< 11.3 ng/L.

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).

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).

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

Glycosylated Hemoglobin (HbA_{1 c})

Analysis. HbA_{1 c} is determined either by chromatography (high-performance liquid chromatography, cation exchange chromatography, or affinity chromatography) or by immunoassay.

Interpretation. HbA_{1 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 HbA_{1 c} provides information on the glycemic state during the last 6–8 weeks.

HbA_{1 c} Pitfalls

Total fraction of HbA₁. It is important to distinguish between the HbA_{1 c} content and the total fraction of HbA₁

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