases.

About 5% of all increases in ESR remain unresolved, and in up to 70% of these cases the ESR normalizes spontaneously.

ESR Pitfalls

In acute inflammation, the ESR increases only after 24–48 hours, and it has a long half-life of 4–6 days. There is thus quite a lag in its response.

Angiotensin-Converting Enzyme (ACE)

Physiology. ACE is a monomeric zinc metalloprotease that is ubiquitous in the body. It is bound to the cell membrane, with its catalytic center on the extracellular side. ACE is found on the luminal surfaces of vascular endothelial cells and also inside the cells of the monocyte–macrophage system; it is particularly plentiful in organs with a large vascular bed, like the lung. ACE is part of the renin–angiotensin system, for it converts angiotensin I into the vasosuppressive form angiotensin II. It also inactivates the vasodilator bradykinin.

Indication. Elevated plasma ACE activity is found particularly in sarcoidosis (Boeck's disease), and also in hyperthyroidism, diabetes mellitus, berylliosis, silicosis, asbestosis, Gaucher's disease, and chronic alcoholism.

Preanalytical requirements. ACE in serum or plasma is stable for 1 week in the refrigerator. If the patient is taking ACE inhibitors (e. g., captopril), the medication must be discontinued 4 weeks prior to determination of ACE activity. Zinc chelators (e. g., EDTA) are unsuitable as anticoagulants.

Analysis. ACE is determined in plasma. Unfortunately, the many possible methods all have different reference ranges, and each patient's result can therefore only be interpreted against the reference range of the laboratory concerned. The common feature of these methods is that ACE converts synthetic aryl-oligopeptides:

• Lieberman method: measures the release of hippuric acid from hippuryl-histidyl-leucine after extraction with ethyl ether.

• Neels method: measures the release of glycyl-glycine from hippuryl-glycyl-glycine by subsequent chromogenic reaction with nitrobenzene sulfonate.

• Ryan radioactive method: uses tritium-labeled hippurylglycyl-glycine as a substrate and measures the amount of the tritium-labeled hippuric acid released.

• Silverstein method: uses spectral fluorometry to determine the histidyl-leucine released after formation of a fluorescent complex with orthophenylenediamine.

Reference range for ACE in serum/heparin plasma (according to method):

image Lieberman: 10–35 U/L

image Neels: 115–420 U/L

image Ryan radioactive: 44–138 U/L

image Silverstein: 12–52 U/L

Interpretation. ACE activity differs greatly among patients because of a polymorphism of the ACE gene. Individuals with an insertion of 250 base pairs in the gene (genotype II) have about half the ACE activity of those who have a deletion (genotype DD).

In active sarcoidosis, ACE activity is increased. ACE determination allows:

• Estimation of the granuloma burden of the body.

• Assessment of the course of the disease.

• Assessment of the success of corticosteroid treatment.

Another good marker for the course of sarcoidosis is sCD 25 (see above).

Ceruloplasmin (Cp)

Physiology. This plasma protein is predominantly produced as an α2-globulin in the liver. Its main functions are the transport of copper in plasma and the oxidation of divalent to trivalent iron.

Indication. Elevated ceruloplasmin concentrations are found in acute and chronic inflammation, cholestasis, and pregnancy, or in persons taking oral contraceptives or estrogens.

Lowered ceruloplasmin levels are found in Menkes syndrome (kinky hair syndrome) and also in liver cirrhosis and nephrotic syndrome. Other causes of low ceruloplasmin concentrations include a rare hereditary defect in ceruloplasmin synthesis and a secondary ceruloplasmin deficiency in Wilson's disease or copper malnutrition (e. g., due to long-term parenteral nutrition).

Analysis. Ceruloplasmin is determined by either immunonephelometry or immunoturbidimetry, or radial immunodiffusion. Determination of ceruloplasmin is standardized, so results are comparable.

Reference range for ceruloplasmin in serum/plasma:

image 0.2–0.6 g/L.

Interpretation. Ceruloplasmin is an acute-phase protein; it is therefore nonspecifically elevated in acute inflammation. In Wilson's disease, ATPase, which controls the incorporation of copper atoms in hepatocytes, is absent; copper is therefore not incorporated and accumulates in the tissue. In the initial phase of Wilson's disease, copper diffusely accumulates in the hepatocytes. Laboratory tests during this phase show that the copper content is elevated only in liver tissue. Later on, when a critical copper concentration is reached in the hepatocytes, copper is redistributed into lysosomes and thus also enters the plasma. The binding of copper to ceruloplasmin lowers the total copper concentration and the serum concentration of ceruloplasmin, while the concentration of free copper (not bound to ceruloplasmin) and the renal elimination of copper bound to albumin are increased.

Folic Acid and Vitamin B12 (Cobalamins)

Pathophysiology. Folic acid is the synthetic form of folates. Like cobalamins (vitamin B12), folates belong to the essential vitamins and must be taken up with the food (folates are mainly contained in liver and vegetables, and vitamin B12 also in fish). Vitamin B12 is a water-soluble vitamin. Both vitamin B12 deficiency and folate deficiency are morphologically recognized by disturbed cell maturation, manifesting itself in megaloblastic anemia [macrocytic: with increased mean corpuscular volume (MCV); hyperchromic: with increased mean corpuscular hemoglobin (MCH)] and granulocytopenia (hypersegmentation of neutrophils).

Preanalytical requirements. Plasma folate levels vary with the amount of folic acid taken in food; it therefore makes sense to collect blood after fasting.

Analysis. Folates and vitamin B12 are determined in immunological tests (ligand assays).

Reference ranges in serum/plasma:

image Folates: 3–30 nmol/L.

image Vitamin B12: 200–750 pmol/L.

Folates may also be determined in erythrocytes. The advantage of this is that intraerythrocytic folate levels are independent of short-term nutritional effects; the disadvantage is that this method is more cumbersome, and it is therefore less often used.

Reference range for folates in erythrocytes:

image 66–198 nmol/L.

Interpretation. Folate deficiency is one of the most common vitamin deficiencies worldwide. Half of the folate in the human body is stored in the liver; when folate intake is low but folate storage is full, therefore, the first clinical symptoms of folate deficiency appear only after 3–6 months. Clinical symptoms of folate deficiency include paleness, weakness, and poor memory. Neuropsychiatric symptoms occur only in connection with vitamin B12 deficiency. Nutritional vitamin B12 deficiency is rare except in vegetarians and elderly persons; deficiency is usually due to insufficient intestinal absorption (autoantibodies to parietal cells, deficiency of intrinsic factor, autoantibodies to intrinsic factor, damaged ileal mucosa). Neurological symptoms of vitamin B12 deficiency include loss of sensory functions, disturbed vision, funicular myelosis, and hallucination.

Diabetes Markers

Fasting Glucose Measurement

Preanalytical requirements. The material used for glucose determination varies. Plasma glucose measurement is considered the reference method. Glucose in whole blood is slowly degraded by glycolysis. To avoid falsely low glucose levels, the sample should either be centrifuged as soon as possible and the supernatant separated from the sediment, or the blood should be collected in a tube containing a glycolysis inhibitor (e. g., sodium fluoride).

Analysis. Fasting glucose can be determined using three different enzyme systems:

• Hexokinase.

• Glucose dehydrogenase.

• Glucose oxidase.

The results obtained with these systems agree very well.

Reference range for fasting plasma glucose:

image 70–110 mg/dL (3.9–5.8 mmol/L).

Reference range for fasting capillary whole blood glucose:

image 70–100 mg/dL (3.9–5.5 mmol/L).

Oral Glucose Tolerance Test (oGTT)

Preanalytical requirements. The patient should fast for at least 12 hours prior to the test and should have carbohydrate-rich food during the 3 days before the test. If possible, any medication affecting the test should be discontinued 3 days beforehand (e. g., diuretics, glucocorticoids, contraceptives, salicylates).

Analysis. After determination of fasting glucose levels, the patient must be given a glucose solution to drink (75 g in 300 mL). The glucose content in the blood is then determined after 1 hour and again after 2 hours. During this time, the patient should rest without eating or smoking.

Interpretation. If after 2 hours the glucose level is higher than 200 mg/dL (11 mmol/L), this is considered to indicate diabetes according to the WHO criteria. Levels between 140 and 200 mg/dL (7.7–11 mmol/L) are considered to indicate impaired glucose tolerance.

Fructosamine

Preanalytical requirements. The results vary with the composition and concentration of serum proteins. Venous congestion and body position must therefore be borne in mind when taking the blood.

Analysis. Fructosamines are determined by measuring the reduction of a chromogen to a colored compound.

Reference range for fructosamines in serum/plasma:

image 205–285 μmol/L.

Interpretation. Fructosamines are glycosylated serum proteins. They are made up of 80% glycosylated albumin and 20% glycosylated immunoglobulin G. Since these two proteins have about the same half-lives (approximately 3 weeks), measuring them provides information about the glycemic state during the last 2–3 weeks.

Fructosamine Pitfalls

Hemolysis and hyperbilirubinemia yield falsely high concentrations. In addition, the specificity of the test is not very high.

Glycosylated Hemoglobin (HbA1 c)

Analysis. HbA1 c is determined either by chromatography (high-performance liquid chromatography, cation exchange chromatography, or affinity chromatography) or by immunoassay.

Reference range for HbA1 c in EDTA whole blood:

image < 6.1%.

Interpretation. HbA1 c is a glycosylated hemoglobin fraction. Glucose is taken up into erythrocytes by diffusion—independently of insulin. As the mean lifespan of erythrocytes is about 120 days, the determination of HbA1 cprovides information on the glycemic state during the last 6–8 weeks.

HbA1 c Pitfalls

image Total fraction of HbA1. It is important to distinguish between the HbA1 c content and the total fraction of HbA1; some laboratories also determine the latter. The HbA1 total fraction represents the total glycosylated hemoglobin and contains the subfractions HbA1 a, HbA1 b, and HbA1 c. The HbA1 total fraction can be up to 2% higher.

image Falsely high values. Hemoglobinopathies (e. g., sickle cell hemoglobin, HbS) may falsify the results of chromatography. Patients with severe renal insufficiency also have a fraction of carbamoylated hemoglobin, which also may lead to falsely high results.

image Falsely low values. Hemolytic conditions (hemolytic anemia) result in low values.

Alcohol Markers

Carbohydrate-Deficient Transferrin (CDT)

Pathophysiology. Transferrin is the iron transport protein in the blood. It consists of a polypeptide chain divided into two similar domains (N-terminal, C-terminal). The C-terminal domain carries two branched carbohydrate chains, with a sialic acid residue at the end of each branch. Separation of iron-saturated transferrin by chromatography or isoelectric focusing yields four isoforms that differ in the number of sialic acid residues: pentasialo-, tetrasialo-, trisialo-, and disialo-transferrin. The disialo fraction is normally only 1%, but this can rise markedly with alcohol consumption. In addition, an asialo isoform may also occur with high alcohol consumption. Together, the disialo and asialo fractions are known as carbohydrate-deficient transferrin (CDT).

Indication. The traditional markers for chronic alcoholism, such as γ-glutamyltransferase (γGT) or raised MCV, are nonspecific. Determination of CDT has a better diagnostic specificity.

Analysis. The CDT fraction is determined by chromatography or isoelectric focusing, although immunological tests may also be used. The results are best expressed as a percentage for comparability, since results given in U/L depend on the method used.

Reference range for CDT in serum/plasma:

image < 1.0%.

Specificity and sensitivity. Diagnostic specificity and sensitivity vary from study to study, but the values are usually around 70% for sensitivity and 95% for specificity.

CDT Pitfalls

In patients with congenital disorders of glycolysation (CDG), carbohydrate side chains are also absent. This condition is an inherited disorder; it affects the nervous system and causes growth retardation and liver dysfunction in childhood. The transferrin isoforms found here correspond to those obtained by chromatography or isoelectric focusing of CDT fractions from alcoholics. Severe liver disease and other genetic transferrin variants also lead to elevated CDT levels.

Endocrine Markers

Some autoimmune diseases are accompanied by dysfunction of the thyroid gland (Hashimoto's thyroditis). For this reason, analysis of the basic markers of thyroid function is indicated.

Thyroid-Stimulating Hormone (TSH, Thyrotropin)

Indication. Measurement of TSH is regarded as the first step in assessing thyroid function.

Analysis. TSH is measured in the laboratory by immunological tests. These tests need to be ultrasensitive to identify hyperthyroidism.

Reference range for TSH in serum/plasma:

image Newborns: < 20 mU/L.

image Children and adults: 0.4–4.0 mU/L.

Interpretation. If TSH values are normal, manifest hypo- or hyperthyroidism can be excluded. Clearly pathological findings require confirmation by analysis of triiodothyronine (T3), free T3 (FT3), thyroxine (T4), and free T4 (FT4).

T3, T4, FT3, FT4

Pathophysiology. The concentrations of T3 and T4 depend on the binding capacity of thyroxine-binding globulin (TBG). If TBG is raised (e. g., because of estrogen intake, pregnancy, hepatitis), raised levels of T3 and T4 are also found, which are unrelated to thyroid function. For this reason, it is advisable to determine the levels of the free thyroid hormones, FT3 and FT4.

Preanalytical requirements. Blood should be collected at least 24 hours after the last intake of thyroid hormones.

Analysis. Thyroid hormones are measured by immunoassay.

Reference ranges for thyroid hormones in serum/plasma (adults):

image Total T4: 55–110 μg/L (77–142 nmol/L).

image Free T4: 8–18 ng/L (10–23 pmol/L).

image Total T3: 0.9–1.8 μg/L (1.4–2.8 nmol/L).

image Free T3: 3.5–8.0 pg/mL (5.4–12.3 pmol/L).

Atherosclerosis Markers

Triglycerides

Physiology. Triglycerides are glycerol esters with three fatty acid residues. They are poorly soluble in water and are transported in the blood bound to apolipoproteins.

Analysis. Triglyceride concentrations are measured by determining the amount of free glycerol generated by hydrolytic cleavage of the triglycerides.

Reference range for triglycerides in serum/plasma:

image < 200 mg/dL or < 2.3 mmol/L.

image Higher values may be found in elderly patients.

Interpretation. Very high triglyceridemia occurs mainly in patients with chylomicronemia. In healthy persons, a small amount of glycerol is also present in free form (about 10 mg/dL); this can be ignored. However, higher concentrations may occur in diabetes or liver disease, and also after administration of glycerol-containing infusions, and this leads to the calculation of falsely high triglyceride values. In such cases, the free glycerol needs to be determined and deducted from the total glycerol content.

Triglyceride Pitfalls

Hemoglobin levels above 200 mg/dL and bilirubin levels above 20 mg/dL result in falsely low triglyceride values.

Cholesterol

Physiology. Cholesterol is an important constituent of the cell membrane and also a precursor in the synthesis of steroid hormones and bile acids. The sterol ring of cholesterol cannot be broken down further. It is therefore very important that cholesterol is transported by apolipoproteins to the liver, where some of it is converted to bile acid. In plasma 25–40% of cholesterol is present as free, non-esterified cholesterol. The rest is esterified with unsaturated fatty acids.

Analysis. In determining total cholesterol, no distinction is made between the esterified and nonesterified forms. By the addition of cholesterol esterase to the sample, cholesterol esters are converted to free cholesterol, which is then oxidized by cholesterol oxidase to cholestenone. This oxidation generates hydrogen peroxide, which is detected by various photometric methods. The most commonly used test is the Trinder reaction, in which peroxidase catalyzes the formation of a red dye from phenol and 4-aminoantipyrine.

Reference range for cholesterol in serum/plasma:

image < 200 mg/dL or < 5.2 mmol/L.

image Higher values are found in elderly patients.

Interpretation. If cholesterol levels are elevated, the next step is to determine the lipoprotein fraction (see below).

Cholesterol Pitfalls

Hemoglobin levels above 200 mg/dL and bilirubin levels above 42 mg/dL interfere with the enzymatic measurement of cholesterol.

Lipoprotein Fractions

Physiology. Lipoproteins are lipid–protein complexes which transport lipids in the blood. They include verylow-density (VLDL), low-density (LDL), intermediate-density (IDL), and high-density lipoproteins (HDL). Lipoproteins may be rich in triglycerides (chylomicrons and VLDL) or rich in cholesterol (LDL and HDL); they also differ in the composition of the apolipoproteins. Lipoprotein metabolism is divided into an exogenous pathway (dietary lipids) and an endogenous pathway (lipids originating in the liver). The exogenous pathway starts with the absorption of fats in the small intestine. The fats are then transported in chylomicrons by the lymphatic circulation to the liver, where they are taken up by apoE receptors. In the liver, VLDL particles are formed and secreted into the blood. On their way to the various tissue cells, VLDL particles are broken down by triglyceride-degrading lipoprotein lipase (LPL) into IDL particles, which at the same time become cholesterol-rich by absorbing cholesterol from HDL particles. Thus, LDL forms from VLDL via IDL, and is then taken up by the LDL receptors on the cells of the body.

Analysis. The various lipoproteins can be separated by ultracentrifugation. There are now also immunological tests for determining the HDL and LDL fractions. At triglyceride levels of less than 400 mg/dL (4.5 mmol/L), LDL cholesterol can be calculated from the levels of total cholesterol, total triglycerides, and HDL cholesterol using the Friedewald formula:

LDL cholesterol = total cholesterol – HDL cholesterol – (total triglycerides/5)

Reference ranges for lipoprotein fractions of serum/plasma:

image HDL cholesterol: > 35 mg/dL (> 0.9 mmol/L).

image LDL cholesterol: < 155 mg/dL (< 4.0 mmol/L).

Interpretation. When the test results are within the above limits, there is no risk of atherosclerosis. LDL cholesterol levels above 190 mg/dL (4.9 mmol/L) represent a high risk factor for atherosclerosis.

Lipoprotein (a), Lp (a)

Physiology. Lipoprotein (a) is a dimer composed of an LDL particle linked by a disulfide bridge to apolipoprotein (a). Apolipoprotein (a) shares partial structural homology with plasminogen, the zymogen of plasmin; it therefore binds to fibrin clots, thus competing with plasminogen. However, Lp (a) has no fibrinolytic properties and thus has an atherogenic effect – and it is indeed found in atherosclerotic plaques.

Although Lp (a) thus shares homology with LDL, its metabolism is independent of LDL. This is obvious from the fact that dietary measures can certainly reduce LDL levels, but reduce Lp (a) levels only slightly. Lipid-lowering drugs also have little effect on the concentration of Lp (a). Raised Lp (a) levels are considered an independent risk factor for atherosclerosis.

Analysis. The concentration of Lp (a) is determined by immunological methods using automated nephelometry. However, there are also other tests that use ELISA and (electro) immunodiffusion.

Reference range for Lp (a) in serum/plasma:

image < 300 mg/L.

Interpretation. Elevated Lp (a) levels are found in patients with nephrotic syndrome, hypothyroidism, poorly controlled diabetes, uremia under hemodialysis, and acute myocardial infarction.

Decreased concentrations are found in patients with hyperthyroidism and patients undergoing treatment with estrogens, niacin, or neomycin.

Homocysteine

Pathophysiology. Homocysteine is considered an independent risk factor for atherosclerosis. This amino acid is formed intracellularly from methionine. Homocysteine undergoes either remethylation to methionine (using vitamin B12 as a cofactor) or degradation to cysteine (using vitamin B6 as a cofactor). Excess homocysteine is secreted into the plasma. Increased formation of atherosclerotic plaques is promoted by defoliation of epithelial cells and oxidation of LDL particles, which are then no longer taken up by the LDL receptors on the surface of cells and are thus degraded via the scavenger cell pathway. This process is further enhanced by reduced protein C activity and increased factor V activity.

Preanalytical requirements. Blood for homocysteine determination should be collected after the patient has been fasting. The test requires EDTA blood, which should be stored in ice water until centrifugation.

Analysis. Determination of homocysteine is by HPLC or FPIA.

Reference range for homocysteine in EDTA plasma:

image < 10 μmol/L (gray area up to 12 μmol/L).

Interpretation. Elevated homocysteine levels often result from deficiency in vitamin B12 or vitamin B6 and can be returned to normal by administering these vitamins.

Creatine Kinase (CK)

Physiology. Creatine kinase is an enzyme that plays an important role in intracellular energy metabolism and the regulation of energy transfer. Creatine kinase is composed of two subunits. Three isozymes, CK-MM, CK-MB, and CKBB, are present in the cytoplasm, and mitochondria contain an additional isoform, CK-MiMi. Creatine kinase is almost ubiquitous, but the isozyme composition of individual organs differs considerably.

Analysis. Creatine kinase is measured kinetically by determining the rise in NADH2.

Reference ranges for creatine kinase in serum/plasma (measured at 37°C):

image Men: < 171 U/L.

image Women: < 145 U/L.

Interpretation. An increase in creatine kinase activity is found in myocardial disease, where the activity of CK-MB is more than 6% of the total CK activity. Creatine kinase activity increases considerably in musculoskeletal diseases, with CK-MM dominating. If the blood–brain barrier is damaged (e. g., neurosurgical intervention, subarachnoid hemorrhage, craniocerebral trauma), CK-BB is transiently detectable in the blood. This should be confirmed by CK isozyme electrophoresis. If CK-MB is measured in these patients by enzyme immunoinhibition assay, falsely high values will be obtained.

Neuron-Specific Enolase (NSE) and S-100 Protein

Neuron-specific enolase is present in neurons and neuroendocrine cells and plays an important role in glycolysis. However, it is also found in erythrocytes and thrombocytes (see Chap. 3, “Brain Proteins in the Blood”). S-100 is an acidic calcium-binding protein consisting of two subunits. It is present at high concentrations in glial cells and Schwann cells, and also in striated muscle, heart, and kidney. Its biological function is still unclear.

Analysis. Both proteins are determined by immunological tests. Only serum should be used.

Reference ranges in serum:

image NSE (depending on the test used): < 20 μg/L.

image S-100: < 0.11 μg/L.

Interpretation. Serum NSE levels are a good follow-up marker in patients with small-cell lung cancer. Elevated levels are also found in patients with brain injury (hypoxic cerebral damage, see Chap. 12, “Cerebral Hypoxia”; cerebral infarction, see Chap. 12, “Ischemic Cerebral Infarction;” and cerebral edema) (Schaarschmidt et al., 1994). NSE analysis in serum is advantageous compared to S100B as it has a 1000-fold higher concentration in the brain and CSF (Table 21.6) and therefore a much higher sensitivity for detection of pathological brain processes in blood.

The serum concentration of S-100 also indicates the extent of cerebral damage. After brain injury, S-100 is released from destroyed cells into the CSF and is detectable in the serum at increased concentrations. S-100 is also found in tumor tissues, e. g., in melanoma, glioma, and highly differentiated neuroblastoma. S-100 is therefore also considered a follow-up and prognostic marker in melanoma patients.

NSE Pitfalls

In the presence of hemolysis NSE testing yields falsely high NSE serum values.

References

Bruhn HD, Fölsch UR. Lehrbuch der Labormedizin. Stuttgart: Schattauer; 1999

Burtis CA, Ashwood ER, Bruns DE (eds). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders; 2006

Dörner K. Klinische Chemie und Hämatologie. 5th ed. Stuttgart: Thieme; 2003

Keil E, Fiedler H. Klinische Chemie systematisch. Bremen: Uni-MED; 2000.

Thomas L. Labor und Diagnose. 6th ed. Frankfurt am Main: TH-Books; 2005

Schaarschmidt H, Prange H, Reiber H. Neuron specific enolase concentrations in blood as a prognostic parameter in cerebrovascular diseases. Stroke 1994;25:558–565