Glucose is the major carbohydrate present in the peripheral blood. Oxidation of glucose is the major source of cellular energy in the body. Glucose derived from dietary sources is converted to glycogen for storage in the liver or to fatty acids for storage in adipose tissue. The concentration of glucose in blood is controlled within narrow limits by many hormones, the most important of which are produced by the pancreas. The most frequent cause of hyperglycemia is diabetes mellitus resulting from a deficiency in insulin secretion or action. A number of secondary factors also contribute to elevated blood glucose levels. These include pancreatitis, thyroid dysfunction, renal failure, and liver disease. Hypoglycemia is less frequently observed. A variety of conditions may cause low blood glucose levels such as insulinoma, hypopituitarism, or insulin induced hypoglycemia. Glucose measurement in urine is used as a diabetes screening procedure and to aid in the evaluation of glucosuria, to detect renal tubular defects, and in the management of diabetes mellitus. Glucose measurement in cerebrospinal fluid is used for evaluation of meningitis, neoplastic involvement of meninges, and other neurological disorders.

Transferrin is a glycoprotein with a molecular weight of 79570 daltons. It consists of a polypeptide strand with two N‑glycosidically linked oligosaccharide chains and exists in numerous isoforms. The rate of synthesis in the liver can be altered in accordance with the body’s iron requirements and iron reserves. Transferrin is the iron transport protein in serum. In cases of iron deficiency, the degree of transferrin saturation appears to be an extremely sensitive indicator of functional iron depletion. The ferritin levels are depressed when there is a deficiency of storage iron. In sideropenia, an iron deficiency can be excluded if the serum transferrin concentration is low, as in inflammations or - less commonly - in cases of ascorbic acid deficiency. In screening for hereditary hemochromatosis, transferrin saturation provides a better indication of the homozygous genotype than does ferritin. The treatment of anemia with erythropoietin in patients with renal failure is only effective when sufficient depot iron is present. The best monitoring procedure is to determine transferrin saturation during therapy. Transferrin saturation in conjunction with ferritin gives a conclusive prediction of the exclusion of iron overloading in patients with chronic liver disease. A variety of methods are available for determining transferrin including radial immunodiffusion, nephelometry and turbidimetry. The Roche transferrin assay is based on the immunological agglutination principle.

Vitamin B12, also referred to as cobalamin․ It is a water-soluble vitamin which is synthesized by microorganisms. It cannot be synthesized in the human body and is seldom found in products of plant origin. Main sources of vitamin B12 are meat, fish, eggs and dairy products.1 The uptake in the gastrointestinal tract depends on intrinsic factor, which is synthesized by the gastric parietal cells, and on the “cubam receptor” in the distal ileum. The most frequent cause of severe vitamin B12 deficiency is a lack of intrinsic factor due to autoimmune atrophic gastritis. The disease is historically called “pernicious anemia”, even though many patients present with mainly neurologic manifestations. Examples of other causes for vitamin B12 deficiency are malabsorption due to gastrectomy, inflammatory bowel disease or dietary deficiency, e.g. in strict vegetarians (vegans).2 Thus, vitamin B12 is important for DNA synthesis, regenerating methionine for protein synthesis and methylation, as well as for the development and initial myelination of the central nervous system (CNS) and for the maintenance of normal CNS function.2,3 Vitamin B12 deficiencies are common in wealthier countries principally among the elderly and are most prevalent in poorer populations. In general the prevalence increases with age.4,5 Vitamin B12 deficiency impacts red blood cell synthesis, resulting in megaloblastic anemia due to abnormal DNA synthesis.3 In addition it impairs neurological function, in particular demyelination of nerves in part due to abnormal methylation, leading to peripheral neuropathy, dementia, poor cognitive performance, and depression.3 Other effects of vitamin B12 deficiency or depletion are increased risk of neural tube defects, osteoporosis, cerebrovascular and cardiovascular diseases.3 Early diagnosis is essential, because of the latent nature of this disorder and the risk of permanent neurological damage.3,5 Generally, the primary test performed to confirm the diagnosis of vitamin B12 deficiency is measurement of serum vitamin B12 level.2 Recent publications suggest that in addition the following biomarkers should be measured to improve the specificity of diagnosis: folate, methylmalonic acid (MMA), homocysteine and holotranscobalamin.

Ferritin is known as iron-storage protein and is synthesized by many body cells. It is found mainly in the liver, spleen, muscle and bone marrow, with only a small fraction found in blood. The amount of ferritin in serum serves as an indicator of the iron reserves, showing if too little (e.g., iron deficiency anemia) or too much iron is available (e.g., hemochromatosis).1,2 The protein is involved in the cellular uptake, storage and release of iron. Ferritin has a double function: storage of iron in its bioavailable form and at the same time protection of the cells from the toxic effects of iron, caused by iron’s capacity to generate reactive species which can directly damage DNA and proteins.2,3 The iron-free protein, apoferritin, consists of 24 subunits and has a molecular weight of approximately 450 kDa. The iron core of ferritin can contain up to approximately 4500 iron atoms in the form of Fe3+ ions.4,5 Iron-loaded ferritin and hemosiderin, an insoluble iron-protein complex, represent the iron store of each cell and of the entire organism.2,4 A number of different isoforms of ferritin exist which are composed of different subunits that are partially tissue specific.1,4 Under steady-state conditions, the serum ferritin concentration is proportional to the total body iron stores: 1 ng of serum ferritin per mL corresponds to 10 mg of total iron stores.6,7,8 Therefore, in the literature, the measurement of serum ferritin levels is proposed as the best and most convenient laboratory test to estimate iron stores and diagnose iron deficiency or iron related disorders.6,8,5,9 It has substituted the invasive and semiquantitative histochemical examination of bone marrow aspirate or biopsy as the gold standard for diagnosis of iron deficiency anemia.2,9 Serum ferritin is a good indicator of storage iron in the body; however it does not provide information about the amount of iron actually available for erythropoiesis. Decreased serum ferritin concentrations of 400 µg/L) may have many implications: Ferritin being an acute phase reactant, elevated serum ferritin values can occur in patients with infections, acute or chronic inflammation and malignant tumors, despite acute iron deficiency. Elevated ferritin levels unrelated to iron stores are also seen in case of alcoholic or viral hepatitis, and chronic renal failure. Diagnosis should be made looking at the entire clinical situation of the individual patient.

Folate belongs to the family of B‑group vitamins․ Folate (folic acid) is vital for normal cellular functions and plays an essential role in nucleic acid synthesis, methionine regeneration, shuttling and redox reactions of one‑carbon units required for normal metabolism and regulation.1,2 The folate metabolism can be exemplified as a cycle, where folate facilitates the transfer of one‑carbon units from one molecule to another required in various biochemical reactions․ The methionine cycle is highly sensitive to folate deficiency: with a low folate status, the ability of the cell to re-methylate homocysteine is impaired and this results in increased homocysteine concentrations in plasma.2 Folate also plays an essential role in the synthesis of purine and pyrimidine precursors of nucleic acids. Altered distribution of methyl groups and impaired DNA synthesis play an essential role in the development of cancers. Abnormal folate status has also been linked with the development of diseases like cardiovascular diseases, neural tube defects, cleft lip and palate, late pregnancy complications, neurodegenerative and psychiatric disorders.1,2 Folate belongs to the group of essential vitamins, i.e. it cannot be synthesized by the human organism and therefore must be absorbed from diet. Primary sources of folates are green and leafy vegetables, sprouts, fruits, brewer’s yeast and liver.1,2 Folate deficiency can be caused by decreased nutritional intake, poor absorption of ingested folate in the intestine or increased demand of folate, for example during physical activity or pregnancy. Deficiency of folate can also be a result of liver diseases or impaired folate metabolism due to genetic defects or drug interactions.2 A clinical manifestation of both folate and vitamin B12 deficiency is the so called megaloblastic (macrocytic) anemia: due to the affected DNA synthesis and cell maturation, especially involving the cells of erythropoiesis, the total count of erythrocytes is significantly reduced. The hemoglobin synthesis capacity however is normal, which leads to abnormally large erythrocyte precursors (“macrocytes” or “megaloblasts”), which have an elevated hemoglobin content (“hyperchromic anemia”).3,4 Because vitamin B12 and folate are closely interrelated in the cellular one‑carbon unit metabolism, and also hematologic and clinical consequences of the two vitamin deficiency states might be similar, it is advisable to determine both parameters simultaneously in patients with the relevant symptoms of vitamin deficiency.

Activated Partial Thromboplastin Time (APTT) • The activated partial thromboplastin time (APTT) is a general coagulation screening test of the coagulation factors XII, XI, IX, VIII, X, V, II and fibrinogen. • A prolongation of the APTT is encountered in the following situations (8): – Congenital Deficiencies ◊ If the prothrombin time (PT) is normal, the following factors may be deficient: - factor VIII (STA® - Deficient VIII, REF 00725) - factor IX (STA® - Deficient IX, REF 00724) - factor XI (STA® - Deficient XI, REF 00723) - factor XII (STA® - Deficient XII, REF 00722). ◊ If all these factors are normal, a deficiency in the following should be considered: - prekallikrein (Fletcher factor) - HMW kininogen (Fitzgerald factor). – Acquired Deficiencies and Abnormal Conditions ◊ Liver diseases ◊ Consumptive coagulopathy ◊ Fibrinolysis ◊ Circulating anticoagulants (LA type or circulating anticoagulant against a factor) ◊ During heparin or oral anticoagulant therapy ◊ Treatments with thrombin inhibitors (e.g., hirudin, argatroban...).

Lupus anticoagulants (LA) are associated with numerous clinical states: systemic lupus erythematosus (13), recurrent spontaneous abortions, thromboses (9), infections (13). The diagnosis of LA is often difficult because of variable reagent sensitivity and the intrinsic heterogeneity of LA (9). Lupus anticoagulants are antibodies directed against phospholipid/ protein complexes (12). They have the ability to prolong the clotting times of the phospholipid dependent tests (9). In practice, factor deficient plasmas are easily identified with APTT, since the addition of normal citrated plasma restores normal in vitro clotting time. However additional tests are necessary to provide clear-cut differentiation between LA and anti-coagulation factor antibodies and/or heparin (5, 10). • The presence of circulating anticoagulants of the LA type leads to the prolongation of the APTT, and this prolongation is much longer with the PTT-LA reagent than with regular APTT reagents that are not sensitized to the LA. The PTT-LA reagent is used for the LA screening only.

D-Dime The specific degradation of fibrin (i.e., fibrinolysis) is the reactive mechanism responding to the formation of fibrin (14). Plasmin is the fibrinolytic enzyme derived from the inactive plasminogen. Plasminogen is converted into plasmin by plasminogen activators. The main plasminogen activators are the tissue plasminogen activator (tPA) and the pro-urokinase which is activated into urokinase (UK) by, among others, the contact system of coagulation (1). In the bloodstream, plasmin is rapidly and specifically neutralized by α2-antiplasmin (1) thereby restricting its fibrinogenolytic activity and localizing the fibrinolysis on the fibrin clot. On the fibrin clot plasmin degrades fibrin into various products. Antibodies specific of these products, which do not recognize fibrinogen, have been developed (13). The presence of these various fibrin degradation products, among which D-dimer is the terminal product, is proof that the fibrinolytic system is in action in response to coagulation activation.

The thrombin time is a rapid and simple test designed for the assessment of fibrin formation. The thrombin time remains normal in deficiencies of factor XIII (fibrin stabilizing factor) (1, 2). Thrombin time should first be performed before any another specific assays are attempted, when a prolongation of the overall tests (PT, APTT) cannot be explained. • Prolongation of the thrombin time indicates (1, 2): – an abnormality of fibrinogen ◊ qualitative: dysfibrinogenaemia ◊ quantitative: severe hypofibrinogenaemia or congenital afibrino- genaemia acquired hypofibrinogenaemia (DIC, fibrinolysis, liver diseases) – the presence of antithrombins ◊ therapeutic: heparin, hirudin, argatroban... ◊ abnormal: FDP appearing during myelomas.

Antithrombin is a single chain glycoprotein which is synthesized in the liver and has a molecular weight of approx. 58 200 daltons. Antithrombin is a progressive inhibitor and inactivates not only thrombin (factor IIa), but also other serine proteases: predominantly factor Xa and to a lesser extent IXa, XIa, XIIa, as well as plasmin and kallikrein. Heparin greatly accelerates the inactivation of IIa and Xa. The antithrombin concentration plays an important role in the maintenance of a hemostatic equilibrium. Hereditary antithrombin deficiency with concomitant thromboembolic complications was first reported in 1965. Hereditary antithrombin deficiency is inherited in an autosomally dominant fashion and is prevalent in both men and women to approximately the same amount. One differentiates between two types: ▪ Type I-deficiency: Antithrombin concentrations and activities are lowered to the same extent due to decreased synthesis in the liver. ▪ Type II-deficiency: Antithrombin concentration remains normal, but its biological activity is reduced due to an altered molecular structure. Acquired antithrombin deficiency is much more common than hereditary antithrombin deficiency, but nevertheless much more rarely causes an increased risk of thrombosis. Antithrombin concentration and activity are both decreased to the same extent. Antithrombin deficiency can be caused by: ▪ Decreased synthesis due to a restricted (hepatic disease) or immature functioning of the liver (newborn, premature babies). In general, all liverdependent coagulation factors and inhibitors are decreased to the same extent. Because of the overall balanced hemostatic equilibrium, an increased tendency to thrombosis does not arise. ▪ Intravasal antithrombin loss due to its relatively small molecular weight: - renal loss in the course of a nephrotic syndrome - enteral loss in the course of protein-loss enteropathies - increased permeation into the extravasal space due to increased blood vessel permeability. ▪ Increased loss due to elevated activation of the coagulation process or activation of coagulation over a longer period of time, e.g. - postoperatively - continuous intravenous heparin therapy - consumptive coagulopathy, DIC. ▪ In the course of septic infection there is a direct relationship between antithrombin activity loss and the severity of the infection or the course of the sepsis. When septic disease is clinically suspected, early determination and determination in the context of disease monitoring of antithrombin activity is indicated to ensure early detection of DIC.

The prothrombin time is a coagulation screening test. It measures, as a whole, the activity of the coagulation factors II, V, VII, X and I. • A prolonged PT has been observed in the following clinical states: – congenital or acquired deficiencies of factor II, V, VII, X or fibrinogen (1) – liver failure (cirrhosis, hepatitis) (1) – treatments with vitamin K antagonists (1) – hypovitaminosis K: nutritional intake deficiency, disorders in absorption or metabolism of vitamin K (hemorrhagic disease of the newborn, cholestasis, treatment with antibiotics) (5) – fibrinolysis (1) – DIC (1). • The PT is commonly used for monitoring vitamin K antagonist therapy (3) because of its sensitivity to variations in the concentration of the vitaminK dependent factors II, VII and X. Consequently, the comparability of results of this test is essential for finding the therapeutical range. It is well known that the PT value of a plasma may vary according to the origin of the thromboplastin reagent and to the instrument used to measure it (4). A solution for standardization adopted by the World Health Organization is a “system of international reference standards for thromboplastins permitting the definition of an international scale for the intensity of anticoagulant therapy” (4). In this system the PT ratio is converted into the International Normalized Ratio (INR). The INR value corresponds to the value of the ratio of the patient’s PT to that of the standard PT raised to the ISI (International Sensitivity Index) power of the thromboplastin used: INR = ( ) The ISI value of a given thromboplastin is determined by testing normal plasmas and coumadin-treated patient plasmas with that thromboplastin and with the International Reference Preparation for thromboplastin. The PT values obtained with the two thromboplastins are plotted on log-log graph paper, and the orthogonal regression line is drawn. The slope of this line multiplied by the ISI value of the reference thromboplastin represents the ISI value of the thromboplastin of interest (3). The use of the INR is recommended for the assessment of the vitamin K antagonist therapy in patients (6, 8).

Protein C belongs to the group of vitamin K-dependent proteins (3). It is synthesized in the liver (6). Like the coagulation factors, protein C is present in plasma as a proenzyme: its transformation into an active enzyme requires the presence of thrombin, calcium and phospholipids; this thrombin-dependent activation of protein C is potentiated by an endothelial factor, thrombomodulin (2). In the activated state protein C regulates the coagulation process by neutralizing the procoagulant activities of the factors Va and VIIIa in the presence of protein S, itself also a vitamin K-dependent protein and is a cofactor of activated protein C (6, 9). Protein C is inhibited by activated protein C inhibitor (PCI) and by α1- antitrypsin (8). • Protein C Deficiencies There is a clinical interest in determining the protein C level because of the existence of protein C deficiencies, either acquired or congenital. Acquired protein C deficiencies are found in cases of hepatic disorders (1, 4) (hepatitis, cirrhosis, etc.), during vitamin K antagonists therapies (4) and in disseminated intravascular coagulation (DIC) (4). On the other hand, congenital protein C deficiencies, which are characterized by recurrent venous thromboses (4), exist in two different types: type I and type II. Type I deficiency is identified by a simultaneous decrease of both the functional and antigenic levels of protein C (8). Type II deficiency, which is rarer, is characterized by a depressed functional protein C level, but a normal immunological protein C level (8).

Protein S is a vitamin K-dependent protein that does not possess any esterase function (1). Protein S is synthesized in the liver as an inactive precursor. The active form is obtained after carboxylation of glutamic residues by a vitamin K-dependent carboxylase and contains the γ-carboxyglutamic residues thus allowing the molecule to fix calcium (1). Physiologically, protein S has an essential anticoagulant function. It acts as the cofactor of activated protein C, with which it appears to form a stoichiometric complex (1). In the presence of calcium, this complex binds strongly to the phospholipid surfaces and thus regulates the coagulation process, inactivating by proteolysis thrombin-activated factors V and VIII (3). Protein S greatly potentiates the anticoagulant function of protein C, probably by increasing the affinity of protein C for phospholipid membranes (8). The thrombin-sensitive region of the protein S molecule may be subjected to a limited proteolysis by thrombin: in this case, protein S retains its affinity for phospholipids, but loses its anticoagulant function as the cofactor of activated protein C (8). The biochemistry of protein S appears to be quite complex by the fact that it forms a dynamic equilibrium with the protein that binds the C4b of complement, i.e., the C4b-binding protein (C4b-BP). This C4b-BP is present in plasma in two different forms (8): one low molecular weight form (about 20 % of total weight) without any affinity for protein S and one high molecular weight form (about 80 % of total weight), which can complex with protein S in a 1 to 1 ratio. In physiological conditions, an equilibrium is reached, in which two different forms of protein S exist: – the free protein S form which acts as the cofactor of activated protein C and it represents about 40 % of total protein S – the high molecular weight C4b-BP bound protein S form which exhibits no activity as a cofactor of activated protein C (8) and it represents about 60 % of total protein S. • The congenital deficiencies of protein S are classified in three types (10): – type I deficiencies correspond to reduced antigen levels of both total and free protein S – type II deficiencies are characterized by a reduced protein S activity but with normal antigen levels of both total and free protein S – type III deficiencies are defined by a reduced antigen level and activity of free protein S but the antigen level of total protein S remains normal. Acquired protein S deficiencies can be found in several clinical states: inflammatory syndromes due to the increase of C4b-BP (8), hepatic disorders (6), nephrotic syndrome (6), treatments with oral anticoagulants (4), oral contraceptives (11), L-asparaginase (6). The congenital or acquired deficiency of protein S increases the risk of thromboembolism, owing to a decrease of blood anticoagulant potential. It may produce recurrent thrombotic episodes (2).

Fibrinogen is a glycoprotein of a molecular weight of approximately 340000 daltons (6), present in plasma at a concentration in the range of 2 to 4 g/l (200-400 mg/dl) (2). It is synthesized in the liver (1.7 to 5 g/day) (6). The synthesis of fibrinogen is controlled by the gene which codes for the β chain synthesis (3). Due to the existence of a genetic polymorphism for this gene, the plasma level of fibrinogen varies among individuals (3). The half-life of fibrinogen is about 3-5 days (6). Fibrinogen is composed of six chains: 2 Aα, 2 Bβ and 2γ (5). Thrombin (factor IIa) breaks up the fibrinogen molecule to split out 2 fibrinopeptide A (FPA) fragments from the Aα chains and 2 fibrinopeptide B (FPB) fragments from the Bβ chains (5). The fibrin monomers that are produced from these reactions then aggregate to form fibrin, which is subsequently stabilized by factor XIIIa (3, 5). The first step of this stabilization consists of the binding of two γ chains of two fibrin monomers (5). This binding is the origin of D-dimer, the degradation product that is specific of fibrin (5). An increase of fibrinogen level is found in cases of diabetes, inflammatory syndromes, obesity (3, 6); a decrease of the fibrinogen level is observed in DIC, fibrinogenolysis (3). Furthermore, fibrinogen seems to be involved in the pathogenicity of thrombotic cardiovascular events (3, 6).

squamous cell carcinoma antigen is a glycoprotein. Most often, the definition of this marker is used to monitor the course and effectiveness of therapy for squamous cell carcinoma of the cervix (sensitivity 70-85%), nasopharynx and ear. SCC is the marker of choice for monitoring the course and effectiveness of therapy for squamous cell carcinoma of the cervix uteri (Meier W. et al., 1989). Determination of the level of SCC in the blood allows not only to detect a relapse at an early stage, but also reflects the reaction of an already detected carcinoma to the therapy. Elevated SCC levels are found in 17% of non-small cell carcinomas and 31% of squamous cell carcinoma of the lungs (95% specificity). Smoking has no effect on SCC levels.

Calcium is the most abundant mineral element in the body with about 99 percent in the bones primarily as hydroxyapatite. The remaining calcium is distributed between the various tissues and the extracellular fluids where it performs a vital role for many life sustaining processes. Among the extra skeletal functions of calcium are involvement in blood coagulation, neuromuscular conduction, excitability of skeletal and cardiac muscle, enzyme activation, and the preservation of cell membrane integrity and permeability. Serum calcium levels and hence the body content are controlled by parathyroid hormone (PTH), calcitonin, and vitamin D. An imbalance in any of these modulators leads to alterations of the body and serum calcium levels. Increases in serum PTH or vitamin D are usually associated with hypercalcemia. Increased serum calcium levels may also be observed in multiple myeloma and other neoplastic diseases. Hypocalcemia may be observed e.g. in hypoparathyroidism, nephrosis, and pancreatitis.

Albumin ALB Albumin is a carbohydrate-free protein, which constitutes 55‑65 % of total plasma protein. It maintains plasma oncotic pressure, and is also involved in the transport and storage of a wide variety of ligands and is a source of endogenous amino acids. Albumin binds and solubilizes various compounds, e.g. bilirubin, calcium and long-chain fatty acids. Furthermore albumin is capable of binding toxic heavy metal ions as well as numerous pharmaceuticals, which is the reason why lower albumin concentrations in blood have a significant effect on pharmacokinetics. Hyperalbuminemia is of little diagnostic significance except in the case of dehydration. Hypoalbuminemia occurs during many illnesses and is caused by several factors: compromised synthesis due either to liver disease or as a consequence of reduced protein uptake; elevated catabolism due to tissue damage (severe burns) or inflammation; malabsorption of amino acids (Crohn’s disease); proteinuria as a consequence of nephrotic syndrome; protein loss via the stool (neoplastic disease). In severe cases of hypoalbuminemia, the maximum albumin concentration of plasma is 2.5 g/dL. Due to the low osmotic pressure of the plasma, water permeates through blood capillaries into tissue (edema). The determination of albumin allows monitoring of a controlled patient dietary supplementation and serves also as an excellent test of liver function.

Bilirubin is formed in the reticuloendothelial system during the degradation of aged erythrocytes. The heme portion from hemoglobin and from other heme-containing proteins is removed, metabolized to bilirubin, and transported as a complex with serum albumin to the liver. In the liver, bilirubin is conjugated with glucuronic acid for solubilization and subsequent transport through the bile duct and elimination via the digestive tract. Diseases or conditions which, through hemolytic processes, produce bilirubin faster than the liver can metabolize it, cause the levels of unconjugated (indirect) bilirubin to increase in the circulation. Liver immaturity and several other diseases in which the bilirubin conjugation mechanism is impaired cause similar elevations of circulating unconjugated bilirubin. Bile duct obstruction or damage to hepatocellular structure causes increases in the levels of both conjugated (direct) and unconjugated (indirect) bilirubin in the circulation.

Bilirubin is formed in the reticuloendothelial system during the degradation of aged erythrocytes. The heme portion from hemoglobin and from other heme-containing proteins is removed, metabolized to bilirubin, and transported as a complex with serum albumin to the liver. In the liver, bilirubin is conjugated with glucuronic acid for solubilization and subsequent transport through the bile duct and elimination via the digestive tract. Diseases or conditions which, through hemolytic processes, produce bilirubin faster than the liver can metabolize it, cause the levels of unconjugated (indirect) bilirubin to increase in the circulation. Liver immaturity and several other diseases in which the bilirubin conjugation mechanism is impaired cause similar elevations of circulating unconjugated bilirubin. Bile duct obstruction or damage to hepatocellular structure causes increases in the levels of both conjugated (direct) and unconjugated (indirect) bilirubin in the circulation.

Plasma proteins are synthesized predominantly in the liver, plasma cells, lymph nodes, the spleen and in bone marrow. In the course of disease the total protein concentration and also the percentage represented by individual fractions can significantly deviate from normal values. Hypoproteinemia can be caused by diseases and disorders such as loss of blood, sprue, nephrotic syndrome, severe burns, salt retention syndrome and Kwashiorkor (acute protein deficiency). Hyperproteinemia can be observed in cases of severe dehydration and illnesses such as multiple myeloma. Changes in the relative percentage of plasma proteins can be due to a change in the percentage of one plasma protein fraction. Often in such cases the amount of total protein does not change. The A/G ratio is commonly used as an index of the distribution of albumin and globulin fractions. Marked changes in this ratio can be observed in cirrhosis of the liver, glomerulonephritis, nephrotic syndrome, acute hepatitis, lupus erythematosus as well as in certain acute and chronic inflammations. Total protein measurements are used in the diagnosis and treatment of a variety of diseases involving the liver, kidney, or bone marrow, as well as other metabolic or nutritional disorders.

Creatinine Chronic kidney disease is a worldwide problem that carries a substantial risk for cardiovascular morbidity and death. Current guidelines define chronic kidney disease as kidney damage or glomerular filtration rate (GFR) less than 60 mL/min per 1.73 m2 for three months or more, regardless of cause. The assay of creatinine in serum or plasma is the most commonly used test to assess renal function. Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body (depending on muscle mass). It is freely filtered by the glomeruli and, under normal conditions, is not re-absorbed by the tubules to any appreciable extent. A small but significant amount is also actively secreted. Since a rise in blood creatinine is observed only with marked damage of the nephrons, it is not suited to detect early stage kidney disease. A considerably more sensitive test and better estimation of glomerular filtration rate (GFR) is given by the creatinine clearance test based on creatinine’s concentration in urine and serum or plasma, and urine flow rate. For this test a precisely timed urine collection (usually 24 hours) and a blood sample are needed. However, since this test is prone to error due to the inconvenient collection of timed urine, mathematical attempts to estimate GFR based only on the creatinine concentration in serum or plasma have been made. Among the various approaches suggested, two have found wide recognition: that of Cockroft and Gault and that based on the results of the MDRD trial. While the first equation was derived from data obtained with the conventional Jaffé method, a newer version of the second is usable for IDMS-traceable creatinine methods. Both are applicable for adults. In children, the Bedside Schwartz formula should be used. 6,7,8,9 In addition to the diagnosis and treatment of renal disease, the monitoring of renal dialysis, creatinine measurements are used for the calculation of the fractional excretion of other urine analytes (e. g. albumin, α‑amylase). Numerous methods were described for determining creatinine. Automated assays established in the routine laboratory include the Jaffé alkaline picrate method in various modifications, as well as enzymatic tests.

Homocysteine (Hcy) is a thiol-containing amino acid produced by the intracellular demethylation of methionine. Total homocysteine (tHcy) represents the sum of all forms of Hcy including forms of oxidized, proteinbound and free. Elevated levels of tHcy has emerged as an important risk factor in the assessment of cardiovascular disease.1,2,3 Excess Hcy in the blood stream may cause injuries to arterial vessels due to its irritant nature, and result in inflammation and plaque formation, which may eventually cause blockage of blood flow to the heart. Elevated tHcy levels are caused by four major factors, including: 1. genetic deficiencies in enzymes involved in Hcy metabolism․ nutritional deficiency in B vitamins such as B6, B12 and folate; 3. renal failure for effective amino acid clearance; and 4. drug interactions, that interfere with Hcy metabolism. Elevated levels of tHcy are also linked with Alzheimer’s disease4 , neuropsychiatric diseases5 and Osteoporosis.

Uric Acid UA Uric acid is the final product of purine metabolism in the human organism. Uric acid measurements are used in the diagnosis and treatment of numerous renal and metabolic disorders, including renal failure, gout, leukemia, psoriasis, starvation or other wasting conditions, and of patients receiving cytotoxic drugs. The oxidation of uric acid provides the basis for two approaches to the quantitative determination of this purine metabolite. One approach is the reduction of phosphotungstic acid in an alkaline solution to tungsten blue, which is measured photometrically. The method is, however, subject to interference from drugs and reducing substances other than uric acid. A second approach, described by Praetorius and Poulson, utilizes the enzyme uricase to oxidize uric acid; this method eliminates the interferences intrinsic to chemical oxidation. Uricase can be employed in methods that involve the UV measurement of the consumption of uric acid or in combination with other enzymes to provide a colorimetric assay. Another method is the colorimetric method developed by Town, et al. The sample is initially incubated with a reagent mixture containing ascorbate oxidase and a clearing system. In this test system it is important that any ascorbic acid present in the sample is eliminated in the preliminary reaction; this precludes any ascorbic acid interference with the subsequent POD indicator reaction. Upon addition of the starter reagent, oxidation of uric acid by uricase begins. The Roche assay described here is a slight modification of the colorimetric method described above. In this reaction, the peroxide reacts in the presence of peroxidase (POD), TOOS, and 4‑aminophenazone to form a quinoneimine dye. The intensity of the red color formed is proportional to the uric acid concentration and is determined photometrically.

Urea is the major end product of protein nitrogen metabolism. It is synthesized by the urea cycle in the liver from ammonia which is produced by amino acid deamination. Urea is excreted mostly by the kidneys but minimal amounts are also excreted in sweat and degraded in the intestines by bacterial action. Determination of blood urea nitrogen is the most widely used screening test for renal function. When used in conjunction with serum creatinine determinations it can aid in the differential diagnosis of the three types of azotemia: prerenal, renal, and postrenal. Elevations in blood urea nitrogen concentration are seen in inadequate renal perfusion, shock, diminished blood volume (prerenal causes), chronic nephritis, nephrosclerosis, tubular necrosis, glomerular-nephritis (renal causes), and urinary tract obstruction (postrenal causes). Transient elevations may also be seen during periods of high protein intake. Unpredictable levels occur with liver diseases.

Immunoglobulin A (IGA) Immunoglobulins protect the human body against invading organisms and agents. Immunoglobulins contain an antigen binding part (Fab portion) and a Fc portion of which the latter can interact with cells of the immune system and the complement factors. The immunoglobulin Fab part recognizes antigens in solution (e.g. toxins) and antigens associated with microorganisms (e.g. bacteria, viruses). The antigen binding site may initiate the direct neutralization of toxins, the sensitization of immunocompetent cells, the reduction of viral infectivity, or the development of an inflammatory reaction. As a normal result of infections all immunoglobulin classes increase in serum. Raised IgA levels are found in skin, gut, respiratory, and renal infections. Malignant cell proliferation of an immunoglobulin producing cell (plasma cell) causes an increased serum level of a single immunoglobulin (plasmocytoma). Immunoglobulin deficiencies may be due to protein loss syndromes, inherited deficiencies or may be secondary to lymphoid malignancies. Due to the slow onset of IgA synthesis, the IgA concentration in serum of infants is lower than in adults. It is known that the so-called paraproteins secreted in monoclonal gammopathies (monoclonal immunoglobulinemia) may differ from the respective immunoglobulins of polyclonal origin by amino acid composition and size. This may impair the binding to the antibody, and hence impair accurate quantitation.

Immunoglobulin E IgE Immunoglobulin E (IgE) plays an important role in immunological protection against parasitic infections and in allergy (type 1 hypersensitivity). Type 1 hypersensitivity is characterized by the occurrence of allergic reactions immediately following re-exposure to an allergy-initiating antigen (allergen) such as encountered in atopic disorders (e.g., allergic asthma), insect venom or latex and in some food allergies. The binding of the allergen to sensitized tissue mast cells or blood basophilic cells leads to cross-linking of the IgE on the cell membrane. This in turn causes cell degranulation and the release of inflammation mediators (e.g. histamine, serotonin, lipid mediators, proteases and cytokines), which produce the typical symptoms of type 1 hypersensitivity, an exaggerated immune response to foreign antigens, such as pollen, dust mites, and certain foods.1,2,3,4,5 The IgE concentration in serum is normally very low as IgE is the least abundant antibody in serum (0.05 % of the IgG concentration). The IgE concentration is age-dependent, with the lowest values being measured at birth. Its concentration gradually increases and becomes stabilized between the age of 5‑7, although the IgE values vary greatly within particular age groups.1,6 Elevated IgE concentrations can be found in patients with allergic diseases such as hay fever, atopic bronchitis and dermatitis.4 Normal IgE values do not, however, mean that an allergic disease can be ruled out. For this reason the quantitative determination of serum IgE concentrations is useful for clinical differentiation between atopic (i.e., predisposition to excessive IgE reaction) and non-atopic (non-IgE mediated) allergic diseases only in combination with other clinical findings.1,6,7 Elevated serum IgE concentrations can also occur in non-allergic diseases, e.g. congenital immunodeficiency syndromes, HIV infection, graft-versushost disease, severe burns and parasitic diseases.

Immunoglobulins protect the human body against invading organisms and agents. Immunoglobulins contain an antigen binding part (Fab portion) and a Fc portion of which the latter can interact with cells of the immune system and the complement factors. The immunoglobulin Fab part recognizes antigens in solution (e.g. toxins) and antigens associated with microorganisms (e.g. bacteria, viruses). The antigen binding site may initiate the direct neutralization of toxins, the sensitization of immunocompetent cells, the reduction of viral infectivity, or the development of an inflammatory reaction. As a normal result of infections all immunoglobulin classes increase in serum. In addition, raised IgG levels are found during autoimmune diseases and chronic hepatitis. Malignant cell proliferation of an immunoglobulin producing cell (plasma cell) causes a serum level increase of a single immunoglobulin (plasmocytoma). Immunoglobulin deficiencies may be due to protein loss syndromes, inherited deficiencies or may be secondary to lymphoid malignancies. Infants show a decrease of IgG between 3 and 6 months because maternal IgG is at first only partly compensated by active IgG synthesis of the newborn. IgG determination in cerebrospinal fluid (CSF) is used for evaluation of infections involving the central nervous system, neoplasms, or primary neurologic diseases (in particular, multiple sclerosis). Increased CSF IgG concentration may occur either because of increased permeability of the blood-brain barrier, or increased local production of IgG, or both. In order to identify intrathecal production specifically, correction for increased permeability is necessary. To accurately determine the intrathecal IgG production, the IgG fraction caused by increased permeability can be corrected by making calculations as follows:4 Abbreviated ratio name: IGGR2 (0‑179) Ratio = IgGCSF (mg/L) / AlbuminCSF (mg/L) A ratio > 0.27 indicates increased intrathecal IgG synthesis. Abbreviated ratio name: IGGI2 (0‑180) IgG index = IgGCSF (mg/L) × AlbuminSer (g/L) / IgGSer (g/L) / AlbuminCSF (mg/L) Index values > 0.7 are considered indicative of increased IgG synthesis. In > 80 % of multiple sclerosis cases, the index exceeds 0.7. It is known that the so-called paraproteins secreted in monoclonal gammopathies (monoclonal immunoglobulinemia) may differ from the respective immunoglobulins of polyclonal origin by amino acid composition and size. This may impair the binding to the antibody, and hence impair accurate quantitation.

Immunoglobulin M IGM Immunoglobulins protect the human body against invading organisms and agents. Immunoglobulins contain an antigen binding part (Fab portion) and a Fc portion of which the latter can interact with cells of the immune system and the complement factors. The immunoglobulin Fab part recognizes antigens in solution (e.g. toxins) and antigens associated with microorganisms (e.g. bacteria, viruses). The antigen binding site may initiate the direct neutralization of toxins, the sensitization of immunocompetent cells, the reduction of viral infectivity, or the development of an inflammatory reaction. As a normal result of infections all immunoglobulin classes increase in serum. Raised IgM levels are found during liver cell diseases, (e.g. hepatitis, liver cirrhosis), autoimmune diseases, and especially during acute and chronic viral infections. Malignant cell proliferation of an immunoglobulin producing cell (plasma cell) causes a serum level increase of a single immunoglobulin (plasmacytoma). Immunoglobulin deficiencies may be due to protein loss syndromes, inherited deficiencies, or may be secondary to lymphoid malignancies. Due to the slow onset of IgM synthesis, the IgM concentration in serum of infants is lower than in adults. It is known that the so-called paraproteins secreted in monoclonal gammopathies (monoclonal immunoglobulinemia) may differ from the respective immunoglobulins of polyclonal origin by amino acid composition and size. This may impair the binding to the antibody, and hence impair accurate quantitation.

C3C Activation of the complement system takes place via a classical and an alternative route. The two pathways come together in a joint terminal path. As complement factor C3 is a factor common to both pathways, the concentration of C3 and its degradation products (including C3c) can be evaluated as a parameter for activation of the complement system. Lowered values are indicative of activation. Additional differentiation can be made by determining C4. If the C4 level is normal, then activation of the alternative route is likely. Depressed values are observed in a number of inflammatory and infectious diseases. Primary causes are systemic lupus erythematosus (SLE), rheumatoid arthritis, subacute bacterial endocarditis, viremia, parasitic infections or bacterial sepsis. A considerable decrease in C3 can be found in patients with partial lipodystrophy or membranoproliferative glomerulonephritis when the C3-nephritis factor is present. As an acute phase protein, C3 is produced to an increased extent during inflammatory processes. It is elevated in systemic infections, non-infectious chronic inflammatory conditions (primarily chronic polyarthritis) and physiological states (pregnancy). The elevation rarely exceeds twice the normal value and can mask a reduction in the current consumption.

C4 The complement system can be activated via the classical and the alternative route. Complement factor C4 participates in activation by the classical route. A decrease in C4 is common, but complete absence is rare. A lowered concentration or the complete absence of C4 occurs in immunocomplex diseases, systemic lupus erythematosus (SLE), autoimmune thyroiditis and juvenile dermatomyositis. The commencement of SLE in patients with C4‑deficiencies can often be detected at a very early stage, and the course of the disease is milder than in patients with normal complement levels. Infections such as bacterial and viral meningitis, streptococcal and staphylococcal sepsis and pneumonia are associated with a fall in C4. Additional differentiation can be obtained by the determination of C4 when the level of complement factor C3 is low. If in such cases the concentration of C4 is normal, then an activation of the alternative route is likely. The main use of C4 determinations is in assessing the course of hypocomplement conditions. As an acute phase protein, C4 is produced to an increased extent during inflammatory processes. It is elevated in systemic infections, noninfectious chronic inflammatory conditions (primarily chronic polyarthritis) and physiological states (pregnancy). The elevation rarely exceeds twice the normal value and can mask a reduction in the current consumption. A variety of methods, such as nephelometry, radial immunodiffusion and turbidimetry, are available for the determination of complement factor C4.

Adrenocorticotropic hormone (ACTH) or corticotropin is a peptide hormone consisting of 39 amino acids. It is produced in the anterior pituitary of the brain as part of the precursor molecule pro‑opiomelanocortin (POMC). Tissue-specific cleavage results in ACTH and a range of related peptides.1,2 ACTH stimulates formation and secretion of glucocorticoids (especially cortisol) by the adrenal cortex. The glucocorticoid production is regulated by various factors.3,4,5,6 After stimulation (e.g. by physical effort or by the internal body clock), the hypothalamus secretes CRH (corticotropin releasing hormone). CRH acts on the pituitary, which in turn synthesizes and secretes ACTH. Finally, ACTH stimulates secretion of the glucocorticoids by the adrenals. High concentrations of glucocorticoids in the blood inhibit secretion of CRH and ACTH via a negative feedback mechanism. ACTH concentrations show a diurnal variation with high levels in the morning and low levels in the evening. Therefore, as with cortisol, it is important to know the collection time of the plasma sample for interpretation of the results. Plasma ACTH measurements are useful in the differential diagnosis of Cushing's disease (ACTH hypersecretion), autonomous ACTH producing pituitary tissue (e.g. Nelson's syndrome), hypopituitarism with ACTH deficiency and ectopic ACTH syndrome.7,8 In addition to cortisol measurements, ACTH determinations can be used together with suppression or stimulation tests to diagnose the origin of glucocorticoid overproduction. Similarly, ACTH measurements can be employed to facilitate differential diagnosis of adrenocortical insufficiency (Addison’s disease).9 ACTH not produced by the pituitary gland is known as ectopic ACTH;10 this is often associated with small cell carcinoma of the lung. In rare cases ectopic ACTH can be caused by thymic tumors, pancreatic adenocarcinomas, or bronchial carcinoids. These tumors often secrete ACTH precursors (POMC and pro‑ACTH). The Elecsys ACTH assay employs two monoclonal antibodies specific for ACTH (9‑12) and for the C‑terminal region (ACTH 36‑39). Due to common antigenic structure, the antibodies recognize intact biologically active ACTH 1‑39 and the ACTH precursors POMC and pro‑ACTH.

Cortisol is quantitatively the major glucocorticoid product of the adrenal cortex.1 The main reason to measure cortisol is to diagnose overproduction of cortisol in Cushing’s syndrome (CS), deficiency of adrenal steroid excretion in Addison’s disease, and for therapy monitoring (e.g. dexamethasone suppression test in Cushing's syndrome and hormone replacement therapy in Addison's disease).1 Cortisol plays an important role in the regulation of many essential physiological processes, including energy metabolism, maintenance of electrolyte balance and blood pressure, immunomodulation and stress responses, cell proliferation as well as cognitive functions. The major fraction of cortisol circulates bound to plasma proteins as corticosteroid binding globulin and albumin.2 The biologically active free fraction comprises only 2‑5 % of the total hormone concentration.1,2 Elevated serum levels can be found in stress responses, psychiatric diseases, obesity, diabetes, alcoholism and pregnancy, which may cause diagnostic problems in patients with Cushing's syndrome. Low levels of cortisol are seen in patients with rare adrenal enzyme defects and after long-lasting stress. For diagnostic purposes the following analyses are used: total and free cortisol in serum and midnight saliva.1 The secretion of cortisol is mainly controlled by the hypothalamic-pituitary-adrenal axis (HPA). When cortisol levels in the blood are low, a group of cells in a region of the brain called the hypothalamus release corticotropin-releasing hormone (CRH) which causes the pituitary gland to secrete another hormone, adrenocorticotropic hormone (ACTH), into the bloodstream. High levels of ACTH are detected in the adrenal glands and stimulate the secretion of cortisol, causing blood levels of cortisol to rise. As the cortisol levels rise, they start to block the release of CRH from the hypothalamus and ACTH from the pituitary.2 Normally, the highest cortisol secretion happens in the second half of the night with peak cortisol production occurring in the early morning. Following this, cortisol levels decline throughout the day with lowest levels during the first half of the night.3 Therefore the circadian variations of cortisol secretion and the influence of stress have to be considered for the sampling conditions in serum, plasma and saliva.4 The Elecsys Cortisol II assay makes use of a competition test principle using a monoclonal antibody which is specifically directed against cortisol. Endogenous cortisol which has been liberated from binding proteins with danazol competes with exogenous cortisol derivative in the test which has been labeled with ruthenium complex) for the binding sites on the biotinylated antibody.

HCG+β hCG is produced in the placenta during pregnancy. In non‑pregnant women, it can also be produced by tumors of the trophoblast, germ cell tumors with trophoblastic components and some non‑trophoblastic tumors.3 The biological action of hCG serves to maintain the corpus luteum during pregnancy. It also influences steroid production. The serum of pregnant women contains mainly intact hCG.5 Elevated values here serve as an indication of chorionic carcinoma, hydatidiform mole or multiple pregnancy. Depressed values indicate threatening or missed abortion,6 ectopic pregnancy, gestosis or intra‑uterine death. Measurement of hCG+β makes also a contribution to the risk assessment for trisomy 21 (Down syndrome) in the second trimester of pregnancy together with AFP (Alpha‑fetoprotein) and other parameters, such as exact gestational age and maternal weight. In a trisomy 21 affected pregnancy the maternal serum concentration of AFP is decreased whereas the maternal serum hCG+β concentration is approximately twice the normal median. Elevated hCG concentrations not associated with pregnancy are found in patients with diseases such as tumors of the germ cells, ovaries, bladder, pancreas, stomach, lungs, and liver.2,15 In the following the prevalence (%) of elevated serum hCG + hCG+β values in various malignancies is listed: Testicular or placental choriocarcinoma (100), hydatidiform mole (97), nonseminomatous testicular germ cell tumor (48‑86), seminoma (10‑22), pancreatic cancer adenocarcinoma (11‑80) and islet‑cell carcinoma (22‑50), gastric cancer (0‑52), ovarian cancer, epithelial (18‑41), colon cancer (0‑37), lung cancer (0‑36), breast cancer (7‑25), hepatoma, liver cancer (17‑21), tumors of small intestine (13), and renal carcinoma (10).14,16 hCG assays detecting the intact hCG plus the free β‑subunit are well established markers as an aid in the management of patients with trophoblastic tumors16 and together with AFP in patients with testicular and other germ cell tumors.

Apolipoprotein APO Apolipoproteins are the protein constituents of the lipoproteins. The lipoproteins are classified according to their ultracentrifugal flotation density. Apolipoprotein A‑1 is the major protein constituent of high-density lipoproteins (HDL). HDL are synthesized by the intestines and the liver; they transport excess cellular cholesterol from extrahepatic tissue and peripheral cells to the liver. Additionally, apolipoprotein A‑1 activates the enzyme lecithin-cholesterol acyltransferase (LCAT), which catalyzes the esterification of cholesterol enhancing the lipid carrying capacity of the lipoproteins. Apolipoprotein A‑1 levels increase in pregnancy, liver disease, and as a result of estrogen administration (e.g. contraceptive pills). Apolipoprotein A‑1 levels decrease in inherited hypo-α-lipoproteinemia (e.g. Tangier disease), cholestasis, sepsis, and atherosclerosis. The liver also synthesizes very low density lipoproteins (VLDL). These particles mainly contain triglycerides and cholesterol. In the presence of lipoprotein lipase the triglycerides are hydrolyzed and LDL particles with a high proportion of cholesterol are formed. Apolipoprotein B is the major constituent of LDL. The combined determination of apolipoprotein A‑1 and apolipoprotein B and the calculation of the apolipoprotein B/apolipoprotein A‑1 ratio can reflect a disorder of lipid metabolism and the risk of developing atherosclerosis and coronary heart disease particularly well providing an excellent addition to the classical HDL/LDL cholesterol determination. A high level of apolipoprotein A‑1 (HDL) and a low level of apolipoprotein B (LDL) correlate best with a low risk for these diseases.

Apolipoprotein APO Apolipoproteins are the protein constituents of the lipoproteins. The lipoproteins are classified according to their ultracentrifugal flotation density. Apolipoprotein A‑1 is the major protein constituent of high-density lipoproteins (HDL). HDL are synthesized by the intestines and the liver; they transport excess cellular cholesterol from extrahepatic tissue and peripheral cells to the liver. Additionally, apolipoprotein A‑1 activates the enzyme lecithin-cholesterol acyltransferase (LCAT), which catalyzes the esterification of cholesterol enhancing the lipid carrying capacity of the lipoproteins. Apolipoprotein A‑1 levels increase in pregnancy, liver disease, and as a result of estrogen administration (e.g. contraceptive pills). Apolipoprotein A‑1 levels decrease in inherited hypo-α-lipoproteinemia (e.g. Tangier disease), cholestasis, sepsis, and atherosclerosis. The liver also synthesizes very low density lipoproteins (VLDL). These particles mainly contain triglycerides and cholesterol. In the presence of lipoprotein lipase the triglycerides are hydrolyzed and LDL particles with a high proportion of cholesterol are formed. Apolipoprotein B is the major constituent of LDL. The combined determination of apolipoprotein A‑1 and apolipoprotein B and the calculation of the apolipoprotein B/apolipoprotein A‑1 ratio can reflect a disorder of lipid metabolism and the risk of developing atherosclerosis and coronary heart disease particularly well providing an excellent addition to the classical HDL/LDL cholesterol determination. A high level of apolipoprotein A‑1 (HDL) and a low level of apolipoprotein B (LDL) correlate best with a low risk for these diseases.

Apolipoprotein APO Apolipoproteins are the protein constituents of the lipoproteins. The lipoproteins are classified according to their ultracentrifugal flotation density. Apolipoprotein A‑1 is the major protein constituent of high-density lipoproteins (HDL). HDL are synthesized by the intestines and the liver; they transport excess cellular cholesterol from extrahepatic tissue and peripheral cells to the liver. Additionally, apolipoprotein A‑1 activates the enzyme lecithin-cholesterol acyltransferase (LCAT), which catalyzes the esterification of cholesterol enhancing the lipid carrying capacity of the lipoproteins. Apolipoprotein A‑1 levels increase in pregnancy, liver disease, and as a result of estrogen administration (e.g. contraceptive pills). Apolipoprotein A‑1 levels decrease in inherited hypo-α-lipoproteinemia (e.g. Tangier disease), cholestasis, sepsis, and atherosclerosis. The liver also synthesizes very low density lipoproteins (VLDL). These particles mainly contain triglycerides and cholesterol. In the presence of lipoprotein lipase the triglycerides are hydrolyzed and LDL particles with a high proportion of cholesterol are formed. Apolipoprotein B is the major constituent of LDL. The combined determination of apolipoprotein A‑1 and apolipoprotein B and the calculation of the apolipoprotein B/apolipoprotein A‑1 ratio can reflect a disorder of lipid metabolism and the risk of developing atherosclerosis and coronary heart disease particularly well providing an excellent addition to the classical HDL/LDL cholesterol determination. A high level of apolipoprotein A‑1 (HDL) and a low level of apolipoprotein B (LDL) correlate best with a low risk for these diseases.

Apolipoprotein APO Apolipoproteins are the protein constituents of the lipoproteins. The lipoproteins are classified according to their ultracentrifugal flotation density. Apolipoprotein A‑1 is the major protein constituent of high-density lipoproteins (HDL). HDL are synthesized by the intestines and the liver; they transport excess cellular cholesterol from extrahepatic tissue and peripheral cells to the liver. Additionally, apolipoprotein A‑1 activates the enzyme lecithin-cholesterol acyltransferase (LCAT), which catalyzes the esterification of cholesterol enhancing the lipid carrying capacity of the lipoproteins. Apolipoprotein A‑1 levels increase in pregnancy, liver disease, and as a result of estrogen administration (e.g. contraceptive pills). Apolipoprotein A‑1 levels decrease in inherited hypo-α-lipoproteinemia (e.g. Tangier disease), cholestasis, sepsis, and atherosclerosis. The liver also synthesizes very low density lipoproteins (VLDL). These particles mainly contain triglycerides and cholesterol. In the presence of lipoprotein lipase the triglycerides are hydrolyzed and LDL particles with a high proportion of cholesterol are formed. Apolipoprotein B is the major constituent of LDL. The combined determination of apolipoprotein A‑1 and apolipoprotein B and the calculation of the apolipoprotein B/apolipoprotein A‑1 ratio can reflect a disorder of lipid metabolism and the risk of developing atherosclerosis and coronary heart disease particularly well providing an excellent addition to the classical HDL/LDL cholesterol determination. A high level of apolipoprotein A‑1 (HDL) and a low level of apolipoprotein B (LDL) correlate best with a low risk for these diseases.

HDL‑Cholesterol High density lipoproteins (HDL) are responsible for the reverse transport of cholesterol from the peripheral cells to the liver. In the liver, cholesterol is transformed to bile acids which are then excreted into the intestine via the biliary tract. Monitoring of HDL‑cholesterol in serum or plasma is of clinical relevance as the HDL‑cholesterol concentration is important in the assessment of atherosclerotic risk. Elevated HDL‑cholesterol concentrations protect against coronary heart disease (CHD), whereas reduced HDL‑cholesterol concentrations, particularly in conjunction with elevated triglycerides, increase cardiovascular risk.1 Two cholesterol related variables that are predictive of cardiovascular disease (CVD) have emerged. These are non‑HDL‑cholesterol2,3,4 (= cholesterol ‑ HDL‑cholesterol) and the rate of cholesterol transfer from the macrophages to HDL, also described as cholesterol efflux capacity.5 Whereas both cholesterol and HDL‑cholesterol can be readily determined with high accuracy, currently, non‑HDL‑cholesterol appears to be best suited for patient management. A variety of methods are available to determine HDL‑cholesterol, including ultracentrifugation (reference method in combination with cholesterol measurement by the Abell‑Kendall method), electrophoresis, HPLC, precipitation, and direct methods.6 Of these, the direct methods are used routinely. Roche HDLC4 is also a direct method. The automated HDLC4 assay uses detergents, cholesterol esterase (CHER), cholesterol oxidase (CHOD) and peroxidase to form a colored pigment that is measured optically.7,8 The HDLC4 assay meets the 1998 National Institutes of Health (NIH) / National Cholesterol Education Program (NCEP) goals for precision and accuracy.

Cholesterol CHOL is a steroid with a secondary hydroxyl group in the C3 position. It is synthesized in many types of tissue, but particularly in the liver and intestinal wall. Approximately three quarters of cholesterol are newly synthesized and a quarter originates from dietary intake. Cholesterol assays are used for screening for atherosclerotic risk and in the diagnosis and treatment of disorders involving elevated cholesterol levels as well as lipid and lipoprotein metabolic disorders. Cholesterol analysis was first reported by Liebermann in 1885 followed by Burchard in 1889. In the Liebermann-Burchard reaction, cholesterol forms a blue-green dye from polymeric unsaturated carbohydrates in an acetic acid/acetic anhydride/concentrated sulfuric acid medium. The Abell and Kendall method is specific for cholesterol, but is technically complex and requires the use of corrosive reagents. In 1974, Roeschlau and Allain described the first fully enzymatic method. This method is based on the determination of Δ4‑cholestenone after enzymatic cleavage of the cholesterol ester by cholesterol esterase, conversion of cholesterol by cholesterol oxidase,and subsequent measurement by the Trinder reaction of the hydrogen peroxide formed. Optimization of ester cleavage (> 99.5 %) allows standardization using primary and secondary standards and a direct comparison with the CDC and NIST reference methods. Nonfasting sample results may be slightly lower than fasting results. The Roche cholesterol assay meets the 1992 National Institutes of Health (NIH) goal of less than or equal to 3 % for both precision and bias.

TRIGL Triglycerides Triglycerides are esters of the trihydric alcohol glycerol with 3 long-chain fatty acids. They are partly synthesized in the liver and partly ingested in food. The determination of triglycerides is utilized in the diagnosis and treatment of patients having diabetes mellitus, nephrosis, liver obstruction, lipid metabolism disorders and numerous other endocrine diseases. The enzymatic triglycerides assay as described by Eggstein and Kreutz still required saponification with potassium hydroxide. Numerous attempts were subsequently made to replace alkaline saponification by enzymatic hydrolysis with lipase. Bucolo and David tested a lipase/protease mixture; Wahlefeld used an esterase from the liver in combination with a particularly effective lipase from Rhizopus arrhizus for hydrolysis. This method is based on the work by Wahlefeld using a lipoprotein lipase from microorganisms for the rapid and complete hydrolysis of triglycerides to glycerol followed by oxidation to dihydroxyacetone phosphate and hydrogen peroxide. The hydrogen peroxide produced then reacts with 4‑aminophenazone and 4‑chlorophenol under the catalytic action of peroxidase to form a red dyestuff (Trinder endpoint reaction). The color intensity of the red dyestuff formed is directly proportional to the triglyceride concentration and can be measured photometrically.

LDL-Cholesterol Low Density Lipoproteins (LDL) play a key role in causing and influencing the progression of atherosclerosis and, in particular, coronary sclerosis.1,2 The LDLs are derived from VLDLs (Very Low Density Lipoproteins) rich in triglycerides by the action of various lipolytic enzymes and are synthesized in the liver. The elimination of LDL from plasma takes place mainly by liver parenchymal cells via specific LDL receptors. Elevated LDL concentrations in blood and an increase in their residence time coupled with an increase in the biological modification rate results in the destruction of the endothelial function and a higher LDL-cholesterol uptake in the monocyte/macrophage system as well as by smooth muscle cells in vessel walls. The majority of cholesterol stored in atherosclerotic plaques originates from LDL. The LDLcholesterol value is the most powerful clinical predictor among all of the single parameters with respect to coronary atherosclerosis. Therefore, therapies focusing on lipid reduction primarily target the reduction of LDLcholesterol which is then expressed in an improvement of the endothelial function, prevention of atherosclerosis and reducing its progression as well as preventing plaque rupture.

Iron is mainly absorbed in the form of Fe2+ in the duodenum andupper jejunum. The trivalent form and the heme-bound Fe3+-component of iron in food has to be reduced by vitamin C. About 1 mg of iron is assimilated daily. Upon reaching the mucosal cells, Fe2+ ions become bound to transport substances. Before passing into the plasma, these are oxidized by ceruloplasmin to Fe3+ and bound to transferrin in this form. The transport of Fe ions in blood plasma takes place via transferrin-iron complexes. A maximum of 2 Fe3+ ions per protein molecule can be transported. Serum iron is almost completely bound to transferrin. Iron (non-heme) measurements are used in the diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated with widespread deposit in the tissue of the two iron-containing pigments, hemosiderin and hemofuscin, and characterized by pigmentation of the skin), and chronic renal disease. Iron determinations are performed for the diagnosis and monitoring of microcytic anemia (e.g. due to iron metabolism disorders and hemoglobinopathy), macrocytic anemia (e.g. due to vitamin B12 deficiency, folic acid deficiency and drug-induced metabolic disorders of unknown origin) as well as normocytic anemias such as renal anemia (erythropoetin deficiency), hemolytic anemia, hemoglobinopathy, bone marrow disease and toxic bone marrow damage. Numerous photometric methods have been described for the determination of iron. All have the following in common: ▪ Liberation of Fe3+ ions from the transferrin complex using acids or detergents. ▪ Reduction of Fe3+ ions to Fe2+ ions. ▪ Reaction of the Fe2+ ions to give a colored complex. The method described here is based on the FerroZine method without deproteinization.

Magnesium along with potassium is a major intracellular cation. Mg2+ is a cofactor of many enzyme systems. Thus, all ATP dependent enzymatic reactions require Mg2+ as a cofactor in the ATP-magnesium complex. Approximately 69 % of magnesium ions are stored in bone. The rest are part of the intermediary metabolism, about 70 % being present in free form while the other 30 % is bound to proteins (especially albumin), citrates, phosphate, and other complex formers. The Mg2+ serum level is kept constant within very narrow limits (0.65 1.05 mmol/L). Regulation takes place mainly via the kidneys, especially via the ascending loop of Henle. This assay is used for diagnosing and monitoring hypomagnesemia (magnesium deficiency) and hypermagnesemia (magnesium excess). Numerous studies have shown a correlation between magnesium deficiency and changes in calcium , potassium and phosphate homeostasis which are associated with cardiac disorders such as ventricular arrhythmias that cannot be treated by conventional therapy, increased sensitivity to digoxin, coronary artery spasms, and sudden death. Additional concurrent symptoms include neuromuscular and neuropsychiatric disorders. Hypermagnesemia is found in acute and chronic renal failure, magnesium excess, and magnesium release from the intracellular space. In addition to atomic absorption spectrometry (AAS), complexometric methods can also be used to determine magnesium.

The measurement of Proinsulin in serum can provide valuable information for the diagnosis of insulinomas. Proinsulin levels have also been shown to be elevated in non-insulin dependent diabetics (NIDDM), newly diagnosed insulin dependent diabetics (IDDM) and other clinical situations. is synthesized in the ß cells of the pancreas and is the precursor molecule for insulin (1, 2, 3). Most proinsulin is converted to insulin and C-Peptide, which are secreted in equimolar amounts into the blood. About 15 % is not converted and is released as proinsulin. The biological activity of proinsulin is only about 10 % of Insulin, but the half life of proinsulin is three times as long as insulin. The level of proinsulin in serum can be a reflection of ß cell status. Both IDDM and NIDDM are characterized by dysfunction of the pancreatic ß cells. Elevated proinsulin levels have been noted at the onset of IDDM and in healthy siblings of IDDM patients. Proinsulin levels may also be increased in patients with established NIDDM. Increased levels of circulating proinsulin are found in older patients, pregnant or obese diabetics, patients with insulinomas, functional hypoglycemia and hyperinsulinemia, a rare syndrome. Because the structure of proinsulin is similar to insulin, proinsulin may be detected as immunoreactive insulin in the insulin assay. Immunoreactive insulin levels are generally determined in conventional RIA's, which overestimate the insulin level because the methods use antibodies which cross-react with proinsulin. By calculating the molar ratio of proinsulin to true insulin (P/I), a better assessment of ß cell function can be made.

Insulin is secreted by the β‑cells of the islets of Langerhans in the pancreas, and passes into circulation via the portal vein and the liver. Insulin is generally released in pulses.1,2 Insulin is the biosynthetic product of the single‑chain precursor preproinsulin, which is subsequently cleaved to give proinsulin.2,3,4,5 5Specific proteases further cleave proinsulin to produce insulin and the connecting (C)‑peptide which pass into the bloodstream simultaneously in equimolar concentrations. Circulating insulin has a half‑life of 3‑5 minutes and is preferentially retained and degraded in the liver. Therefore only about half of the insulin reaches the systemic circulation. Inactivation or excretion of proinsulin and C‑peptide mainly takes place in the kidney and virtually none of the C‑peptide is retained in the liver. As a result, C‑peptide has a higher plasma concentration than insulin.6 The action of insulin is mediated by specific receptors and primarily consists of facilitation of glucose uptake by the cells of the liver, fatty tissue and musculature; this is the basis of its hypoglycemic action.2,8 Serum insulin determinations are mainly performed on patients with symptoms of hypoglycemia and may be useful in classifying the different types of diabetes.9,10 They are used to ascertain the glucose/insulin quotients and for clarification of questions concerning insulin secretion and β‑cell function, e.g. in the evaluation of oral glucose tolerance tests or hunger provocation tests.11 A disorder in insulin metabolism can have a significant impact on a number of metabolic processes. Low concentrations of free, biologically active insulin can lead to the development of diabetes mellitus. Possible causes of this include destruction of the β‑cells (type I diabetes), reduced activity of insulin or reduced pancreatic synthesis (type II), circulating antibodies to insulin, delayed release of insulin or the absence (or inadequacy) of insulin receptors.

HIV combi PT The human immunodeficiency virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), belongs to the family of retroviruses. HIV can be transmitted through sexual contact, contaminated blood and blood products or from an HIV-infected mother to her child before, during and after birth. Two types of HIV, called HIV‑1 and HIV‑2, have been identified to date.2,3,4,5 HIV‑1 can be divided into 4 distantly related groups: group M (for main), group N (for non‑M, non‑O), group O (for outlier) and group P.6,7,8 Based on their genetic relationship, 9 different subtypes (A to D, F to H, J, K) as well as several circulating recombinant forms (CRFs) have been identified within HIV‑1 group M.9 The large majority of HIV‑1 infections are caused by viruses belonging to group M, while geographical distribution of subtypes and CRFs within this group varies strongly.10 Due to differences in the sequence of immunodominant epitopes, especially in the envelope proteins of HIV‑1 group M, HIV‑1 group O and HIV‑2, specific antigens are necessary to avoid failure in the detection of an HIV infection by immunoassays.11,12 HIV p24 antigen in blood specimen of recently infected patients can be detected as early as 2‑3 weeks after infection.13,14 Anti‑HIV antibodies are detectable in serum from around 4 weeks post infection.13,15 The combined detection of HIV p24 antigen and anti‑HIV antibodies in 4 th generation HIV screening assays leads to improved sensitivity and therefore a shorter diagnostic window compared to traditional anti‑HIV assays.16,17 With the Elecsys HIV combi PT assay the HIV‑1 p24 antigen and antibodies to HIV‑1 and HIV‑2 can be detected simultanously within one determination. For the detection of HIV‑1 p24 antigen specific monoclonal antibodies are used. Repeatedly reactive samples must be confirmed according to recommended confirmatory algorithms. Confirmatory tests include Western Blot and HIV RNA tests.

Syphilis is caused by the intracellular gram-negative spirochete bacterium Treponema pallidum (TP) subspecies pallidum.2 Syphilis is mainly transmitted sexually, but can also be transmitted from mother to fetus during pregnancy or birth. The global incidence of infection in 2008 was approximately 10.6 million and the total number of infections during that year was estimated to be 36.4 million.3 In the USA the national infection rate rose to 6.3 cases per 100000 people, the highest rate since 1994.4 Certain European countries have also seen increases in the rate of infection5,6 and large localized outbreaks.7 Each year, globally, an estimated 2 million pregnancies are affected.8 Congenital syphilis is still common in the developing world, as many women do not receive antenatal care or the scheme does not include syphilis screening.9 Up to 80 % of syphilis infected pregnant women show adverse pregnancy outcomes.8 The World Health Organization recommends all women to be tested at their first antenatal visit and again in the third trimester.8 If they are positive, the recommendation also includes treatment of the partner. Typically, symptoms of syphilis start with a painless ulcer at the site of entry to the body (primary syphilis) followed by a widespread rash as the bacteria disseminate (secondary syphilis). This is followed by a lengthy latent (asymptomatic) period. Eventually, tertiary syphilis ensues, characterized by the development of granulomatous dermal lesions, neurosyphilis, and/or cardiovascular syphilis (which can be fatal).10 The immune response to T. pallidum is the main driver of lesion development.10 The antibody response is directed not only against antigens specific to T. pallidum (treponemal antibodies), but antibodies are also generated against antigens which are not specific (non-treponemal antibodies); for example, antigens released during the cellular damage caused by the organism. Therefore, treponemal and non-treponemal tests co-exist for the diagnosis of syphilis.2 Non‑treponemal tests detect antibodies against lecithin, cholesterol and cardiolipin, which are present in many syphilis patients.2 Treponemal tests detect antibodies directed against T. pallidum antigens such as TpN47, TpN17 and TpN15, for IgM and IgG detection.2 A positive treponemal antibody test result indicates exposure to T. pallidum but cannot distinguish between treated and untreated syphilis. Non-treponemal assays are useful to help distinguish between treated and untreated syphilis and are also used for monitoring the progression of disease and treatment response.

Toxoplasmosis is a relatively common infection caused by the protozoan parasite Toxoplasma gondii. The infection is mainly acquired by ingestion of food or water contaminated by mature oocysts shed by cats or by undercooked meat containing tissue cysts.1,2,3,4 Infection can also be transmitted congenitally if a woman is newly infected during or just prior to pregnancy, and via organ transplant or blood transfusion from an infected donor.4 Primary, acute infection in healthy individuals is mostly mild or even asymptomatic and is followed by life-long latency.3,4 Reactivation of a latent Toxoplasma infection can occur as a result of immunosuppression (e.g. in organ transplant recipients, patients with cancer or HIV) and can be associated with high morbidity and mortality.3,4 Reactivated disease in immunocompromised hosts frequently presents with brain lesions, especially in patients with advanced HIV-related immunosuppression.3,4,5 Primary maternal Toxoplasma infection occurring during pregnancy may have significant implications for the fetus as the parasite can be transmitted across the placenta.3,6 The majority of infants with congenital infection do not present clinical symptoms at birth but may develop severe sequelae later in life such as chorioretinitis, intellectual and psychomotor disabilities, visual and hearing impairment and hearing loss.3,6,7,8 The fetal infection rate increases with gestational age, but the risk of severe clinical manifestations is higher in the case of early maternal infection.3,6,7,8 Early drug therapy in acute infection during pregnancy can prevent congenital damage or ameliorate the severity of clinical manifestations.6,7 The diagnosis of Toxoplasma infection is most commonly made by the detection of anti‑Toxoplasma‑specific IgG and IgM antibodies.3,4,9 The determination of Toxo IgG antibodies is used to assess the serological status of T. gondi infection and their presence is indicative of a latent or acute infection.4,9 Detection of Toxo IgM antibodies is presumptive of an acute or recent Toxoplasma infection.3,4,9 The diagnosis of the acute acquired infection during pregnancy is established by a seroconversion or a significant rise in antibody titers (IgG and/or IgM) in serial samples.

Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight. Vitamin D is biologically inert and must undergo two successive hydroxylations in the liver and kidney to become the biologically active 1,25‑dihydroxyvitamin D.1 Vitamin D is essential for bone health. In children, severe deficiency leads to bone-malformation, known as rickets. Milder degrees of insufficiency are believed to cause reduced efficiency in the utilization of dietary calcium.9 Vitamin D deficiency causes muscle weakness; in elderly, the risk of falling has been attributed to the effect of vitamin D on muscle function.10 Vitamin D deficiency is a common cause of secondary hyperparathyroidism.11,12 Elevations of parathyroid hormone levels, especially in elderly vitamin D deficient adults can result in osteomalacia, increased bone turnover, reduced bone mass and risk of bone fractures.13 Low 25‑hydroxyvitamin D concentrations are also associated with lower bone mineral density.14 In conjunction with other clinical data, the results may be used as an aid in the assessment of bone metabolism. So far, vitamin D has been shown to affect expression of over 200 different genes. Insufficiency has been linked to diabetes, different forms of cancer, cardiovascular disease, autoimmune diseases and innate immunity.2

Prostate‑specific antigen (PSA) is a glycoprotein (molecular weight 30000‑34000 daltons) having a close structural relationship to the glandular kallikreins. It has the function of a serine proteinase.1 The proteolytic activity of PSA in blood is inhibited by the irreversible formation of complexes with protease inhibitors such as alpha‑1‑antichymotrypsin (ACT) and alpha‑2‑macroglobulin.2,3 Beside these complexes, about 10‑30 % of the PSA present in blood occurs in the free form, but is proteolytically inactive.3 Autopsies have shown that prostate cancer is quite common. Among man aged 70‑79 years the incidence was found to be 36‑51 %. Most of these cancers are indolent i.e. without symptoms and relatively benign.4 If PSA is measured and the result is found to be elevated, the decision on further steps must consider the possibility that the condition is indolent. Nevertheless, PSA screening has been found to reduce prostate cancer related mortality.5 Different models have been proposed to improve the predictive accuracy of PSA measurements.6 As PSA is also present in para‑urethral and anal glands, as well as in breast tissue or with breast cancer, low levels of PSA can also be detected in sera from women. PSA may still be detectable even after radical prostatectomy. The main areas in which PSA determinations are employed are the monitoring of progress and efficiency of therapy in patients with prostate carcinoma or receiving hormonal therapy.7,8 The steepness of the rate of fall in PSA down to no‑longer detectable levels following radiotherapy, hormonal therapy or radical surgical removal of the prostate provides information on the success of therapy.8 An inflammation or trauma of the prostate (e.g. in cases of urinary retention or following rectal examination, cystoscopy, coloscopy, transurethral biopsy, laser treatment or ergometry) can lead to PSA elevations of varying duration and magnitude. The two monoclonal antibodies used in the Elecsys total PSA assay recognize unbound PSA and PSA‑ACT on an equimolar basis in the range of 10‑50 % free PSA/total PSA which are the free PSA‑ratios as seen in clinical practice.9

CYFRA 21-1 Cytokeratins are structural proteins that form subunits of epithelial intermediate fibers. Twenty different cytokeratins have been identified to date, CYFRA 21-1 being a fragment of cytokeratin 19, the best known. Intact cytokeratin polypeptides are poorly soluble, but soluble fragments such as CYFRA 21-1 are frequently released into the blood of cancer patients and are detectable in the serum.1 This affects a variety of organs, but most commonly the lungs. CYFRA 21-1 may be considered as the preferred biomarker in non-small cell lung cancer (especially squamous cell and large cell carcinoma).2,3,4,5,6 In lung adenocarcinoma, the combination of CYFRA 21-1 and carcinoembryonic antigen (CEA) has been found to be most useful.7,8 The main indication for the CYFRA 21-1 test is to monitor the progression of NSCLC.9 Successful therapy is accompanied by a rapid decrease in serum levels to the normal range.9,10 Elevated CYFRA 21-1 levels have also been described in benign diseases (pneumonia, sepsis)11,12,13 and renal dysfunction.14 Although renal function testing (i.e., serum creatinine measurement) should be performed in cases of high CYFRA 21-1 levels, it is not corresponding to the diagnostic and clinical characteristics of the patient. Using two specific monoclonal antibodies (KS 19.1 and BM 19.21), the Elecsys CYFRA 21‑1 assay measures cytokeratin 19 fragments with a molecular weight of approximately 30,000 Da.

Alpha1‑fetoprotein (AFP), an albumin‑like glycoprotein, is formed in the yolk sac during fetal life, in non‑differentiated liver cells, and the fetal gastro‑intestinal tract.1,2 Tumors that synthesize AFP are mainly testicular non-seminomatous germ cell tumors (NSGCT), yolk sac tumors of the ovary and hepatocellular carcinoma (HCC). Moreover AFP is an important part in the risk assessment for trisomy 21 in the second trimester of pregnancy together with hCG+β and other parameters.3 Testicular cancer Careful monitoring of the serum tumor markers AFP and human chorionic gonadotropin (hCG) is essential in the management of patients with germ cell tumors (GCT), as these markers are important for diagnosis, as prognostic indicators, in monitoring treatment response, and in the detection of early relapse.4 In addition, hCG and AFP are important parameters for estimating the survival rate of patients with advanced NSGCTs and are also recommended by the National Academy of Clinical Biochemistry for the management of such patients.5 Hepatocellular carcinoma Hepatocellular carcinoma (HCC) is frequently the result of advanced liver disease and can develop in patients with and without cirrhosis.6 AFP has long been recognized as a biomarker for HCC, and has played a prominent role in the diagnosis of HCC. Substantially elevated AFP values can indicate primary liver cell carcinoma and it has been shown that AFP levels increase with tumor size.7 Diagnosis of HCC has primarily relied on the presence of typical features seen on contrast-enhanced imaging studies, histopathological assessment, and serum AFP levels.8 While AFP is elevated during hepato‑carcinogenesis, it can also be found in other tumors such as testicular, embryonic or gastric cancer.9,10 AFP has reported sensitivities ranging from 39 to 65 %, and specificities from 76 to 94 % in HCC patients.11 The divergence in sensitivity and specificity of AFP in these studies is probably due to a variety of factors including different etiologies, variable study designs, and different cutoff values. As the AFP values can also rise during regeneration of the liver, moderately elevated values are found in alcohol‑mediated liver cirrhosis and acute viral hepatitis.12 Surveillance of patients at risk for developing HCC by abdominal ultrasonography in combination with AFP is recommended by several clinical practice guidelines.13,14,15 Trisomy 21 Measurement of AFP makes a contribution to the risk assessment for trisomy 21 (Down syndrome) in the second trimester of pregnancy together with hCG+β and other parameters, such as exact gestational age and maternal weight.3 In a trisomy 21 affected pregnancy the maternal serum concentration of AFP is decreased whereas the maternal serum hCG+β concentration is approximately twice the normal median.16 The risk for a trisomy 21 affected pregnancy in the second trimester can be calculated by a suitable software (see “Materials required, but not provided” section) using the algorithm as described by Cuckle et al.17 and the respective assay specific parameters.

Human calcitonin (hCT) is secreted primarily by the parafollicular C cells of the thyroid gland.1 It is metabolized in the liver and kidney and regulated by serum calcium levels. Physiologically hCT has effects on calcium and phosphorus metabolism. It is an inhibitor of bone resorption to prevent bone loss at times of calcium stress (e.g. pregnancy, lactation and growth).2,3 Serum hCT levels are relatively high in infants, decline rapidly and are relatively stable from childhood through adult life. In general hCT serum levels are higher in men than in women whereas smoking may lead to an additional increase in serum calcitonin levels.4,5,6 The most prominent clinical syndrome associated with a disordered hypersecretion of hCT is the medullary thyroid carcinoma (MTC), a tumor of the calcitonin secreting cells of the thyroid, which comprises 5‑10 % of all thyroid cancers Moderately elevated calcitonin levels can be falsely positive for either technical reasons or the presence of other rare pathological conditions (i.e. other neuroendocrine tumors, hyperparathyroidism, renal failure etc.).

PIVKA II Hepatocellular carcinoma (HCC) is the 6 th most common cancer worldwide and accounts for more than 90 % of primary liver cancer.1,2 It is the 2 nd most common cause of death from cancer in males and the 6 th in females worldwide. Major risk factors of developing HCC are chronic infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) as indicated by the strong correlation between the prevalence of HCC and chronic hepatitis B and C.3 As most HCCs develop in cirrhotic livers,5 ultrasound surveillance of patients with advanced chronic liver disease is recommended.6,7,8,9 However, as ultrasound performance is operator-dependent, degrades in overweight and obese patients and is sub-optimal for early detection of HCC,10,11 addition of biomarkers is recommended.12 α1 fetoprotein (AFP) is the most commonly used marker for primary liver tumors worldwide. While AFP is elevated during hepato-carcinogenesis, it can also be found in other tumors such as testicular, embryonic13 or gastric cancer.14 AFP has reported sensitivities ranging from 39 to 65 %, and specificities from 76 to 94 % in HCC patients.15 The divergence in sensitivity and specificity of AFP in these studies is probably due to a variety of factors including different etiologies, variable study designs, and different cutoff values. Protein induced by vitamin K absence or antagonist-II (PIVKA II, also known as des γ carboxy prothrombin [DCP]), as well as AFP L3% (Lens culinaris agglutinin-reactive fraction of α fetoprotein [AFP L3] expressed as a percentage of AFP) have been identified as promising biomarkers, which may have utility in the surveillance, diagnosis, and management of HCC.16,17 PIVKA II is an abnormal form of prothrombin secreted into the bloodstream when the activity of vitamin K-dependent carboxylase in the liver is inhibited as a result of the absence of vitamin K or the presence of vitamin K antagonists.16,18 Serum PIVKA II was found to have sensitivities of 48 62 %, specificities of 81 98 %, and an accuracy of 59 84 % in diagnosing HCC in several studies, mostly from Asian cohorts.19,20,21,22 According to recent data, PIVKA II has better diagnostic effectiveness than AFP in differentiating HCC from non-HCC hepatic diseases. In addition, the combination of the two markers could significantly improve the diagnostic performance.23 In another study which compared PIVKA II, AFP and AFP L3%, PIVKA II was found to be significantly superior to the others in differentiating primary liver cancer from cirrhosis (sensitivity 86 % and specificity 93 %).24 PIVKA II is an independent predictor of HCC presence and a better diagnostic biomarker than AFP in discriminating between neoplastic and non-neoplastic lesions in cirrhotic patients with initial ultrasound evidence of suspicious liver nodules.

The human epididymal protein 4 (HE4, also known as WFDC2) belongs to the family of whey acidic four-disulfide core (WFDC) proteins with suspected trypsin inhibitor properties.1,2 In its mature glycosylated form the protein has a molecular weight of approximately 20‑25 kDa and consists of a single peptide chain containing two WFDC domains.3,4 HE4 expression was originally described to be specific to the epididymis.4,5 Recent findings show that HE4 has low expression in the epithelia of respiratory and reproductive tissues, but high expression in ovarian cancer tissue.6 High secretion levels can also be found in the serum of ovarian cancer patients.7 Ovarian cancer is the seventh cancer-related cause of death in women worldwide.8 It is the most lethal form of gynecological cancer, but potentially curable if diagnosed early9 and treated by surgeons familiar with the management of ovarian cancer.9,10 However, the symptoms of ovarian cancer are often vague and unspecific. Thus, the majority of ovarian cancers are detected at a late stage, and the 5‑year patient survival rate decreases from 90 % in stage I to below 20 % in stage IV.11 As a single tumor marker, HE4 had high sensitivity for ovarian cancer, especially in stage I disease, the early non-symptomatic stage. Combined, CA 125 and HE4 yielded the highest sensitivity with 76.4 % at 95 % specificity.12,13 The combination of CA 125 and HE4 can help to determine whether a pelvic mass is benign or malignant in pre- and post-menopausal women. The dual marker combination CA 125 and HE4 is a more accurate predictor of malignancy than either alone.12 Huhtinen et al. reported a 78.6 % sensitivity at 95 % specificity in ovarian carcinoma vs. endometriotic cysts.14 Moore et al. reported 94 % accuracy in identifying malignant vs. benign pelvic masses when combining CA 125 and HE4 values in an algorithm called ROMA (Risk of Ovarian Malignancy Algorithm).15 In addition, HE4 levels correlate with clinical response to therapy and recurrence status in women with diagnosis of ovarian carcinoma as determined by CT imaging. HE4 could thus be an important early indicator for disease recurrence.

CA 125 - Glycoprotein present in serous membranes in tissues. The concentration of antigen increases with diseases of these tissues, pregnancy and menstruation. A significant increase in the level of CA 125 in the blood is sometimes observed in various benign gynecological tumors, as well as in inflammatory processes involving the appendages. A slight rise in the level of this marker is also detected in the first trimester of pregnancy, with various autoimmune diseases, hepatitis, chronic pancreatitis and liver cirrhosis. It is used mainly for monitoring ovarian cancer and diagnosing its recurrence. Regression of the tumor or its removal by surgery is accompanied by a decrease in the content of CA 125 in the blood. CA 125 correlates with disease remission after chemo- and chemoradiotherapy. An increase in the level of CA 125 in the blood is associated with the progression of the tumor process. Tumors of the gastrointestinal tract, carcinoma of the bronchi and carcinoma of the breast can also, in some cases, cause a significant rise in CA 125 levels.

The CA 15‑3 (Cancer Antigen 15‑3) is derived from glycoprotein Mucin‑1 (MUC‑1).1 The antigen is normally found in the luminal secretion of glandular cells and does not circulate in the blood. When cells become malignant and their basal membranes permeable, the antigen is detectable in serum. 5 Overexpression of MUC1 plays an important role in epithelial to mesenchymal transition; an important and complex phenomenon that determines cancer progression.6

CA 19‑9 is a biomarker which is primarily used in the management of pancreatic cancer patients in addition to other diagnostic methods.1 The CA 19‑9 antibody binds to the Lewis (a) antigen on a mucin.2,3 Elevated concentrations are frequently present in the blood of patients with various gastrointestinal conditions, such as pancreatic‑, colorectal‑, gastric‑, hepatocellular‑ and cholangiocellular carcinomas.4 No data exist today which support the use of CA 19‑9 in screening for malignancies5 also concerning the fact that approximately 6 % of the population belong to the Lewis (a‑/b‑) blood group, lacking the antigenic determinant CA 19‑9 and will therefore not release CA 19‑9 even when a malignancy is present. This must be taken into account when interpreting the findings.6 Among non‑malignant conditions, obstructive jaundice is frequently associated with increases in CA 19‑97 and unspecific elevation of CA 19‑9 in serum reflects both inflammatory hypersecretion and leakage of biliary mucins into serum.8 CA 19‑9 levels have also been reported in benign diseases like cystic fibrosis, hydronephrosis, and Hashimoto’s thyroiditis.9 In addition, there is a strong correlation between the serum CA 19‑9 concentration and the degree of cholestasis as well as the levels of alkaline phosphatase and bilirubin during acute liver failure, acute hepatitis or chronic liver diseases.10,11 The common underlying mechanism for elevations in non‑malignant conditions is probably inflammatory hypersecretion of CA 19‑9 by epithelial cells. In pancreatic cancer, levels > 100 U/mL are highly suggestive of unresectablity or metastatic disease and levels <100 U/mL imply a likely resectable disease.12 The European Group of Tumor Markers (EGTM) advise that CA 19‑9 may be used as a diagnostic aid and for monitoring therapy in patients with pancreatic adenocarcinoma.13 CA 19‑9 has been found to be prognostic for survival following resection of pancreatic ductal adenocarcinoma.14 In hepatobiliary carcinoma, CA 19‑9 independently predicted a 2.6‑fold increased mortality in a prospectively collected group of HCC patients in a multivariable analysis.15 In colorectal cancer, CA 19‑9 is described as an additional marker for disease monitoring in patients without an increase in CEA.16

CA 72-4 The tumor associated glycoprotein (TAG) 72, also known as CA 72‑4 is a mucin protein of high molecular weight (approximately 200‑400 kD) and found on the surface of many cancer cells, including stomach, ovary, breast, colon and pancreatic cells.1 An antibody construct directed against TAG 72 has been proposed as an anti‑tumor agent against ovarian and prostate cancer.2 Elevated serum levels are primarily found in gastric cancer patients,3,4 but can also be found in certain non-malignant diseases like pneumonia, pancreatitis, liver cirrhosis and ovarian cysts.5 The most important advantage of CA 72‑4 is its ability to discriminate between malignant and non-malignant gastric and ovarian diseases. 3,6 Gastric and Ovarian Cancer: For gastric cancer, a diagnostic sensitivity of 33 % was reported for CA 72‑4.7 Monitoring treatment and disease course in patients with gastric and ovarian cancer is the main indication for CA 72‑4. After surgical intervention, CA 72‑4 levels return to normal and remain within the normal range in cases where tumor tissue is no longer present.8 A diagnostic sensitivity of 47‑76 % has been reported in ovarian carcinoma.9 Especially for mucinous ovarian cancer, the diagnostic sensitivity of CA 72‑4 is greater than that of CA 125.

Antistreptolysin O ASO Group A streptococci cause different infections: skin diseases or angina tonsillaris that may be followed by glomerulonephritis, acute endocarditis, Sydenham’s Chorea, and acute rheumatic fever, when the upper respiratory tract is infected. These infections can later lead to damage of the heart or the kidneys. Early diagnosis, efficient treatment and monitoring of the patient can reduce these risks. Several metabolites of β‑hemolyzing streptococci are exogenous toxins for the human body, e.g. NAD‑glycohydrolase, streptodornases (ADNases), and hyaluronidase which induce immunological defense reactions. The most clinically important antibody reactions are found against streptolysin O, streptococcaldeoxyribonuclease and streptococcal-hyaluronidase. Immunological testing for specific antibodies provides useful information about the degree of the streptococcal infection and the course of disease. The determination of the level of antistreptolysin O antibodies (ASO) is the most widely used. Eighty-five percent of patients with acute rheumatic fever show increased ASO levels. ASO levels should be monitored several times at weekly intervals to obtain useful data. The titer development can indicate either a successful antibiotic treatment or the persisting antigen stimulus even if the clinical signs of the infection have already disappeared.

Rheumatoid factors are a heterogeneous group of autoantibodies directed against the antigenic determinants on the Fc‑region of IgG molecules. They are important in the diagnosis of rheumatoid arthritis, but can also be found in other inflammatory rheumatic diseases and in various non-rheumatic diseases. They are also found in clinically healthy persons over 60 years of age. Despite these restrictions, the detection of rheumatoid factors is a diagnostic criterion of the American College of Rheumatology for classifying rheumatoid arthritis. The autoantibodies occur in all the immunoglobulin classes, although the usual analytical methods are limited to the detection of rheumatoid factors of the IgM type. The classic procedure for the quantitation of rheumatoid factors is by agglutination with IgG‑sensitized sheep erythrocytes or latex particles. Particular problems of these semiquantitative methods are the poor between‑laboratory precision and reproducibility, together with standardization difficulties. For these reasons, new assay methods such as nephelometry, turbidimetry, enzyme‑immunoassays and radioimmunoassays have been developed. The Roche RF assay is based on the immunological agglutination principle with enhancement of the reaction by latex.

Interleukin 6 (IL 6) is a pleiotropic cytokine with a wide range of functions. It was first described as interferon β2, plasmacytoma growth factor, and hepatocyte stimulating factor. Later on it was described as human B cell stimulating factor 2 (BSF2). In 1988 it was proposed to name it IL 6 as further studies had demonstrated that the protein shows activities not only on B cells but also on T cells, hematopoietic stem cells, hepatocytes and brain cells. IL 6 production is rapidly induced in the course of acute inflammatory reactions associated with injury, trauma, stress, infection, brain death, neoplasia, and other situations.2 IL 6 concentrations in trauma patients may predict later complications from additional surgical stress or indicate missed injuries or complications.4,5 Sequential measurements of IL 6 in serum or plasma of patients admitted to the ICU (intensive care unit) showed to be useful in evaluating the severity of SIRS (systemic inflammatory response syndrome), sepsis and septic shock and to predict the outcome of these patients.6,7,8 IL 6 is also useful as an early alarm marker for the detection of neonatal sepsis.9,10,11,12 IL 6 also plays a role in chronic inflammation e.g. rheumatoid arthritis.

C‑reactive protein is the classic acute phase protein in inflammatory reactions. It is synthesized by the liver and consists of five identical polypeptide chains that form a five‑membered ring having a molecular weight of 105000 daltons. CRP is the most sensitive of the acute phase reactants and its concentration increases rapidly during inflammatory processes. Complexed CRP activates the classical complement pathway. The CRP response frequently precedes clinical symptoms, including fever. In normal healthy individuals CRP is a trace protein with a range up to 5 mg/L. After onset of an acute phase response the serum CRP concentration rises rapidly and extensively. The increase begins within 6 to 12 hours and the peak value is reached within 24 to 48 hours. Levels above 100 mg/L are associated with severe stimuli such as major trauma and severe infection (sepsis). CRP response may be less pronounced in patients suffering from liver disease. CRP assays are used to detect systemic inflammatory processes; to assess treatment of bacterial infections with antibiotics; to detect intrauterine infections with concomitant premature amniorrhexis; to differentiate between active and inactive forms of disease with concurrent infection, e.g. in patients suffering from SLE or Colitis ulcerosa; to therapeutically monitor rheumatic disease and assess anti‑inflammatory therapy; to determine the presence of post‑operative complications at an early stage, such as infected wounds, thrombosis and pneumonia, and to distinguish between infection and bone marrow rejection. Postoperative monitoring of CRP levels of patients can aid in the recognition of unexpected complications (persisting high or increasing levels). Measuring changes in the concentration of CRP provides useful diagnostic information about how acute and how serious a disease is. It also allows judgements about the disease genesis. Persistence of a high serum CRP concentration is usually a grave prognostic sign which generally indicates the presence of an uncontrolled infection.

Procalcitonin (PCT) is a 116 amino acid prohormone with a molecular weight of approximately 12.7 kDa. PCT is expressed by neuroendocrine cells (C cells of the thyroid, pulmonary and pancreatic tissues) and successively enzymatically cleaved into (immature) calcitonin, katacalcin, and an N terminal region. The blood of healthy individuals contains only low levels of PCT.1,2 It was discovered that PCT increases during bacterial infection. It is probable that multiple tissues express PCT throughout the body in response to sepsis.3 PCT circulating in septic patients consists of only 114 amino acids lacking the N terminal dipeptide Ala Pro.4 Increased PCT levels are often found in patients suffering from bacterial sepsis, especially severe sepsis and septic shock.5,6,7,8,9,10 PCT is considered as a prognostic marker to support outcome prediction in sepsis patients.8,11,12,13 In acute pancreatitis PCT was found to be a reliable indicator of severity and of major complications.14,15 In patients suffering from community-acquired respiratory tract infections or ventilator induced pneumonia PCT has been proposed as a guide for the decision of antibiotic treatment necessity and to monitor treatment success.

Thyroid‑stimulating hormone (TSH, thyrotropin) is a glycoprotein. TSH is formed in specific basophil cells of the anterior pituitary. The hypophyseal release of TSH (thyrotropic hormone) is the central regulating mechanism for the biological action of thyroid hormones. TSH has a stimulating action in all stages of thyroid hormone formation and secretion. The determination of TSH serves as the initial test in thyroid diagnostics. Even very slight changes in the concentrations of the free thyroid hormones bring about much greater opposite changes in the TSH level. Accordingly, TSH is a very sensitive and specific parameter for assessing thyroid function and is particularly suitable for early detection or exclusion of disorders in the central regulating circuit between the hypothalamus, pituitary and thyroid.

Antibodies to TSH receptor (Anti-TSHR) Hyperthyroidism in Graves' disease (autoimmune hyperthyroidism) is typically caused by autoantibodies to the thyroid stimulating hormone receptor (TSHR), and measurement of these TSHR antibodies (TRAb) can be useful in disease diagnosis and management.1,2,3,4,5 TSH autoantibodies can be classified as stimulating, blocking or neutral depending on their mechanism of action. Despite having actions similar to TSH, TSHR stimulating antibodies are not subject to the negative feedback mechanisms associated with TSH, leading to prolonged activation of the TSHR. This results in the elevated thyroid hormone levels and clinical thyrotoxic state associated with Graves’ disease.6,7 Indications for TRAb determination include: ▪ the detection or exclusion of autoimmune hyperthyroidism and its differentiation from disseminated autonomy of the thyroid gland. The presence of TRAb indicates that the patient's thyrotoxicosis is of autoimmune etiology rather than due to toxic nodular goiter.8,9 Because the aim of treatment for Graves' disease may differ from the treatment of other forms of thyrotoxicosis, an initial TRAb determination is clearly of value. ▪ monitoring the therapy of Graves' disease patients and prediction of relapse, thereby constituting an important decision-making aid in treatment management. TRAb levels tend to fall during antithyroid drug therapy for Graves' disease. Low levels or the absence of TRAb after a course of drug treatment may indicate disease remission, and therefore the withdrawal of therapy can be considered.10,11,12 ▪ TRAb measurement during the last trimester of pregnancy. Because TRAb are IgG‑class antibodies, they cross the placenta and can cause neonatal thyroid disease. The measurement of TRAb during pregnancy in patients with a history of thyroid disease is therefore important in assessing the risk of thyroid disease in the neonate.

Free T4 Thyroxine (T4) is the main thyroid hormone secreted into the bloodstream by the thyroid gland. Together with triiodothyronine (T3) it plays a vital role in regulating the body's metabolic rate, influences the cardiovascular system, growth and bone metabolism, and is important for normal development of gonadal functions and nervous system.1 T4 circulates in the bloodstream as an equilibrium mixture of free and serum bound hormone. Free T4 (fT4) is the unbound and biologically active form, which represents only 0.03 % of the total T4. The remaining T4 is inactive and bound to serum proteins such as thyroxine binding globulin (TBG) (75 %), pre-albumin (15 %), and albumin (10 %).2,3,4,5 The determination of free T4 has the advantage of being independent of changes in the concentrations and binding properties of these binding proteins; additional determination of a binding parameter (T‑uptake, TBG) is therefore unnecessary. Thus free T4 is a useful tool in clinical routine diagnostics for the assessment of the thyroid status. It should be measured together with TSH if thyroid disorders are suspected and is also suitable for monitoring thyrosuppressive therapy.

Антитела к рецептору ТГ Тиреоглобулин (ТГ) вырабатывается клетками щитовидной железы и является основным компонентом коллоида в просвете фолликула щитовидной железы. В сочетании с тиреопероксидазой (ТПО) тиреоглобулин играет важную роль в йодировании L‑тирозина и выработке гормонов щитовидной железы T4 и T3.1 ТГ, как и ТПО, является потенциальным аутоантигеном.2,3 Повышенные концентрации антител к ТГ (ТГ-аутоантитела) в сыворотке крови наблюдаются у пациентов с аутоиммунным тиреоидитом.2,3 Высокие концентрации анти‑ТГ в сочетании с анти‑ТПО отмечаются у большинства пациентов с хроническим лимфоцитарноинфильтративным тиреоидитом (тиреоидит Хашимото).3 Частота обнаружения антител к тиреоглобулину составляет приблизительно 50‑80 % у пациентов с аутоиммунным тиреоидитом, включая пациентов с тиреоидитом Хашимото, и приблизительно 30‑50 % у пациентов с диффузным токсическим зобом.3,4,5,6 Тест на анти‑ТГ также может быть полезен для контроля течения тиреоидита Хашимото и для дифференциальной диагностики.3,7 Сюда относятся случаи подозрения на аутоиммунный тиреоидит неизвестной этиологии с отрицательными результатами теста на анти‑ТПО8,9 и случаи, когда необходимо дифференцировать тиреоидит Хашимото от узлового нетоксического зоба или других форм тиреоидита.4 Анти‑ТГ также считаются полезным суррогатным диагностическим маркером дифференцированного рака щитовидной железы при отрицательном значении ТГ в сыворотке крови10 и позволяют исключить влияние аутоантител к ТГ при измерении уровня ТГ в сыворотке крови с помощью теста на ТГ.11,12 Хотя чувствительность диагностики можно увеличить за счет одновременного определения дополнительных тиреоидных антител (анти‑ТПО антитела и антитела к рецептору ТТГ), отрицательный результат однозначно не исключает возможности наличия аутоиммунного заболевания. Титр антител не связан с клинической активностью заболевания. Первоначально повышенные титры могут стать отрицательными, если болезнь продолжается в течение длительного времени или в случае наступления ремиссии. Если после ремиссии антитела появляются вновь, возможен рецидив заболевания.

T3 Triiodothyronine (3,5,3'‑triiodothyronine or T3) is the thyroid hormone principally responsible for the regulation of metabolism of the various target organs. T3 is mainly formed extrathyroidally, particularly in the liver, by enzymatic 5'‑deiodination of T4 (thyroxine). Accordingly, the T3 concentration in serum is more a reflection of the functional state of the peripheral tissue than the secretory performance of the thyroid gland.1 A reduction in the conversion of T4 to T3 results in a decrease in the T3 concentration. It occurs under the influence of medicaments such as propranolol, glucocorticoids or amiodarone and in severe non-thyroidal illness (NTI), and is referred to as “low T3 syndrome”. As with T4, over 99 % of T3 is bound to transport proteins. However, the affinity of T3 to them is around 10‑fold lower, but T3 has a 15‑fold higher affinity for thyroid receptor compared to T4.1,2,3 The determination of T3 is utilized in the diagnosis of T3‑hyperthyroidism, the detection of early stages of hyperthyroidism and for indicating a diagnosis of thyrotoxicosis factitia.

EBNA IgG Epstein‑Barr virus, also known as human herpesvirus 4 (HHV4) is one of the 8 known human herpes viruses, infecting about 90 % of the world population already at young age and generally causing little complications. The majority of these infections are either asymptomatic or manifest with only minor unspecific symptoms.1 The most common EBV-linked disease is the symptomatic acute primary infection called infectious mononucleosis (IM), mainly affecting adolescents and young adults. IM is characterized by the triad of fever, pharyngitis and cervical lymphadenopathy, and is generally a self-limiting disease with supportive therapy as the mainstay of treatment.2 Yet, early and accurate diagnosis is valuable as EBV is highly communicable, and in rare cases complications may develop, posing serious health risks.1 Following the lytic replication during primary infection, EBV remains latent for life, mainly in B‑cells.3 EBV infection has been associated to various autoimmune diseases as well as several distinct malignant diseases including both lymphomas and carcinomas.4 Immunosuppression can result in post-transplant lymphoproliferative disorder (PTLD), a frequently fatal disorder of uncontrolled B‑cell proliferation.5 EBV is mainly transmitted by saliva, but sexual transmission, and transmission via solid-organ and hematopoietic-stem-cell transplantation has been reported.6 Various viral, bacterial, and parasitic diseases can cause mononucleosislike symptoms, especially in early infection.7 A combination of biomarkers is commonly used for differential diagnosis, to rule out other infections or conditions with similar symptoms, such as acute HIV or CMV infection or toxoplasmosis. EBV serology is also used for the determination of the immune status of transplant donors and recipients assessing the risk of a patient to develop PTLD, that can be caused by a reactivation or a new EBV infection in the previously EBV naïve patient.8,9,10,11 Serologic tests specific for EBV are routinely used to confirm the diagnosis of an acute EBV infection, as clinical signs and symptoms are not very sensitive or specific.2 3 different biomarkers are routinely used in combination to determine the stage of EBV infection: IgM antibodies to EBV antigens, IgG antibodies to EBV viral capsid antigens (VCA), and IgG antibodies to EBV nuclear antigen‑1 (EBNA‑1).12,13 Anti‑EBV IgM and anti‑EBV VCA IgG antibodies are typically detectable at the clinical onset of illness. IgM may remain positive until 2 to 6 months after primary infection, and VCA IgG antibodies typically show lifelong persistence. EBNA‑1 IgG antibodies usually appear within 6‑12 weeks after primary infection and persist lifelong. Therefore, the presence of IgM and VCA IgG antibodies, and the absence of EBNA‑1 IgG, in combination with the typical clinical presentation are indicative for acute infection. The absence of IgM antibodies and presence of VCA IgG and EBNA‑1 IgG antibodies are indicative for past infection and a state of latency.12,13 For EBV-monitoring in cancer, transplantation, HIV/AIDS and autoimmune syndromes, specific rules may apply, that differ per disease condition.

HSV-1 IgG Herpes simplex viruses 1 and 2 (HSV‑1 and HSV‑2) are two members of the family Herpesviridae. The prevalence of HSV‑1 infections in the general population is estimated to be around 70‑80 %, for HSV‑2 around 17‑25 %.1,2 Transmission of HSV‑1 and HSV‑2 depends on intimate, personal contact between a seronegative individual and someone excreting the virus.3 Infection with HSV‑1 and HSV‑2 can produce a wide spectrum of symptoms, e.g. mucous membrane and skin lesions and ocular, visceral, and central nervous system (CNS) disease. In immunosuppressed patients HSV infection can be associated with severe and extensive lesions.4 Although HSV‑1 and HSV‑2 are usually transmitted by different routes and involve different areas of the body, much overlap is seen between the epidemiology and clinical manifestations of these two viruses.2,5,6,7 Primary HSV‑1 infections are typically acquired during childhood. Following oropharyngeal infection, the trigeminal ganglion becomes colonized and harbors latent virus. A major manifestation of HSV‑1 infection in young children is gingivostomatitis, a serious infection of the gums, tongue, mouth, lip, facial area, and pharynx. In older people infected with HSV‑1 upper respiratory tract infections and mononucleosis-like syndrome are very common.2 Recurrent skin lesions are the hallmark of HSV pathogenesis. Nearly all people with clinically recognized HSV‑1 infection develop at least one recurrent episode within 1 year after the primary infection. Reactivation is associated with mucosal ulcerations or lesions at the mucocutaneous junction of the lips.8 Genital herpes can be induced by either HSV‑1 or HSV‑2.9 Approximately 85 % of the symptomatic primary genital HSV infections are caused by HSV‑2, the rest is caused by HSV‑1. Genital HSV‑1 results from selfinoculation or from oral sexual practices.10 Neonatal herpes – which can be caused by HSV‑1 as well as HSV‑2 – has the most severe implications and is usually acquired during the intrapartum period through exposure in the genital tract.7,11 In most cases the mothers have no reported history of HSV infection.12 Neonatal HSV infections may remain localized to the site of infection (skin, eye, mouth), extend to the CNS, or disseminate to multiple organs.13 Neonates have the highest frequency of visceral and CNS involvement of all HSV‑infected patients.14,15,16 HSV infection is frequently not recognized. Subclinical viral shedding and unrecognized infections seem to be major factors in transmission.12 Genital HSV infection is frequently not recognized and diagnosis based on the clinical presentation alone has a low sensitivity.8 Serologic tests have been recommended for pregnant women with active HSV lesions at delivery in order to guide patient management and when there is a high risk for infection.17,18 Type-specific serologic tests allow the identification of silent carriers of HSV‑2 infection in patients with or without pre-existing antibodies to HSV‑1.19,20․

HSV-1 IgG Herpes simplex viruses 1 and 2 (HSV‑1 and HSV‑2) are two members of the family Herpesviridae. The prevalence of HSV‑1 infections in the general population is estimated to be around 70‑80 %, for HSV‑2 around 17‑25 %.1,2 Transmission of HSV‑1 and HSV‑2 depends on intimate, personal contact between a seronegative individual and someone excreting the virus.3 Infection with HSV‑1 and HSV‑2 can produce a wide spectrum of symptoms, e.g. mucous membrane and skin lesions and ocular, visceral, and central nervous system (CNS) disease. In immunosuppressed patients HSV infection can be associated with severe and extensive lesions.4 Although HSV‑1 and HSV‑2 are usually transmitted by different routes and involve different areas of the body, much overlap is seen between the epidemiology and clinical manifestations of these two viruses.2,5,6,7 Primary HSV‑1 infections are typically acquired during childhood. Following oropharyngeal infection, the trigeminal ganglion becomes colonized and harbors latent virus. A major manifestation of HSV‑1 infection in young children is gingivostomatitis, a serious infection of the gums, tongue, mouth, lip, facial area, and pharynx. In older people infected with HSV‑1 upper respiratory tract infections and mononucleosis-like syndrome are very common.2 Recurrent skin lesions are the hallmark of HSV pathogenesis. Nearly all people with clinically recognized HSV‑1 infection develop at least one recurrent episode within 1 year after the primary infection. Reactivation is associated with mucosal ulcerations or lesions at the mucocutaneous junction of the lips.8 Genital herpes can be induced by either HSV‑1 or HSV‑2.9 Approximately 85 % of the symptomatic primary genital HSV infections are caused by HSV‑2, the rest is caused by HSV‑1. Genital HSV‑1 results from selfinoculation or from oral sexual practices.10 Neonatal herpes – which can be caused by HSV‑1 as well as HSV‑2 – has the most severe implications and is usually acquired during the intrapartum period through exposure in the genital tract.7,11 In most cases the mothers have no reported history of HSV infection.12 Neonatal HSV infections may remain localized to the site of infection (skin, eye, mouth), extend to the CNS, or disseminate to multiple organs.13 Neonates have the highest frequency of visceral and CNS involvement of all HSV‑infected patients.14,15,16 HSV infection is frequently not recognized. Subclinical viral shedding and unrecognized infections seem to be major factors in transmission.12 Genital HSV infection is frequently not recognized and diagnosis based on the clinical presentation alone has a low sensitivity.8 Serologic tests have been recommended for pregnant women with active HSV lesions at delivery in order to guide patient management and when there is a high risk for infection.17,18 Type-specific serologic tests allow the identification of silent carriers of HSV‑2 infection in patients with or without pre-existing antibodies to HSV‑1.19,20․

Toxoplasmosis is a relatively common infection caused by the protozoan parasite Toxoplasma gondii. The infection is mainly acquired by ingestion of food or water contaminated by mature oocysts shed by cats or by undercooked meat containing tissue cysts.1,2,3,4 Infection can also be transmitted congenitally if a woman is newly infected during or just prior to pregnancy, and via organ transplant or blood transfusion from an infected donor.4 Primary, acute infection in healthy individuals is mostly mild or even asymptomatic and is followed by life-long latency.3,4 Reactivation of a latent Toxoplasma infection can occur as a result of immunosuppression (e.g. in organ transplant recipients, patients with cancer or HIV) and can be associated with high morbidity and mortality.3,4 Reactivated disease in immunocompromised hosts frequently presents with brain lesions, especially in patients with advanced HIV-related immunosuppression.3,4,5 Primary maternal Toxoplasma infection occurring during pregnancy may have significant implications for the fetus as the parasite can be transmitted across the placenta.3,6 The majority of infants with congenital infection do not present clinical symptoms at birth but may develop severe sequelae later in life such as chorioretinitis, intellectual and psychomotor disabilities, visual and hearing impairment and hearing loss.3,6,7,8 The fetal infection rate increases with gestational age, but the risk of severe clinical manifestations is higher in the case of early maternal infection.3,6,7,8 Early drug therapy in acute infection during pregnancy can prevent congenital damage or ameliorate the severity of clinical manifestations.6,7 The diagnosis of Toxoplasma infection is most commonly made by the detection of anti‑Toxoplasma‑specific IgG and IgM antibodies.3,4,9 The determination of Toxo IgG antibodies is used to assess the serological status of T. gondi infection and their presence is indicative of a latent or acute infection.4,9 Detection of Toxo IgM antibodies is presumptive of an acute or recent Toxoplasma infection.3,4,9 The diagnosis of the acute acquired infection during pregnancy is established by a seroconversion or a significant rise in antibody titers (IgG and/or IgM) in serial samples.

EBV VCA IgG Epstein‑Barr virus, also known as human herpesvirus 4 (HHV4) is one of the 8 known human herpes viruses, infecting about 90 % of the world population already at young age and generally causing little complications. The majority of these infections are either asymptomatic or manifest with only minor unspecific symptoms.1 The most common EBV-linked disease is the symptomatic acute primary infection called infectious mononucleosis (IM), mainly affecting adolescents and young adults. IM is characterized by the triad of fever, pharyngitis and cervical lymphadenopathy, and is generally a self-limiting disease with supportive therapy as the mainstay of treatment.2 Yet, early and accurate diagnosis is valuable as EBV is highly communicable, and in rare cases complications may develop, posing serious health risks.1 Following the lytic replication during primary infection, EBV remains latent for life, mainly in B‑cells.3 EBV infection has been associated to various autoimmune diseases as well as several distinct malignant diseases including both lymphomas and carcinomas.4 Immunosuppression can result in post-transplant lymphoproliferative disorder (PTLD), a frequently fatal disorder of uncontrolled B‑cell proliferation.5 EBV is mainly transmitted by saliva, but sexual transmission, and transmission via solid-organ and hematopoietic-stem-cell transplantation has been reported.6 Various viral, bacterial, and parasitic diseases can cause mononucleosislike symptoms, especially in early infection.7 A combination of biomarkers is commonly used for differential diagnosis, to rule out other infections or conditions with similar symptoms, such as acute HIV or CMV infection or toxoplasmosis. EBV serology is also used for the determination of the immune status of transplant donors and recipients assessing the risk of a patient to develop PTLD, that can be caused by a reactivation or a new EBV infection in the previously EBV naïve patient.8,9,10,11 Serologic tests specific for EBV are routinely used to confirm the diagnosis of an acute EBV infection, as clinical signs and symptoms are not very sensitive or specific.2 3 different biomarkers are routinely used in combination to determine the stage of EBV infection: IgM antibodies to EBV antigens, IgG antibodies to EBV viral capsid antigens (VCA), and IgG antibodies to EBV nuclear antigen‑1 (EBNA‑1).12,13 Anti‑EBV IgM and anti‑EBV VCA IgG antibodies are typically detectable at the clinical onset of illness. IgM may remain positive until 2 to 6 months after primary infection, and VCA IgG antibodies typically show lifelong persistence. EBNA‑1 IgG antibodies usually appear within 6‑12 weeks after primary infection and persist lifelong. Therefore, the presence of IgM and VCA IgG antibodies, and the absence of EBNA‑1 IgG, in combination with the typical clinical presentation are indicative for acute infection. The absence of IgM antibodies and presence of VCA IgG and EBNA‑1 IgG antibodies are indicative for past infection and a state of latency.12,13 For EBV-monitoring in cancer, transplantation, HIV/AIDS and autoimmune syndromes, specific rules may apply, that differ per disease condition.

Toxoplasmosis is a relatively common infection caused by the protozoan parasite Toxoplasma gondii. The infection is mainly acquired by ingestion of food or water contaminated by mature oocysts shed by cats or by undercooked meat containing tissue cysts.1,2,3,4 Infection can also be transmitted congenitally if a woman is newly infected during or just prior to pregnancy, and via organ transplant or blood transfusion from an infected donor.4 Primary, acute infection in healthy individuals is mostly mild or even asymptomatic and is followed by life-long latency.3,4 Reactivation of a latent Toxoplasma infection can occur as a result of immunosuppression (e.g. in organ transplant recipients, patients with cancer or HIV) and can be associated with high morbidity and mortality.3,4 Reactivated disease in immunocompromised hosts frequently presents with brain lesions, especially in patients with advanced HIV-related immunosuppression.3,4,5 Primary maternal Toxoplasma infection occurring during pregnancy may have significant implications for the fetus as the parasite can be transmitted across the placenta.3,6 The majority of infants with congenital infection do not present clinical symptoms at birth but may develop severe sequelae later in life such as chorioretinitis, intellectual and psychomotor disabilities, visual and hearing impairment and hearing loss.3,6,7,8 The fetal infection rate increases with gestational age, but the risk of severe clinical manifestations is higher in the case of early maternal infection.3,6,7,8 Early drug therapy in acute infection during pregnancy can prevent congenital damage or ameliorate the severity of clinical manifestations.6,7 The diagnosis of Toxoplasma infection is most commonly made by the detection of anti‑Toxoplasma‑specific IgG and IgM antibodies.3,4,9 The determination of Toxo IgG antibodies is used to assess the serological status of T. gondi infection and their presence is indicative of a latent or acute infection.4,9 Detection of Toxo IgM antibodies is presumptive of an acute or recent Toxoplasma infection.3,4,9 The diagnosis of the acute acquired infection during pregnancy is established by a seroconversion or a significant rise in antibody titers (IgG and/or IgM) in serial samples.

Toxoplasmosis is a relatively common infection caused by the protozoan parasite Toxoplasma gondii. The infection is mainly acquired by ingestion of food or water contaminated by mature oocysts shed by cats or by undercooked meat containing tissue cysts.1,2,3,4 Infection can also be transmitted congenitally if a woman is newly infected during or just prior to pregnancy, and via organ transplant or blood transfusion from an infected donor.4 Primary, acute infection in healthy individuals is mostly mild or even asymptomatic and is followed by life-long latency.3,4 Reactivation of a latent Toxoplasma infection can occur as a result of immunosuppression (e.g. in organ transplant recipients, patients with cancer or HIV) and can be associated with high morbidity and mortality.3,4 Reactivated disease in immunocompromised hosts frequently presents with brain lesions, especially in patients with advanced HIV-related immunosuppression.3,4,5 Primary maternal Toxoplasma infection occurring during pregnancy may have significant implications for the fetus as the parasite can be transmitted across the placenta.3,6 The majority of infants with congenital infection do not present clinical symptoms at birth but may develop severe sequelae later in life such as chorioretinitis, intellectual and psychomotor disabilities, visual and hearing impairment and hearing loss.3,6,7,8 The fetal infection rate increases with gestational age, but the risk of severe clinical manifestations is higher in the case of early maternal infection.3,6,7,8 Early drug therapy in acute infection during pregnancy can prevent congenital damage or ameliorate the severity of clinical manifestations.6,7 The diagnosis of Toxoplasma infection is most commonly made by the detection of anti‑Toxoplasma‑specific IgG and IgM antibodies.3,4,9 The determination of Toxo IgG antibodies is used to assess the serological status of T. gondi infection and their presence is indicative of a latent or acute infection.4,9 Detection of Toxo IgM antibodies is presumptive of an acute or recent Toxoplasma infection.3,4,9 The diagnosis of the acute acquired infection during pregnancy is established by a seroconversion or a significant rise in antibody titers (IgG and/or IgM) in serial samples.

CMV IgG Avidity Cytomegalovirus (CMV), a member of the herpes virus family, is ubiquitous in all human populations, causing infections which are followed by life‑long latency in the host with occasional reactivations.1,2 The seroprevalence of antibodies in adults ranges from 40‑100 % with inverse correlation to socioeconomic status.1,2,3 CMV is transmitted through body fluids, including blood, genital secretions and breast milk. Saliva and urine of infected individuals also represent a prominent source of infection, and children, especially those attending day care facilities, are an important vector for viral spread.2,3,4,5,6 In immunocompetent individuals primary CMV infection is usually mild or asymptomatic.2,5 Patients commonly present with a mononucleosis‑like syndrome, including fever, sore throat, cervical lymphadenopathy, malaise, headache, muscle ache and joint pains.2,3,4,5,7 During pregnancy, CMV can cause congenital infection which may result in permanent physical and/or neurological sequelae in the child.5 CMV infection can be primary, i.e. newly acquired, or secondary, i.e. due to reactivation of the latent virus or re-infection with a different viral strain.3,5 Primary CMV infection is reported in 1‑4 % of seronegative women during pregnancy and the risk of transmission to the fetus is estimated to be about 30‑40 %.3,4 Reactivation of CMV infection during pregnancy is reported in 10‑30 % of seropositive women and, in this circumstance, the risk of transmission of the virus is about 1‑3 %.3,4,5 Overall, prenatal CMV infection occurs in 0.6‑0.7 % of all life births in the developed world.4,5,8 The majority of babies born with congenital CMV infection are asymptomatic at birth.8,9,10 Of these 5‑15 % still develop irreversible impairments, most frequently hearing loss, that can occur several months or even years after birth.5,8,9,10 For babies symptomatic at birth, prognosis is very poor, as they are likely to develop severe mental impairment and/or hearing loss.5,8,9,10 Different studies have shown that the risk of symptomatic congenital disease in the fetus or newborn infant is high, when maternal primary infection takes place in early pregnancy before week 20 of gestation, and lower thereafter.4,5 The congenital CMV infection caused by recurrent maternal infection seldom leads to symptomatic disease at birth.4,5 At risk for CMV infection and disease are also immunocompromised patients such as transplant recipients and HIV infected patients where the virus can cause life-threatening diseases.11,12 The CMV status of transplant donors and recipients is very important, as it will determine prophylactic and pre‑emptive treatment strategies against CMV. CMV-negative transplant recipients should receive donations from CMV-negative individuals or leukocyte depleted blood products. The CMV status can still be determined by testing for CMV IgG antibodies. Within the appropriate clinical context, the first step in diagnosing acute primary CMV infection is most commonly made by the detection of anti‑CMV‑specific IgG and IgM antibodies.5 Samples being reactive for IgM antibodies indicate an acute, recent or reactivated infection.2,4,5,12 For further analysis of a primary CMV infection the determination of the CMV IgG avidity is used as an aid.2,4,5,12 The CMV IgG avidity assay measures the functional binding affinity of CMV IgG antibodies in response to infection. The antibodies produced during the primary response have lower antigen avidity than the antibodies produced later on.2,5,10 Low avidity is encountered approximately up to 18‑20 weeks after onset of symptoms in immunocompetent subjects.5,10 However, individual variation does exist in the rate of avidity maturation. In rare cases low avidity results can be observed up to 6 months or even longer after the onset of infection. The avidity testing should be performed early in gestation. Low avidity CMV IgG antibodies detected before the 16th‑18th week of pregnancy in combination with positive CMV IgM result is a strong evidence for recent primary infection.3,5,10 A high avidity result later after gestation (after 20th week of gestation) cannot rule out a primary infection earlier in gestation when low avidity CMV IgG may have been present.3 A high avidity index during the first 12‑16 weeks of pregnancy can be considered indicative of past infection.3,5,7,10

Cytomegalovirus (CMV), a member of the herpes virus family, is ubiquitous in all human populations, causing infections which are followed by life-long latency in the host with occasional reactivations.1,2 The seroprevalence of antibodies in adults ranges from 40‑100 % with inverse correlation to socioeconomic status.1,2,3 CMV is transmitted through body fluids, including blood, genital secretions and breast milk. Saliva and urine of infected individuals also represent a prominent source of infection, and children, especially those attending day care facilities, are an important vector for viral spread.2,3,4,5,6 In immunocompetent individuals primary CMV infection is usually mild or asymptomatic.2,5 Patients commonly present with a mononucleosis-like syndrome, including fever, sore throat, cervical lymphadenopathy, malaise, headache, muscle ache and joint pains.2,3,5,7 During pregnancy, CMV can cause congenital infection which may result in permanent physical and/or neurological sequelae to the child.5 CMV infection can be primary, i.e. newly acquired, or secondary, i.e. due to reactivation of the latent virus or re-infection with a different viral strain.3,5 Primary CMV infection is reported in 1‑4 % of seronegative women during pregnancy and the risk of transmission to the fetus is estimated to be about 30‑40 %.3,4 Reactivation of CMV infection during pregnancy is reported in 10‑30 % of seropositive women and, in this circumstance, the risk of transmission of the virus is about 1‑3 %.3,4,5 Overall, prenatal CMV infection occurs in 0.6‑0.7 % of all life births in the developed world.4,5,8 The majority of babies born with congenital CMV infection are asymptomatic at birth.8,9,10 Of these 5‑15 % still develop irreversible impairments, most frequently hearing loss, that can occur several months or even years after birth.5,8,9,10 For babies symptomatic at birth, prognosis is very poor, and the vast majority will develop severe mental impairment and/or hearing loss.5,8,9,10 Different studies have shown that the risk of symptomatic congenital disease in the fetus or newborn infant is high, when maternal primary infection takes place in early pregnancy before week 20 of gestation, and lower thereafter.4,5 The congenital CMV infection caused by recurrent maternal infection seldom leads to symptomatic disease at birth.4,5 At risk for CMV infection and disease are also immunocompromised patients such as transplant recipients and HIV infected patients where the virus can cause life-threatening diseases.11,12 The CMV status of transplant donors and recipients is very important, as it will determine prophylactic and pre-emptive treatment strategies against CMV. Within the appropriate clinical context, the first step in diagnosing acute primary CMV infection is most commonly made by the detection of anti‑CMV‑specific IgG and IgM antibodies.5 Samples being reactive for IgM antibodies indicate an acute, recent or reactivated infection.2,4,5,12 For further analysis of a primary CMV infection the determination of the CMV IgG avidity is used as an aid.2,4,5,12 A positive IgM result in combination with a low avidity index for IgG is a strong indication of a recent primary CMV infection.4,5,12 Seroconversion to CMV IgM and IgG may also indicate a recent CMV infection.

Testosterone in serum is the most important laboratory parameter for confirming a suspected endocrine testicular disorder, for documenting an androgen deficiency and for monitoring testosterone replacement.

Serum 17-α-hydroxyprogesterone (17-OH PROG) (adrenogenital syndrome test) 17-OH PROG is a steroid precursor of cortisol with natriuretic effect. This hormone is produced in the adrenal glands, ovaries, testicles and placenta. 17-OH PROG is converted to cortisol by hydroxylation. Determination of 17-OH PROG levels in blood plays a leading role in the diagnosis of adrenogenital syndrome, which is accompanied by hyperproduction of one group of hormones by the adrenal cortex and a decrease in the secretion of the other. Adrenogenital syndrome (AGS) is caused by a hereditary deficiency of various enzymes involved in the biosynthesis of steroid hormones. There are several forms of AGS, and their clinical manifestations depend on which particular enzyme is lacking. Common to all forms of AGS is impaired synthesis of cortisol, which regulates the secretion of ACTH via a negative feedback loop. Low levels of cortisol in the blood result in an increased secretion of ACTH by the anterior lobe of the pituitary gland, leading to hyperfunction of the adrenal glands, their hyperplasia and an increase in the secretion of steroid precursors for androgens synthesis. However, high levels of androgens do not suppress pituitary ACTH secretion via a negative feedback loop. As a result, an excessive amount of 17-α-hydroxyprogesterone accumulates in the adrenal cortex, both due to its insufficient conversion into cortisol, and due to its increased formation.

DHEA‑S is a steroid hormone for which the adrenal gland is the sole source in females and the principle source in males. DHEA‑S is found in the fetus but declines rapidly in the first year of life. Around 5‑7 years of age, DHEA‑S production slowly resumes, increases during puberty and reaches a maximum between 20 and 30 years of age. Thereafter DHEA‑S levels steadily decline to approximately 10 % of peak levels by the age of 80.1,2 DHEA‑S has a relatively long half‑life of 7‑10 hours and its concentration is approximately constant over the day.1 Measurement of DHEA‑S can be useful in the diagnostic work‑up of female patients presenting with clinical symptoms of hyperandrogenism.3 Elevated DHEA‑S levels are indicative of an involvement of the adrenal gland. A decrease of DHEA‑S and total serum testosterone by more than 50 % upon dexamethasone suppression, is seen as confirmation of hyperandrogenism of the adrenal gland.4 The most common cause is missense mutations in the 21‑hydroxylase gene resulting in a mild or adult‑onset or non‑classical congenital adrenal hyperplasia (NCCAH). It has been estimated that the incidence of NCCAH is around 1 % in the population of New York.4 In rare cases the cause is an adrenal tumor; in a study by Carmina et al.,5 the incidence of an adrenal tumor was 0.2 % (2 out of 950 women with hyperandrogenism). Tumor relevant values in women are those values exceeding 700 µg/dL DHEA‑S.6 The Elecsys DHEA‑S assay makes use of a competition test principle using a polyclonal antibody (rabbit) specifically directed against DHEA‑S. Endogenous DHEA‑S in the sample competes with added DHEA‑S derivative labeled with a ruthenium complex) for the binding sites on the biotinylated antibody.

Estrogens are responsible for the development of the secondary female sex characteristics. Together with gestagens they control all the important female reproductive processes. The biologically most active estrogen is 17β‑estradiol. Estrogens are produced primarily in the ovary (follicle, corpus luteum), but small quantities are also formed in the testes and in the adrenal cortex. During pregnancy, estrogens are mainly formed in the placenta.1 In human plasma the bulk of estradiol is bound specifically to SHBG (= sex hormone binding globulin) and non-specifically to human serum albumin.2 Estrogen secretion is biphasic during the menstrual cycle. The determination of estradiol is utilized clinically in the elucidation of fertility disorders in the hypothalamus‑pituitary‑gonad axis, gynecomastia, estrogen‑producing ovarian and testicular tumors. Further clinical indications are the monitoring of fertility therapy and determining the time of ovulation within the framework of in vitro fertilization (IVF).

LH (luteinizing hormone), together with FSH (follicle stimulating hormone), belongs to the gonadotropin family. LH and FSH regulate and stimulate the growth and function of the gonads (ovaries and testes) synergistically.1,2 In women, the gonadotropins act within the hypothalamus‑pituitary‑ovary regulating circuit to control the menstrual cycle.4,5 LH and FSH are released from the gonadotropic cells of the anterior pituitary and pass via the bloodstream to the ovaries. Here the gonadotropins stimulate the growth and maturation of the follicle and hence the biosynthesis of estrogens and progesterones. The highest LH‑concentrations occur during the mid‑cycle peak and induce ovulation and formation of the corpus luteum, the principal secretion product of which is progesterone. In the Leydig cells of the testes, LH stimulates the production of testosterone.1 Determination of the LH concentration is used in the elucidation of dysfunctions within the hypothalamus‑pituitary‑gonads system. The determination of LH in conjunction with FSH is utilized for the following indications: congenital diseases with chromosome aberrations (e.g. Turner's syndrome), polycystic ovaries (PCO), clarifying the causes of amenorrhea, menopausal syndrome, and suspected Leydig cell insufficiency.

Gonadotropins – FSH (follicle-stimulating hormone) and LH (luteinizing hormone) – are glycoproteins secreted by cyanophilic cells of the anterior pituitary gland under the influence of the hypothalamic releasing factor. The target organs are the gonads. Regulation of FSH and LH secretion is carried out by the type of negative feedback effect. In men, high levels of testosterone in the blood have an inhibitory effect on LH secretion. Regulation of gonadotropin secretion in women is much more complex. During the menstrual cycle in women, hormone concentrations in the blood are subject to certain rhythmic changes. The menstrual cycle has a duration of 28±4 days and is divided into:

Progesterone PROG The gestagen progesterone is a steroid hormone which is mainly formed in the cells of the corpus luteum and during pregnancy in the placenta. The progesterone concentration correlates with the development and regression of the corpus luteum. Whereas progesterone is barely detectable in the follicular phase of the female cycle, a rise in the progesterone level is observed one day prior to ovulation. Increased progesterone synthesis occurs during the luteal phase. In the second half of the cycle pregnanediol is excreted in urine as the main degradation product of progesterone.1 Progesterone brings about the conversion of the uterine mucosa into a tissue rich in glands (secretion phase), in order to prepare for the intrauterine implantation of the fertilized ovum. During pregnancy, progesterone inhibits the contraction of the myometrium. In the mammary gland, progesterone (together with estrogens) promotes the proliferation, secretion and disposition of the alveoli.1,2,3,4 The determination of progesterone is utilized in fertility diagnosis for the detection of ovulation and assessment of the luteal phase.

Prolactin is synthesized in the anterior pituitary and is secreted in episodes. The hormone is made up of 198 amino acids and has a molecular weight of approximately 22‑23 kDa. Prolactin appears in serum in three different forms. The biologically and immunologically active monomeric form predominates, followed by the biologically inactive dimeric form and the tetrameric form having low biological activity.1,2 The target organ for prolactin is the mammary gland, the development and differentiation of which is promoted by this hormone. High concentrations of prolactin have an inhibiting action on steroidogenesis of the ovaries and on hypophyseal gonadotropin production and secretion. During pregnancy the concentration of prolactin rises under the influence of elevated estrogen and progesterone production. The stimulating action of prolactin on the mammary gland leads postpartum to lactation. Prolactin further affects glucose and lipid metabolism and may be involved in the manifestation of insulin resistance.3,4,5 Hyperprolactinemia (in men and women) is a cause of fertility disorders.6 The determination of prolactin is utilized in the diagnosis of hyperprolactinemia7,8 and peritoneal endometriosis.9 The Elecsys Prolactin II assay uses two monoclonal antibodies specifically directed against human prolactin.10 Both antibodies show a low reactivity with most forms of macroprolactin.

Sex hormone‑binding globulin (SHBG) is the blood transport protein for testosterone and estradiol (E2). SHBG is produced mainly by the liver and its synthesis and secretion are regulated by estrogen and negatively influenced by liver fat content and inflammatory cytokines.4,5,6,7,8,9 Decreased SHBG serum levels are associated with conditions where elevated androgen levels are present or where the effect of androgen on its target organs is excessive. This explains the gender‑related differences seen between men and women, especially during puberty. However, decreased SHBG levels are also seen in inflammation and in case of a diet leading to fat build‑up in the liver e.g. rich in monosaccharides, particularly fructose. Low serum SHBG concentrations may correlate with cardiovascular disease risk,10,11 type 2 diabetes,12,13 as well as breast cancer.14 High serum SHBG concentrations have been proposed to be associated with change of diet resulting in loss of weight. Moreover, there is increasing evidence that plasma SHBG levels could serve as a biomarker for diseases with chronic inflammation and as indicator of response to anti-inflammatory treatment.6 Low SHBG titer can be an important indicator of an excessive/chronic androgenic action where androgen levels are normal, but where clinical symptoms would seem to indicate androgen in excess.15 By calculating the free androgen index (FAI), also called free testosterone index (FTI), from the ratio of total testosterone (T) to SHBG [% FAI or FTI = (100 T/SHBG)], it is possible to calculate the approximate amount of free testosterone, as there is a direct correlation between FAI and FT. By additionally taking the non‑specifically albumin-bound testosterone into account, it is possible to calculate the bioavailable testosterone, which is the sum of free testosterone and the albumin‑bound testosterone fraction, calculated via the association constant to albumin.16 A similar calculation can be made for E2.17 Only free testosterone is biologically active, and it best indicates the clinical situation of the patient. Free and bioavailable testosterone are also referred to as non‑SHBG‑bound testosterone and can be obtained by precipitation of the SHBG‑bound‑testosterone with ammonium sulfate, and by equilibrium dialysis.18 Elevated SHBG levels can be seen in elderly men, and are often found in patients with hyperthyroidism and cirrhosis. SHBG levels also increase when oral contraceptives or antiepileptic drugs are taken. Pregnant women have markedly higher SHBG serum concentrations due to their increased estrogen production. Decreased SHBG concentrations are often seen in patients with hypothyroidism, polycystic ovarian syndrome, obesity, hirsutism, elevated androgen levels, alopecia, and acromegaly.

Testosone is regarded as one of the key androgen steroids. It is a steroid secreted from the testis and the adrenal cortex in men and from the adrenal cortex and the ovary in women. It is also produced by the peripheral tissues from androstenedione. In men, testosterone is synthesized almost exclusively by the Leydig cells of the testes. The secretion of testosterone is regulated by luteinizing hormone (LH) and testosterone promotes the development of the secondary sex characteristics, such as the growth of pubic, facial, and axillary hair, or the accessory sex organs. Most of the circulating testosterone is bound to carrier proteins (SHBG = sex hormone‑binding globulin).1,2,3 In women, small quantities of testosterone are formed in the ovaries, adrenal gland, and peripheral fatty tissues, and it has a serum concentration that is approximately 10 times less than in males. In physiological concentrations, androgens have no specific effects in women. Increased production of testosterone in women can cause virilization (depending on the increase).

FSH (follicle stimulating hormone), together with LH (luteinizing hormone), belongs to the gonadotropin family. FSH and LH regulate and stimulate the growth and function of the gonads (ovaries and testes) synergistically.1 In women FSH, in conjunction with LH, stimulates oestrogen secretion and ovulation.2 FSH and LH are released from the gonadotropic cells of the anterior pituitary. The levels of the circulating hormones are controlled by steroid hormones via negative feedback to the hypothalamus. In the ovaries FSH, together with LH, stimulates the growth and maturation of the follicle2 and hence also the biosynthesis of estrogens in the follicles. The FSH level shows a peak at mid-cycle, although this is less marked than with LH. Due to changes in ovarian function and reduced estrogen secretion, high FSH concentrations occur during menopause.3 In men, FSH serves to induce spermatogonium development.2 Determination of the FSH concentration is used in the elucidation of dysfunctions within the hypothalamus‑pituitary‑gonads system. The determination of FSH in conjunction with LH is utilized for the following indications: congenital diseases with chromosome aberrations, polycystic ovaries (PCO), amenorrhea (causes), and menopausal syndrome. Depressed gonadotropin levels in men occur in azoospermia.

Anti-HAV The hepatitis A virus (HAV) is a non-enveloped single stranded RNA-virus that belongs to the family of picornaviruses. To date, just one human serotype and 6 genotypes have been described, 3 of which infect humans (genotypes I, II and III).1 The viral capsid consists of 3 major structural proteins (VP1‑VP3) and a fourth putative protein (VP4) that form an immunodominant structure on the surface of the viral particle, which is highly conserved between all genotypes. After vaccination or natural infection, the immune response is directed against this structure.1,3 HAV is one of the most common causes of infectious jaundice and is transmitted by the fecal-oral route. HAV causes acute hepatitis and is not associated with chronic liver disease because the virus does not persist in the organism.1,3 Total anti‑HAV (anti‑HAV IgM and IgG) is positive at the onset of symptoms and due to the presence of IgM.4 After natural infection, anti‑HAV IgG can usually be detected early in the course of infection and remains detectable throughout a person’s lifetime conferring protection against the disease if the organism is reinfected.4,5 Vaccines against HAV and combined vaccines against hepatitis A and B are available today.3,4 Anti‑HAV IgG can be detected approximately 2 weeks after vaccination against HAV. In the case of complete immunization, protection usually lasts for many years. Assays to detect anti‑HAV antibodies are used to determine an existing or past HAV infection or to observe the immune response after HAV vaccination.1

HBsAg The hepatitis B surface antigen (HBsAg), a polypeptide of varying size, is a component of the external envelope of the hepatitis B virus (HBV) particle.2,3 The blood of persons infected with HBV contains, in addition to intact infectious HBV particles, an excess of smaller non-infectious ‘empty’ envelope particles, or filaments, formed from HBsAg.4 The HBsAg determinant ‘a’, against which the immune response is mainly directed, is common to all HBsAg particles. Within this ‘a’ determinant several HBsAg subtype determinants could be defined as d, y, w1‑w4, r and q.5 Under selective pressure (caused by antiviral therapy or by the action of the immune system itself) the virus can express many different viable HBsAg mutants (so-called ‘escape mutants’). Some mutants might lead to a loss of detection in commercially available HBsAg assays.3,6 The Elecsys HBsAg II assay was specifically developed to detect a multitude of these mutants. HBsAg is the first immunologic marker of HBV infection and is generally present some days or weeks before clinical symptoms begin to appear. Detection of HBsAg in human serum or plasma indicates the presence of acute or chronic HBV infection.7 HBsAg assays are used within the scope of diagnostic procedures to identify persons infected with HBV and prevent the transmission of the virus by blood and blood products.4,8 HBsAg assays can also be used to monitor the course of the disease and the efficacy of therapy in persons with acute or chronic HBV infections.9 In addition, HBsAg assays are recommended as part of prenatal care, in order to initiate suitable measures for preventing as far as possible the transmission of an HBV infection to the newborn child.10

Anti-HCV The hepatitis C virus (HCV), first identified in 1989, is a leading cause of liver disease and a major healthcare concern with over 170 million persons (roughly 3 % of the human population), infected worldwide.2,3 The highest prevalence is found in Africa, the Eastern Mediterranean and Asian regions.3,4 HCV is a member of the Flaviviridae family and has a singlestranded, positive-sense RNA genome.5 Currently over 67 subtypes have been identified and these have been classified into 7 genotypes (1‑7).6 Due to the high rate of asymptomatic infections, clinical diagnosis is difficult and screening assays are of major importance.7 Infection with HCV can lead to acute and chronic hepatitis disease. Approximately 70‑85 % of HCV infections progress to chronic disease, although this varies according to patient gender, age, race and immune status.5,8 Chronic HCV infection may lead to cirrhosis and hepatocellular carcinoma,9 therefore, early anti‑HCV detection is the first step in the management of chronic hepatitis and in the selection of patients needing treatment.7 HCV infection can be detected by measuring the amount of HCV RNA, alanine aminotransferase (ALT) and HCV‑specific immunoglobulins (anti‑HCV) in patient serum or plasma samples. This can also indicate if the infection is acute or chronic.5,8 Anti‑HCV antibody tests are used alone or in combination with other tests (e.g. HCV RNA) to detect an infection with HCV and to identify blood and blood products of individuals infected with HCV. The Elecsys Anti‑HCV II assay uses peptides and recombinant proteins representing HCV core, NS3 and NS4 antigens for the determination of anti‑HCV antibodies.

CHE Cholinesterase Cholinesterase (pseudocholinesterase or cholinesterase II) is found in the liver, pancreas, heart, serum and in the white matter of the brain. This enzyme must not be confused with acetylcholinesterase from erythrocytes (EC 3.1.1.7), which is also referred to as cholinesterase I. The biological function of cholinesterase is unknown. Serum cholinesterase serves as an indicator of possible insecticide poisoning. It is measured as an index of liver function. In pre‑operative screening, cholinesterase is used to detect patients with atypical forms of the enzyme and hence avoid prolonged apnea caused by slow elimination of muscle relaxants. Depressed cholinesterase levels are found in cases of intoxication with organophosphorus compounds and in hepatitis, cirrhosis, myocardial infarction, acute infections and atypical phenotypes of the enzyme. This assay is based on the method published by Schmidt E et al. in 1992.3

AMY-P Two types of α‑amylases can be distinguished, the pancreatic type (P‑type) and the salivary type (S‑type). Whereas the P‑type can be attributed almost exclusively to the pancreas and is therefore organ-specific, the S‑type can originate from a number of sites. As well as appearing in the salivary glands it can also be found in tears, sweat, human milk, amniotic fluid, the lungs, testes and the epithelium of the fallopian tube. Because of the sparsity of specific clinical symptoms of pancreatic diseases, enzymatic determinations are of considerable importance in pancreas diagnostics. The determination of pancreas‑specific α‑amylase instead of total α‑amylase is of advantage here. The determination of pancreatic α‑amylase is suitable for the diagnosis and monitoring of acute pancreatitis and acute attacks during chronic pancreatitis. In terms of clinical sensitivity and specificity, the diagnostic value of pancreatic α‑amylase is comparable to that of lipase, the generally recognized pancreas‑specific enzyme. The sensitivity of pancreatic α‑amylase is 38 % higher than that of total α‑amylase in the diagnosis of acute pancreatitis when - as commonly used - three times the upper normal limit is taken as the criterion

The enzyme alanine aminotransferase (ALT) has been widely reported as present in a variety of tissues. The major source of ALT is the liver, which has led to the measurement of ALT activity for the diagnosis of hepatic diseases. Elevated serum ALT is found in hepatitis, cirrhosis, obstructive jaundice, carcinoma of the liver, and chronic alcohol abuse. ALT is only slightly elevated in patients who have an uncomplicated myocardial infarction. Although both serum aspartate aminotransferase (AST) and ALT become elevated whenever disease processes affect liver cell integrity, ALT is the more liver-specific enzyme. Moreover, elevations of ALT activity persist longer than elevations of AST activity. In patients with vitamin B6 deficiency, serum aminotransferase activity may be decreased. The apparent reduction in aminotransferase activity may be related to decreased pyridoxal phosphate, the prosthetic group for aminotransferases, resulting in an increase in the ratio of apoenzyme to holoenzyme.

Aspartate aminotransferase (AST) is widely distributed in tissue, principally hepatic, cardiac, muscle, and kidney. Elevated serum levels are found in diseases involving these tissues. Hepatobiliary diseases, such as cirrhosis, metastatic carcinoma, and viral hepatitis also increase serum AST levels. Following myocardial infarction, serum AST is elevated and reaches a peak two days after onset. Two isoenzymes of AST have been detected, cytoplasmic and mitochondrial. Only the cytoplasmic isoenzyme occurs in normal serum, while the mitochondrial, together with the cytoplasmic isoenzyme, has been detected in the serum of patients with coronary and hepatobiliary disease. The addition of pyridoxal phosphate to the assay causes an increase in aminotransferase activity. The activation is higher for AST than for ALT. Pyridoxal phosphate activation prevents falsely low aminotransferase activity in patient samples with insufficient endogenous pyridoxal phosphate (vitamin B6 deficiency).

Gamma‑glutamyltransferase is used in the diagnosis and monitoring of hepatobiliary diseases. Enzymatic activity of GGT is often the only parameter with increased values when testing for such diseases, and is one of the most sensitive indicators known. Gamma‑glutamyltransferase is also a sensitive screening test for occult alcoholism. Elevated GGT activities are found in the serum of patients requiring long-term medication with phenobarbital and phenytoin. In 1969, Szasz published the first kinetic procedure for GGT in serum using γ‑glutamyl‑p‑nitroanilide as substrate and glycylglycine as acceptor. In order to circumvent the poor solubility of γ‑glutamyl-p‑nitroanilide, Persijn and van der Slik investigated various derivatives of the compound with respect to solubility. The substrate L‑γ‑glutamyl-3‑carboxy-4‑nitroanilide is superior in terms of stability and solubility. The assay described below uses the water-soluble substrate L‑γ‑glutamyl-3‑carboxy-4‑nitroanilide. The results correlate with those derived using the original substrate.

Lactate dehydrogenase LDH The lactate dehydrogenase (LDH) enzyme is widely distributed in tissue, particularly in the heart, liver, muscles and kidneys. The LDH in serum can be separated into five different isoenzymes based on their electrophoretic mobility. Each isoenzyme is a tetramer composed of two different subunits. These two subunits have been designated heart and muscle, based on their polypeptide chains. There are two homotetramers, LDH‑1 (heart) and LDH‑5 (muscle), and three hybrid isoenzymes. Elevated serum levels of LDH have been observed in a variety of disease states. The highest levels are seen in patients with megaloblastic anemia, disseminated carcinoma and shock. Moderate increases occur in muscular disorders, nephrotic syndrome and cirrhosis. Mild increases in LDH activity have been reported in cases of myocardial or pulmonary infarction, leukemia, hemolytic anemia and non-viral hepatitis. This method is in accordance with the recommendations of the International Federation of Clinical Chemistry (IFCC).

Lipase LIP Lipases are glycoproteins with a molecular weight of 47,000 daltons. They are defined as triglyceride hydrolases which catalyze the breakdown of triglycerides into diglycerides with subsequent formation of monoglycerides and fatty acids. Pancreatic lipase, along with α-amylase, has undoubtedly been the most important clinical biochemistry parameter for the differential diagnosis of pancreatic diseases for many years. Determination of lipase activity is gaining increasing international recognition due to its high specificity and rapid response. In acute pancreatitis, lipase activity increases after 4-8 hours, reaches its maximum values ​​after 24 hours and decreases after 8-14 days. However, there is no correlation between the increase in lipase activity determined in serum and the degree of pancreatic damage. Numerous methods for measuring lipase have been described, based on turbidimetric or nephelometric determination of its decrease in the substrate or determination of degradation products. This method is based on the cleavage of a specific chromogenic substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin) lipase ester, emulsified with bile acids. The activity of pancreatic enzymes is determined specifically by the combination of bile acid and colipase used in this method. In fact, in the absence of colipase, lipase activity is not detectable. Colipase does not activate other forms of lipolytic enzymes in serum except pancreatic lipase. The high concentration of cholates ensures the absence of a reaction of the esterase in serum with the chromogenic substrate due to the high negative charge of the surface.

ALP Alkaline phosphatase in serum consists of four structural genotypes: the liver-bone-kidney type, the intestinal type, the placental type and the variant from the germ cells. It occurs in osteoblasts, hepatocytes, leukocytes, the kidneys, spleen, placenta, prostate and the small intestine. The liver-bonekidney type is particularly important. A rise in the alkaline phosphatase occurs with all forms of cholestasis, particularly with obstructive jaundice. It is also elevated in diseases of the skeletal system, such as Paget’s disease, hyperparathyroidism, rickets and osteomalacia, as well as with fractures and malignant tumors. A considerable rise in the alkaline phosphatase activity is sometimes seen in children and juveniles. It is caused by increased osteoblast activity following accelerated bone growth. The assay method was first described by King and Armstrong, modified by Ohmori, Bessey, Lowry and Brock and later improved by Hausamen et al. In 2011 the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C‑RSE) recommended a reference procedure for the determination of alkaline phosphatase using an optimized substrate concentration and 2‑amino-2-methyl-1-propanol as buffer plus the cations magnesium and zinc at 37 °C. This assay follows the recommendations of the IFCC, but was optimized for performance and stability.