Welcome to
HMR’s Central
Laboratory
Clinical laboratory services
in London, UK
Contract laboratory at
Hammersmith Medicines Research
Welcome to
HMR’s Central
Laboratory
Clinical laboratory services
in London, UK
Contract laboratory at
Hammersmith Medicines Research
Tests at central laboratory A – D
If the test you required does not appear on our list, we can set-up and validate the method at your request.
Alternative test name
APTT
Purpose and scope
Activated partial thromboplastin time (APTT) reflects the integrity of the blood coagulation system, and measures factors VIII, IX, XI and XII, as well as factors X, V, prothrombin and fibrinogen. Drugs, and deficiencies and abnormalities of coagulation factors, prolong APTT. In clinical trials of new medicines, APTT serves as a marker for an effect on blood coagulation. In some studies, that effect might be intended.
Method
Absorbance
Sample requirements
109 mmol/L buffered sodium citrate plasma with a 9:1 ratio of blood to citrate. Store at –40°C for up to 2 months.
Maximum turnaround time
24 h
Alternative test name
APC, APCR
Purpose and scope
Protein C is a plasma protein involved in the regulation of blood coagulation. Activated protein C (APC), with free protein S as a cofactor, indirectly inhibits thrombin production. Activated protein C resistance (APCR) is the most common inherited condition associated with increased risk of thrombosis. Also, in clinical trials of new medicines, the degree of APCR may serve as a marker of effects on blood coagulation.
Method
Chromogenic
Sample requirements
109 mmol/L buffered sodium citrate plasma with a 9:1 ratio of blood to citrate For long-term storage, keep samples at –40°C for a maximum of 2 months.
Maximum turnaround time
24 h
Purpose and scope
Adiponectin is a protein hormone that is secreted mainly by adipose cells. It belongs to the soluble defence collagen superfamily, and has a collagen-like domain. Adiponectin is an adipokine that modulates many metabolic processes, including glucose regulation and fatty acid catabolism.
Paradoxically, concentrations of adiponectin in adipose tissue are inversely related to the body mass index. Reduced concentrations of adiponectin in serum are associated with in insulin-resistant states (eg obesity and type 2 diabetes mellitus), and with coronary artery disease. Increased concentrations in serum are associated with type 1 diabetes mellitus, anorexia and chronic renal failure.
Assay of adiponectin is used in clinical practice to help identify patients with type 1 or type 2 diabetes mellitus, obesity, anorexia nervosa and coronary artery disease.
In clinical trials of new medicines, adiponectin serum levels may help indicate the efficacy of medicines designed to treat blood lipids and diabetic disorders.
Method
Competitive enzyme linked immunoassay (EIA, ELISA) (EIA)
Sample requirements
Serum or plasma samples. Samples are stable for 24 h at room temperature, for 1 week at 2–8°C and for 1 year at –80°C.
Maximum turnaround time
Batched
Alternative test name
ACTH
Purpose and scope
Adrenocorticotrophic hormone (ACTH) is a polypeptide hormone produced in the anterior pituitary. ACTH stimulates steroid production by the adrenal cortex. ACTH secretion is in turn controlled by the hypothalamic hormone corticotrophin releasing factor (CRF) and by negative feedback from cortisol.
Circulating ACTH concentrations are valuable in the differential diagnosis of adrenal insufficiency and hypersecretion. Low ACTH concentrations are usually due to a non-ACTH-secreting pituitary tumour, to pituitary infarction, or to excess steroid secretion by the adrenal cortex. For example, assay of ACTH helps to identify the cause of cortisol hypersecretion in Cushing’s syndrome. ACTH concentrations are typically high when Cushing’s syndrome is due to ectopic ACTH production or hypersecretion of ACTH by the pituitary. In Addison’s disease (primary adrenal insufficiency), high ACTH concentrations are usual.
Plasma concentrations of ACTH show significant diurnal variation. It is important, therefore, to standardise the time of collection: reference ranges are typically established from blood samples taken at about 0800–0900 h. Blood samples should therefore be taken at about that time.
In clinical trials of new medicines, circulating ACTH concentration serves mainly as a marker of adrenal suppression by steroid medicines.
Method
2-site sequential chemiluminescent immunometric assay
Sample requirements
EDTA plasma. Collect blood into iced EDTA tubes. Keep samples in an ice bath at or below 4°C at all times after collection.
Maximum turnaround time
24 h
Alternative test name
ALT, GPT, SGPT, EC 2.6.1.2
Purpose and scope
Normally, almost all serum alanine aminotransferase is intracellular. It is released into the circulation after any injury to physiologically active cells in the heart (infarction, carditis, surgery), liver (infective hepatitis, cholangitis, alcohol debauch, poisons, drugs, infectious mononucleosis), lung (infarction), pancreas (pancreatitis), skeletal muscle (trauma, exercise). Equilibration between plasma and interstitial fluid takes 6–18 h.
In clinical trials of new medicines, ALT mainly serves as a test of liver function. An increase in ALT and other tests of liver function, such as serum bilirubin, aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT), might represent drug-induced liver dysfunction. ALT is usually regarded as abnormal only if raised by at least 1.5 or 2 times the upper limit of normal. Results should be interpreted with caution because of the known increases in ALT, AST, and GGT in healthy subjects who are resident and given placebo (Rosenzweig et al 1999; Narjes and Nehmiz, 2000 Mertz et al 1997). The increase in ALT is usually greater than the increase in AST. One possible cause of that increase is an imbalance between reduced physical activity and calorie intake. The composition of the diet may also play a role. Excess calories and a high carbohydrate intake (Porikos and Van Itallie, 1983; Purkins et al 1997) cause increased enzyme levels.
Method
Enzymatic rate method
Sample requirements
Serum or plasma (heparinised or EDTA). Centrifuge the samples within two hours from the time of collection. If assay is not completed within 8 hours, serum or plasma can be stored at 2–8ºC for 48 hours or frozen at –20ºC for longer storage. Thaw frozen samples only once.
Maximum turnaround time
24h
Purpose and scope
Albumin is a small protein synthesized mainly by the liver. Its main function is to maintain the colloidal osmotic pressure in both the vascular and extra-vascular spaces. Albumin also binds and transports a large number of compounds, including hormones, phospholipids, and drugs. Increased levels of albumin are only seen in acute dehydration and are not clinically significant. Decreased levels are caused by a number of conditions including inflammation, and diseases of the liver and kidney. In clinical trials of new medicines, albumin is usually a marker for underlying disease in subjects.
Method
Timed-endpoint method
Sample requirements
Serum or heparinised and EDTA plasma. Centrifuge the samples within two hours from the time of collection. If assay is not completed within 8 hours, serum or plasma can be stored at 2–8ºC for 48 hours or frozen at –20°C for longer storage. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Purpose and scope
Aldolase is an enzyme in the glycolytic pathway that converts fructose 1,6 diphosphate (F-1,6-DP) to glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DAP).
In clinical trials, aldolase may be measured as a marker of heart muscle damage.
Method
Enzymatic reaction rate
Sample requirements
Serum
Maximum turnaround time
24 h
Purpose and scope
Aldosterone is a steroid hormone secreted by the adrenal cortex. Aldosterone is the principal mineralocorticoid that controls sodium and potassium balance and, consequently, the circulating blood volume.
Aldosterone is synthesized from cholesterol in the zona glomerulosa of the adrenal cortex. The renin-angiotensin system is the main regulator of aldosterone secretion. Angiotensin II and potassium stimulate secretion of aldosterone by increasing the synthesis of the hormone. Aldosterone acts on the renal tubule, causing them to reabsorb more sodium and water from the glomerular filtrate. Potassium is secreted into the tubule in exchange for sodium, which is reabsorbed. Aldosterone also acts on the central nervous system to increase thirst, and appetite for salt. Abnormally high levels of aldosterone cause hypertension, sodium retention, high blood pressure, and, via hypokalaemia, heart rhythm irregularities, and muscle weakness. In conjunction with plasma renin concentrations, aldosterone concentrations can be used to differentiate between primary and secondary aldosteronism. In clinical trials of new medicines, aldosterone serves as a measure of pharmacodynamic effects on the renin-angiotensin system.
Method
Competitive, solid-phase, enzyme linked immunoassay (EIA, ELISA)
Sample requirements
Serum. Store serum at –20°C for up to 6 months, before assay
Maximum turnaround time
Batched
Alternative test name
ALP, EC 3.1.3.1
Purpose and scope
An enzyme made in the liver, bone, and the placenta and normally present in high concentrations in growing bone and in bile. Alkaline phosphatase is released into the blood during injury and during such normal activities as bone growth and pregnancy. The enzyme is termed alkaline phosphatase because it works under alkaline conditions.
Alkaline phosphatase is raised in patients with hepatobiliary disorders and bone disease associated with increased osteoblastic activity. ALP is also raised in the third trimester of pregnancy and in children and young adults due to bone growth. In clinical trials of new medicines, ALP mainly serves as a test of liver function. An increase in ALP and other tests of liver function, such as serum bilirubin and transaminases, might represent drug-induced liver dysfunction. ALP is usually regarded as abnormal only if raised by at least 1.5 or 2 times the upper limit of normal.
Method
Kinetic rate method
Sample requirements
Serum or heparinised plasma. Centrifuge the samples within two hours from the time of collection. It is stable for 4 days at room temperature, 2–8ºC or at –20°C. Serum ALP activity increases slowly with storage. Hence it is recommended to analyse samples on day of collection.
Maximum turnaround time
24 h
Alternative test name
BAP, EC 3.1.3.1
Purpose and scope
Bone is a dynamic tissue in which bone formation and bone resorption continue throughout life. In the metabolism of bone, osteoblasts are involved in bone formation, and osteoclasts are involved in resorption. Serum levels of bone alkaline phosphatase (BAP) reflect the metabolic status of osteoblasts. BAP comprises about 50% of circulating alkaline phosphatase (ALP). The measurement of BAP is useful in patients with various forms of metabolic bone disease, in determining the degree of bone turnover, and in monitoring disease activity and therapy.
In clinical trials of new medicines, BAP serves as a measure of pharmacodynamic effects on bone metabolism.
Method
Enzyme linked immunoassay (EIA, ELISA)
Sample requirements
Serum. Bone Alkaline Phosphatase is stable in sample for 48h at 2–8°C. Store at –20°C for upto 2 months.
Maximum turnaround time
Batched
Alternative test name
EC 3.2.1.1
Purpose and scope
Amylase is a hydrolytic enzyme that catalyses the breakdown of 1,4-α-glucosidic linkages in polysaccharides. It is produced mainly by the acinar cells of the pancreas, and also by the salivary glands. Clinically, it is used as a measure of damage to the exocrine pancreas, resulting in pancreatitis. Drugs are a cause of pancreatitis, and therefore assays of plasma amylase are done as a laboratory safety test in studies of drugs that might be toxic to the pancreas, eg if animal studies show evidence of pancreatic toxicity.
Method
Enzymatic rate method
Sample requirements
Serum or heparinised plasma. Centrifuge the samples within 2 hours of collection. It is stable for 7 days at room temperature or 30 days at 2–8ºC. Freezing of the sample is not recommended.
Maximum turnaround time
24 h
Alternative test name
Anti-HAV (IgM)
Purpose and scope
Hepatitis A virus is the most common cause of hepatitis in the UK. The virus is a small, non-enveloped, relatively heat-stable, ribonucleic acid (RNA) virus, and is classified with the enterovirus group of the Picornaviridae family. It is usually spread by hand-to-mouth contact with stool of an infected person. Illness caused by HAV is characterised by fever, nausea, abdominal pain, loss of appetite, and jaundice. Those symptoms appear 10–14 days after ingesting contaminated food or water. However, many HAV infections are asymptomatic.
HAV infection is diagnosed by detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to HAV in human serum. Anti-HAV IgM appears in the acute stage of infection, or in the early convalescent stage. Anti-HAV IgG, which appears shortly after IgM, appears during the course of infection, remains detectable for a person’s lifetime, and confers lifelong protection against the disease.
The anti-HAV IgG test is used to assess immune status in naturally infected and in vaccinated individuals, and in epidemiological studies.
Method
2-site, sequential, chemiluminescent immunometric method
Sample requirements
Serum or plasma (heparinised, sodium citrate or EDTA), samples are stable for 3 days at 2–8°C, or up to 1 year at –20°C.
Maximum turnaround time
24 h
Alternative test name
Total anti-HAV
Purpose and scope
Hepatitis A virus is the most common cause of hepatitis in the UK. The virus is a small, non-enveloped, relatively heat-stable, ribonucleic acid (RNA) virus, and is classified with the enterovirus group of the Picornaviridae family. It is usually spread by hand-to-mouth contact with stool of an infected person. Illness caused by HAV is characterised by fever, nausea, abdominal pain, loss of appetite, and jaundice. Those symptoms appear 10–14 days after ingesting contaminated food or water. However, many HAV infections are asymptomatic.
HAV infection is diagnosed by detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies to HAV in human serum. Anti-HAV IgM appears in the acute stage of infection, or in the early convalescent stage. Anti-HAV IgG, which appears shortly after IgM, appears during the course of infection, remains detectable for a person’s lifetime, and confers lifelong protection against the disease.
Total anti-HAV is useful in determining the immune status of a patient. A reactive total anti-HAV (IgG and IgM) test in serum or plasma indicates past infection, or immunity induced by vaccine.
In research studies, the test may be used to assess the efficacy of vaccines, and the prevalence of immunity in selected populations.
Method
Sequential, competitive, chemiluminescent immunometric method
Sample requirements
Serum or plasma (heparinised, sodium citrate or EDTA), samples are stable for 3 days at 2–8°C or up to 1 year at –20°C.
Maximum turnaround time
24 h
Purpose and scope
Antithrombin inhibits nearly all the reactions of the blood coagulation cascade. When bound to heparin, antithrombin is one of the most important natural anticoagulants. There are 2 types of antithrombin deficiency: type I and type II. In type I, there is insufficient antithrombin to inactivate the coagulation factors. In type II, the level of antithrombin in the blood is normal, but it does not function properly. In clinical trials of new medicines, antithrombin deficiency serves as a marker of effects on blood coagulation.
Method
Chromogenic
Sample requirements
109 mmol/L buffered sodium citrate plasma with a 9:1 ratio of blood to citrate. Add the sample exactly to the line marked on the tube; under- or overfilled-tubes may give erroneous results.
Centrifuge samples for 15 min at 2000G, and test within 2 h of collection, or as specified by study protocols. For long-term storage, keep samples at –40°C for a maximum of 2 months. Thaw frozen specimens to 37°C for at least 15 min; after thawing, do the assay within 2 h.
Maximum turnaround time
Alternative test name
Apo A1
Purpose and scope
Apolipoprotein A is the main form of protein in high density lipoproteins (HDL). Apolipoprotein A1 (Apo A1) activates lecithin cholesterol acyltransferase, leading to removal of free cholesterol from extra-hepatic tissues. Apo A1 is non-atherogenic: there is an inverse relationship between Apo A1 concentration in the blood, and the risk of coronary artery disease.
In clinical trials of new medicines, Apo A1 serves as a marker of drug effects on lipid metabolism.
Method
Enzymatic reaction rate
Sample requirements
Serum. Sample is stable at 15–25°C for up to 24 h, or at 2–8°C for up to 8 days.
Maximum turnaround time
24 h
Alternative test name
Apo B
Purpose and scope
Apolipoprotein B (Apo B) is the main form of protein in low density lipoproteins (LDL). There are 2 forms of human Apo B: B-100 (the most common), which is the Apo B in lipoproteins synthesised in the liver; and B-48, which is probably synthesised in the intestinal wall. Apo B is the main cholesterol-carrying protein in the blood, and is the ligand involved in the uptake of cholesterol into cells by the LDL-receptor pathway. Patients with coronary artery disease usually have increased blood concentrations of Apo B.
In clinical trials of new medicines, Apo B serves as a marker of drug effects on lipid metabolism.
Method
Enzymatic reaction rate
Sample requirements
Serum. Sample is stable for 24 h if stored at 15–25°C, or for 8 days if stored at 2–8°C.
Maximum turnaround time
24 h
Alternative test name
Apo C-III
Purpose and scope
Lipoproteins are large complexes of molecules that transport lipids, mainly triglycerides and cholesterol, in blood. Apolipoproteins (Apo) are proteins on the surface of the lipoprotein complex that bind to specific enzymes or transport proteins on the cell membrane. That directs the lipoprotein to the proper site of metabolism.
Apo C-III is a 79-amino-acid protein synthesised mainly in the liver and, to a lesser extent, in the intestine. It circulates in the plasma in association with triglyceride-rich lipoproteins and HDL. Apo C-III modulates the uptake of triglyceride-rich lipoproteins by the LDL receptor-related protein, through inhibition of lipoprotein lipase. Elevated concentrations of Apo C-III are associated with both primary and secondary hypertriglyceridaemia. High concentrations of Apo C-III occur in many conditions, including Type 2 diabetes, hyperbilirubinaemia, renal failure, and decreased thyroid function. In clinical trials of new medicines, Apo C-III serves as a marker of drug effects on lipid metabolism.
Method
Enzymatic reaction rate
Sample requirements
Serum or plasma. Use only the following anticoagulants: sodium heparin; or disodium or dipotassium EDTA.
Maximum turnaround time
24 h
Alternative test name
Apo E
Purpose and scope
Apolipoproteins are proteins on the surface of the lipoprotein complex. They bind to specific enzymes or transport proteins on the cell membranes, to direct the lipoproteins to the proper site of metabolism. Apolipoprotein E (Apo E) is a 299 amino acid protein, synthesised mainly in the liver and, to a lesser extent, in the brain, spleen, lungs, ovaries, kidneys, muscle cells, and macrophages. Apo E has many roles, including transporting triglycerides to the liver (as part of very low-density lipoprotein (VLDL)), and distribution of cholesterol among cells (as part of high-density lipoprotein (HDL)). Apo E is a ligand for low-density lipoprotein (LDL), and mediates the receptor binding of Apo E lipoprotein to the LDL receptor protein.
Apo E has 3 common variants: apo E2, apo E3 and apo E4. Apo E3 is the most common, although the relative proportions of the 3 isoforms vary among populations. Apo E polymorphism influences the physiological and pathological processes, eg serum concentrations of cholesterol, LDL-cholesterol and Apo B. The polymorphism has been implicated in cardiovascular and neurodegenerative diseases, for example Alzheimer’s. Apo E is associated with type III hyperproteinemia, and does not bind the lipid receptor. Apo E deficiency causes high serum cholesterol and triglyceride concentrations, and leads to premature atherosclerosis. Apo E also affects the formation of atherosclerotic lesions, by inhibiting platelet aggregation.
The polymorphism also influences Apo E concentration, which directly affects the metabolic processes. Biological factors that affect Apo E concentration include the genetic polymorphism, oral contraceptive intake, puberty, and age. The reference range quoted is usually for the most common isoform. If the result falls outside that range, then the genotype should be determined.
In clinical trials of new medicines, Apo E serves as a marker of drug effects on lipid metabolism.
Method
Enzymatic reaction rate
Sample requirements
Serum
Maximum turnaround time
24 h
Alternative test name
AST, GOT, SGOT, EC 2.6.1.1
Purpose and scope
Normally almost all aspartate aminotransferase is intracellular. It is released into the circulation after any injury to physiologically active cells in the heart (infarction, carditis, surgery), liver (infective hepatitis, cholangitis, alcohol debauch, poisons, drugs, infectious mononucleosis), lung (infarction), pancreas (pancreatitis), skeletal muscle (trauma, exercise). Equilibration between plasma and interstitial fluid takes 6–18 hours.
In clinical trials of new medicines, AST mainly serves as a test of liver function. An increase in AST and other tests of liver function, such as serum bilirubin, alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT), might represent drug-induced liver dysfunction. AST is usually regarded as abnormal only if raised by at least 1.5 or 2 times the upper limit of normal. The increase in ALT is usually greater than the increase in AST. Results should be interpreted with caution because of the known increases in ALT, AST, and GGT in healthy subjects who are resident and given placebo (Rosenzweig et al 1999; Narjes and Nehmiz, 2000; Mertz et al 1997). One possible cause of that increase is an imbalance between reduced physical activity and calorie intake. The composition of the diet may also play a role. Excess calories and a high carbohydrate intake (Porikos and Van Itallie, 1983; Purkins et al 1997) cause increased enzyme levels.
Method
enzymatic reactions
Sample requirements
Serum or plasma (heparinised or EDTA). Centrifuge the samples within two hours from the time of collection. It is stable for 3 days at room temperature or 7 days at 2–8ºC. The sample can be frozen at –20ºC for up to 3 months for longer storage. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Alternative test name
Beta HCG, BHCG, bHCG, serum pregnancy test.
Purpose and scope
Pregnant woman are hardly ever recruited for clinical trials of new medicines, because of the risk of fetal abnormalities. To prevent recruitment of pregnant women, a serum pregnancy test can be done. bHCG is produced in increasing amounts by the placenta and corpus luteum after a ovum fertilised has implanted in the endometrium, and can be detected in serum 7 days after conception. A positive result indicates pregnancy, although trophoblastic tumours and some teratomas can also secrete bHCG.
Method
Two-site immunoenzymatic ‘sandwich’ assay
Sample requirements
Serum. The sample is stable for 8 h at room temperature, 48 h at 2–10ºC and for 6 months at –20ºC.
Maximum turnaround time
24 h
Alternative test name
CO2
Purpose and scope
Bicarbonate is one of the normal anions present in plasma; it is involved in buffering the pH of arterial blood within the range 7.35–7.45. Bicarbonate is in equilibrium with carbon dioxide, which is the final product of the metabolism of carbohydrate and fat. Disturbances of acid-base homeostasis, eg diabetic ketoacidosis and renal failure, can result in increased or decreased bicarbonate concentrations. In clinical trials of new medicines, bicarbonate usually serves as a marker of underlying disease. Medicines may disturb acid-base homeostasis (and hence bicarbonate concentration) directly, or indirectly, eg by causing renal damage.
Method
Enzymatic method
Sample requirements
Serum or plasma (heparinised or oxalate/fluoride). Centrifuge the samples within two hours of collection. If assay is not completed within 8 hours, store samples 2–8°C for 2 days. Samples can be frozen at –20°C for longer storage. Frozen samples should be thawed only once. Sample tubes should remain closed as much as possible.
Maximum turnaround time
24 h
Alternative test name
Direct bilirubin, conjugated bilirubin
Purpose and scope
When mature red blood cells are broken down in the reticuloendothelial system, haem is separated from globin and iron. Haem is metabolised to biliverdin, which is reduced to bilirubin. Bilirubin is also formed by the catabolism of other haem-containing proteins, eg myoglobin, cytochromes and catalases. Initially, bilirubin is unconjugated and water insoluble, and is transported to the liver attached to albumin. In the hepatic endoplasmic reticulum, bilirubin is conjugated with glucuronic acid by the microsomal enzyme uridine diphosphoglucuronyl transferase, and is excreted into the bile. Because conjugated bilirubin is water soluble, an excess may appear in urine.
Jaundice occurs when serum bilirubin is elevated. There are 3 types of jaundice: jaundice resulting from congenital hyperbilirubinaemias, haemolytic jaundice, and cholestatic jaundice. Dubin-Johnson and Rotor syndromes are 2 rare congenital hyperbilirubinaemias in which conjugated bilirubin is raised. Gilbert’s syndrome is a relatively common congenital hyperbilirubinaemia, in which unconjugated bilirubin is raised. It is usually detected during screening of healthy subjects.
Cholestatic jaundice, in which conjugated bilirubin is raised, can be intrahepatic or extrahepatic; drugs usually cause intrahepatic jaundice. In clinical trials of new medicines, conjugated bilirubin serves as a test of liver function. An increase in conjugated bilirubin and in other measures of liver function, such as the transaminases, may indicate drug-induced liver dysfunction.
Method
Timed-endpoint Diazo method
Sample requirements
Fresh haemolysis-free serum or plasma. Centrifuge the samples within two hours of collection. Bilirubin is light-sensitive; protect samples and controls from light. Serum or plasma samples are stable for 8 h at room temperature and 2 days at 2–8°C. The separated sample can be frozen at –20°C for up to 6 months. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Alternative test name
Total bilirubin
Purpose and scope
When mature red blood cells are broken down in the reticuloendothelial system, haem is separated from globin and iron. Haem is metabolised to biliverdin, which is reduced to bilirubin. Bilirubin is also formed by the catabolism of other haem-containing proteins, eg myoglobin, cytochromes and catalases. Initially, bilirubin is unconjugated and water insoluble, and is transported to the liver attached to albumin. In the hepatic endoplasmic reticulum, bilirubin is conjugated with glucuronic acid by the microsomal enzyme uridine diphosphoglucuronyl transferase, and is excreted into the bile. Because conjugated bilirubin is water soluble, an excess may appear in urine.
Jaundice occurs when serum bilirubin is elevated. There are 3 types of jaundice: jaundice resulting from congenital hyperbilirubinaemias, haemolytic jaundice, and cholestatic jaundice.
Drugs may cause haemolytic or cholestatic jaundice. In clinical trials of new medicines, direct bilirubin serves as a test of liver function. An increase in bilirubin and in other tests of liver function, such as the transaminases, may represent drug-induced liver dysfunction. Drugs can cause haemolysis, which can lead to elevated levels of (indirect) bilirubin, although the results of other liver tests are normal, and haptoglobulins are low.
Method
Timed-endpoint Diazo method
Sample requirements
Serum or plasma (lithium or sodium). Centrifuge the samples within 2 h from the time of collection. Samples are stable for 4 h at room temperature, or 3 days at 2–8ºC. Samples can be frozen at –20°C for up to 6 months. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Purpose and scope
Proinsulin is the major storage form of insulin. Proinsulin is cleaved to a 31-amino acid connecting C-peptide and insulin. C-peptide is devoid of biological activity, but is necessary to ensure the correct structure of insulin. C-peptide circulates in plasma at a level roughly 5 times that of insulin, even though the 2 molecules are secreted into the portal circulation in equimolar amounts. The liver does not excrete C-peptide, hence C-peptide assays are better indicators of β-cell function than peripheral insulin concentration. C-peptide levels are low where insulin secretion is diminished, and high as a result of increased β-cell activity. C-peptide determinations can be used in the differential diagnosis of hypoglycaemia. Circulating anti-insulin antibodies are commonly seen in patients who have had insulin therapy. These antibodies interfere with immunoassays for insulin. C-peptide measurements are used in such cases to indirectly monitor insulin secretion, and to help decide on an appropriate course of treatment. C-peptide measurement can also be used to assess glucose tolerance.
In clinical trials, C-peptide measurement is used to assess the effect of drugs on endogenous beta cell function or insulin secretion.
Method
chemiluminescent immunometric method
Sample requirements
Serum or heparinised plasma. Assay within 2–3 hours, or store frozen at –20°C for up to 1 week.
Maximum turnaround time
24 h
Alternative test name
CRP
Purpose and scope
C-reactive protein is one of the acute-phase proteins that are released during inflammation and tissue damage. CRP binds to pathogens, and to toxins released from damaged tissues, contributing to their elimination by the immune system. Plasma CRP concentration increases dramatically during the initial stages of inflammation or tissue damage. CRP is therefore an important marker in clinical trials of drugs that cause inflammation or tissue damage, and for monitoring the response of diseases to drugs.
Method
Highly sensitive near infrared particle immunoassay rate method
Sample requirements
Serum or plasma (EDTA, sodium heparin or lithium heparin). It is stable for 4 hours at room temperature or for 3 days at 2–8ºC. Sample can be frozen at –20°C for up to 6 months. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Alternative test name
Ca
Purpose and scope
Calcium is found mainly in the skeleton (99% of the total), with small amounts in soft tissues and extracellular fluid. Calcium measurements are used in the diagnosis and treatment of parathyroid disease, a variety of bone diseases, chronic renal disease and tetany (intermittent muscular contractions or spasms). In clinical practice, low blood concentrations of calcium may be due to low albumin, chronic renal failure, hypoparathyroidism or vitamin D deficiency. High blood concentrations of calcium may be due to primary hyperparathyroidism, malignancy (particularly with bone metastases), or to medicines such as lithium and thiazide diuretics. Urinary calcium measurement is used in the differential diagnosis of absorptive hypercalciuria and hypercalciuria caused by hyperparathyroidism and hyperthyroidism. In clinical trials of new medicines, calcium may be measured to confirm normal blood and urine levels in trials where an abnormal calcium may adversely affect a trial outcome, eg QTc prolongation. Calcium may also be measured when the trial medicine is expected to affect blood and urine levels of calcium.
Method
Indirect potentiometry
Sample requirements
Serum, heparinised plasma or urine. Centrifuge the samples within two hours of collection. It is stable for 7 days at room temperature or for 22 days at 2–8°C. Samples can be frozen at –20°C for up a year for longer storage. Frozen samples should be thawed only once.
For analysis of urine calcium, use a random or timed specimen. Perform the assay within 2 hours of collection. For timed specimens, keep the collection container in the refrigerator or on ice during the time period. To acidify the urine, add 10mL of 6N HCl to the container before collection begins.
Maximum turnaround time
24 h
Purpose and scope
Chloride is the commonest anion in plasma and is important in maintaining the osmotic pressure and electrical neutrality; as such chloride is also intimately involved, along with sodium, with maintaining blood volume and blood pressure. The kidneys and the intestine handle sodium and water in similar ways, and sodium and chloride levels usually mirror each other. In clinical practice the main value of chloride is in determining the anion gap in acid-base disturbances. In trials of new medicines, chloride is used as a marker for underlying disease in volunteers; it can also be useful as a marker for the use of medicines by volunteers, eg a low chloride might be seen in volunteers taking diuretics.
Method
Indirect potentiometry
Sample requirements
Serum or plasma (heparinised or oxalate/fluoride). Centrifuge the samples within two hours of collection. It is stable for 7 days at room temperature or for 4 weeks at 2–8°C. Samples can be frozen at –20°C for longer storage. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Purpose and scope
Fats are insoluble in water, and are transported in the blood stream as protein-lipid complexes (lipoproteins) that contain cholesterol, cholesterol esters and triglycerides. Cholesterol may be of dietary origin (animal products), but most is synthesized from acetyl-CoA in the liver, intestine, adrenal cortex, and skin. It either occurs as free cholesterol or is esterified with fatty acids; that reaction is catalysed by the enzyme lecithin-cholesterol acyltransferase (LCAT). The pathway for synthesis of endogenous cholesterol is a major target for therapeutic agents that suppress enzyme activity to lower cholesterol levels.
Cholesterol is a starting point for many metabolic pathways, including synthesis of cell membranes, vitamin D, steroid hormones, and bile acids. It is also a major factor in disease states, eg atherosclerosis, that lead to coronary heart disease. In chronic liver disease, cholesterol production is reduced. In early clinical trials of new medicines, serum cholesterol levels are measured to identify disorders of cholesterol metabolism, or raised cholesterol levels, which increases the risk of heart disease.
Method
Timed-endpoint enzymatic method
Sample requirements
Serum or heparinised plasma. Centrifuge the samples within 2 hours of collection. If assay is not completed within 8 hours, serum or plasma can be stored at 2–8ºC for 48 hours or frozen at –20°C for up to 3 months. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Alternative test name
Parathyroid secretory protein 1
Purpose and scope
Human chromogranin A (CgA) is an acidic protein consisting of 439 amino acids. It is encoded for by a gene located on chromosome 14.
CgA is found in some normal and some neoplastic neuroendocrine cells. It is co-secreted with a wide variety of peptide hormones and neurotransmitters. Plasma concentrations of CgA may be high in patients with neuroendocrine tumours. Determination of CgA in human plasma is a valuable diagnostic tool, and is used mainly in the diagnosis and monitoring of patients with tumours of neuroendocrine origin.
Non-malignant elevations of CgA may occur in kidney, liver or heart failure. CgA may also be elevated in patients experiencing stress, because CgA is co-stored and co-released with catecholamines from storage granules in the adrenal medulla.
Pancreastatin, a peptide hormone derived from CgA, inhibits gastric, exocrine pancreatic, and insulin secretions. In patients taking proton pump inhibitors (PPI), or other drugs that inhibit gastric acid secretion, circulating CgA levels have been shown to increase markedly. This is thought to be a result of the trophic effect that raised gastrin concentrations have on enterochromaffin-like cells.
CgA assays are useful in clinical trials of some medicines that alter plasma CgA concentrations, particularly those that affect gastric acid secretion.
Method
Simplified double antibody-sandwich assay. It measures human CgA concentrations in the range 5–40 U/L.
Sample requirements
Heparin or EDTA plasma samples. Separated plasma can be stored at 2–8°C, and used within 1 week of collection. If unable to assay within this period, store frozen at –20°C for up to 6 months.
Maximum turnaround time
Batched
Purpose and scope
Corticosterone is a glucocorticoid secreted by the cortex of the adrenal gland. It is produced in response to stimulation of the adrenal cortex by adrenocorticotrophic hormone (ACTH), and is the precursor of aldosterone. It is involved in metabolism of carbohydrate, protein and fat, and has anti-inflammatory and immunosuppressive properties. Corticosterone is a major indicator of stress, since stress increases the production of corticosteroids. It is also believed to play an important role in sleep-wake patterns.
In clinical trials of new medicines, corticosterone is measured to check the effect of the drug on the function of anterior pituitary, and adrenal cortex.
Method
Enzyme linked immunoassay (EIA, ELISA)
Sample requirements
Serum or EDTA plasma. Store samples at 2–8°C for up to 24 h, or at –20°C for up to 3 months.
Maximum turnaround time
Batched
Purpose and scope
Cortisol, which is produced in the fasciculata and reticularis zones of the adrenal glands, is the most important glucocorticoid steroid hormone. Corticotrophin-releasing factor (CRF) is secreted from the hypothalamus, and shows a circadian rhythm that may be disturbed by signals from the higher brain. CRF stimulates the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary; ACTH in turn stimulates the secretion of cortisol. Cortisol feeds back to the hypothalamus and pituitary, to inhibit the release of CRF and ACTH, respectively. Circulating cortisol concentrations are highest in the morning. Stress increases the secretion of CRF, and hence cortisol.
Cortisol has several functions, including: maintaining the extracellular fluid volume and normal blood pressure, stimulating gluconeogenesis, and promoting the breakdown of protein and fat. It can also decrease the immune response to infection, and increase the neutrophil count via demargination.
The causes of reduced cortisol concentrations include Addison’s disease, and other diseases of the adrenal glands; drugs are a rare cause. Raised levels of cortisol are rare, and are usually due to raised ACTH secretion, or to an adrenal tumour.
Cortisol bound to protein is protected from metabolism by the liver. Unbound (or free) cortisol in serum is metabolised by the liver, yielding a wide variety of metabolites. Many of the metabolites are water soluble and rapidly excreted in the urine. A small amount (< 100 μg/24 h) of cortisol, and other extractable metabolites, are also excreted in the urine.
Urine cortisol excretion reflects the amount of unbound serum cortisol, and may aid the diagnosis of adrenal hyperactivity.
In clinical trials of new medicines, cortisol is measured as a screening test (to confirm eligibility for a study), and as a marker of pharmacodynamic or toxic effect of the medicine.
Method
Competitive binding immunoenzymatic assay
Sample requirements
Serum, urine, heparinised plasma or EDTA plasma. Samples are stable for 8 h at room temperature, and for 48 h at 2–8°C. For longer storage, freeze at –20°C.
Maximum turnaround time
24 h
Purpose and scope
Cotinine is the major metabolite of nicotine. It is eliminated much more slowly than nicotine, and can be detected in urine several days after the last cigarette has been smoked. Urine cotinine is considered the most reliable biomarker in active smokers; it rarely produces false positives in passive smokers. Urine cotinine is usually normalised against urine creatinine, and is reported routinely as the cotinine:creatinine ratio. In many of our studies, we require that subjects be non-smokers, and the measurement of urine cotinine provides a more reliable screen of tobacco use than the Smokerlyser.
Method
Enzymatic reaction rate
Sample requirements
Random urine
Maximum turnaround time
24 h
Alternative test name
CPK, CK, Creatine phosphokinase, EC 2.7.3.2
Purpose and scope
Creatine kinase is an enzyme that catalyses the phosphorylation of creatine by adenosine triphosphate. Its activity is greatest in striated and heart muscle, and in brain tissue. In clinical practice, serum total CK may be elevated in a number of conditions that cause inflammation or injury to muscle, eg myocardial infarction, muscular dystrophy, or intramuscular injection. In healthy volunteers who participate in early clinical trials of new medicines, the total CK is often elevated to 2–3 times the upper limit of the reference range. That reflects both the increased muscle mass of these subjects, and exercise. Strenuous exercise may increase the serum total CK to more than 15 times the upper limit of the reference range. That change is often accompanied by a small rise in AST. Certain medicines, eg statins and peroxisome proliferator-activated receptor agonists, may cause myositis. Serial measurement of CK is useful in early trials of such medicines for possible early detection of these effects.
Method
Enzymatic reaction rate
Sample requirements
Serum or plasma (heparinised or EDTA). Centrifuge samples within two hours of collection. It is stable for 4 hours at room temperature or 12 hours at 2–8ºC. The sample can be frozen at –20ºC for up to 3 days only. Once thawed, do not refreeze.
Maximum turnaround time
24 h
Alternative test name
CK-MB
Purpose and scope
CK-MB is an isoform of creatine kinase (CK) – the principal enzyme of muscle metabolism. CK-MB is found mainly in cardiac muscle, and represents about 45% of total CK activity in the body. Prostate, spleen and skeletal muscle also contain significant amounts of CK-MB.
An increase in CK-MB is usually the result of damage to cardiac muscle. Circulating CK-MB concentration rises quickly after a myocardial infarction, reaches a peak after about 24 h, and returns to normal after 48–72 h.
In clinical trials of new medicines, CK-MB serves as a marker of cardiac muscle damage.
Method
2-step immunoenzymatic ‘sandwich’ assay
Sample requirements
Serum. Samples are stable for 8 h at room temperature, and for 48 h at 2–8°C.
Maximum turnaround time
24 h
Alternative test name
CRT
Purpose and scope
Creatinine is elevated in people with renal disease. Creatinine is produced endogenously; the amount produced is nearly constant, and is related to muscle mass. Creatinine excretion is by both glomerular filtration and tubular secretion, although at normal circulating concentrations, the latter is relatively small. In healthy subjects, there is enormous reserve in renal excretory function and serum creatinine does not rise above the normal range until there is a 50% fall in glomerular filtration rate (GFR). Therefore, in clinical trials of new medicines, serum creatinine concentrations alone are of limited value in monitoring the effects of the medicine on renal function. Production and excretion of creatinine are relatively constant; creatinine clearance is therefore a useful estimate of GFR. Creatinine clearance may be estimated from serum (or plasma) creatinine, age, weight and sex using the Cockcroft-Gault equation or measured from serum (or plasma) and urine creatinine concentrations and urine volume over 24 h, or other time periods. It is also a useful measurement in determining the completeness of urine collections, as a marker of whether a urine sample has been diluted and for calculation of creatinine clearance as a measure of renal function.
Method
Modified rate Jaffe procedure
Sample requirements
Serum, heparinised plasma or EDTA plasma. Centrifuge the samples within two hours of collection. It is stable for 5 days at room temperature or 1 month at 2–8ºC. The sample can be frozen at –20°C for longer storage. Once thawed, do not refreeze.
Urine assays should be performed within 2 hours of collection. For timed specimens, keep the collection container in the refrigerator or on ice during the time period.
Maximum turnaround time
24 h
Purpose and scope
Cysteine proteinase inhibitors, of the cystatins superfamily, have been identified in animals, plants and protozoa. Human cystatin C is produced at a constant rate by all nucleated body cells, and is abundant in all body fluids.
Serum cystatin C concentration correlates negatively with glomerular filtration rate (GFR) as well as or better than does creatinine, so has been proposed as a sensitive marker of changes in GFR.
In clinical pharmacology studies, we use serum concentrations of cystatin C as an alternative to creatinine in the estimation of GFR.
Method
Sandwich-enzyme immunoassay
Sample requirements
Serum. Store serum at 2–8°C for up to 72 h, or at –20°C for up to 6 months, before assay.
Maximum turnaround time
Batched
Purpose and scope
After vascular injury and the formation of the fibrin plug, fibrinolysis occurs in which fibrin is broken down by plasmin into fibrin degradation products, including
D-dimers. An increase in D-dimers reflects increased fibrinolysis, and therefore can also indirectly serve as a marker for increased blood coagulation. Drugs and underlying disease cause an increase in D-dimers. In clinical trials of new medicines, D-dimers serve as a marker for an effect on fibrinolysis. In some studies, that effect might be intended.
Method
Latex agglutination
Sample requirements
109 mmol/L buffered sodium citrate plasma with a 9:1 ratio of blood to citrate. For long-term storage, keep samples at –40°C for a maximum of 2 months.
Maximum turnaround time
24 h
Alternative test name
DHEA
Purpose and scope
Dehydroepiandrosterone (DHEA) is a hormone synthesized from cholesterol by the adrenal glands. It is a precursor of testosterone and oestrogen. Dehydroepiandrosterone sulphate (DHEAS) is the sulphated version of DHEA. The conversion of DHEA to DHEAS is catalyzed by sulfotransferase, mainly in the adrenals, liver and small intestine. DHEA has a rapid metabolic clearance rate, compared with DHEAS. Therefore, most DHEA in blood is in the form of as DHEAS, whose concentrations are about 300 times higher than those of unconjugated DHEA. Orally ingested DHEA is converted to its sulphate form during its passage through the intestine and liver. DHEA concentrations show significant diurnal variation, which is dependent on adrenocorticotrophic hormone (ACTH). Serum DHEA concentrations increase in response to exogenous ACTH.
The physiological role of DHEA has not been defined conclusively. DHEA may affect cholesterol and lipid metabolism, insulin sensitivity and secretion, and immune function. Abnormal DHEA concentrations have been reported in schizophrenia and obesity. Serum DHEA concentrations are high in the fetus and neonate, and low during childhood, then increase during puberty. Increased concentrations of DHEA during puberty may contribute to the development of secondary sexual hair. Serum DHEA concentrations progressively decline after the age of 30 years.
Serum DHEA is a useful marker of adrenal androgen synthesis. Abnormally low concentrations may occur in hypoadrenalism, and high concentrations occur in several conditions, including virilising adrenal adenoma and carcinoma, and in some cases of female hirsutism.
In clinical trials of new medicines, DHEA serves as a measure of pharmacodynamic effects on androgen metabolism.
Method
Enzyme linked immunoassay
Sample requirements
Serum or EDTA plasma separated from blood. Store samples at 2–8°C for up to 24 h, or at –20°C or lower for up to 2 months
Maximum turnaround time
Batched
Alternative test name
DHEA-SO4, DHEAS, DHS
Purpose and scope
Measurement of dehydroepiandrosterone sulphate (DHEA-SO4, DHEAS), an adrenal steroid, is important in the investigation of abnormal hair growth (hirsutism) and balding (alopecia) in women. It is also of value in the assessment of adrenarche and delayed puberty.
The DHEA-SO4 in the circulation originates almost entirely from the adrenals, though in men some may also derive from the testes, partly accounting for the sex difference which emerges at about age 15 years. On the other hand, the ovaries do not produce this hormone, even under pathological conditions. DHEA-SO4 is only weakly androgenic, but it can be metabolized to more potent androgens like androstenedione and testosterone, and thus be indirectly a cause of hirsutism or virilisation.
Plasma concentrations of DHEA-SO4 increase steadily from about the seventh year of life, then gradually decline after the third decade.
DHEA-SO4 is measured in trials of IMP that might affect sex hormone secretion.
Method
Competitive binding immunoenzymatic assay
Sample requirements
Serum or plasma (lithium or sodium heparin and EDTA). Samples are stable for 8 h at room temperature and 48 h at 2–8°C. Store samples at –20°C for longer storage.
Maximum turnaround time
24 h