Tests at central laboratory I – L

Purpose and scope

Inhibins are polypeptide hormones. They selectively suppress the secretion of pituitary follicle stimulating hormone (FSH). The fully processed form of the inhibin molecule consists of 2 distinct chains, α and β, linked by disulfide bridges. Inhibin B comprises an α subunit and a βB subunit. It is produced by the Sertoli cells of the testis in men, and the granulosa cells of the ovary in women. Its primary role appears to be in the regulation of gametogenesis via negative feedback on the production of FSH. Measurement of Inhibin B is used for monitoring male and female gonadal function. In clinical trials, we measure Inhibin B when the investigational medicinal product (IMP) might affect blood concentrations of Inhibin B.

Method

Enzyme linked immunoassay (EIA, ELISA)

Sample requirements

Serum. Store sample at 2–8°C for up to 24 h, or at –20°C for up to 30 days.

Maximum turnaround time

Batched

Purpose and scope

Insulin is a protein synthesised by the β-cells of the islets of Langerhans in the pancreas. It is an anabolic hormone that stimulates the uptake of glucose by fat and muscle cells, and promotes the conversion of glucose into glycogen in the liver. Assay of plasma or serum insulin is done mainly in studies of drugs that are designed to increase insulin production by the pancreas, or to improve insulin sensitivity.

Method

Simultaneous one-step immunoenzymatic ‘sandwich’ assay

Sample requirements

Serum or EDTA plasma. Samples are stable for 8 h at room temperature, 48 h at 2–8°C, or for 6 months at –20°C.

Maximum turnaround time

24 h

Alternative test name

Fe

Purpose and scope

Iron is required for the synthesis of the iron-porphyrin proteins haemoglobin, myoglobin, cytochromes, and cytochrome oxidase. Iron is carried in the blood bound to the plasma protein transferrin, and is stored in the tissues as ferritin, an iron protein containing ferric hydroxide and ferric phosphate. Ferritin is found predominantly in the cells of the liver, spleen, and bone marrow. Iron measured in serum is mainly Fe(III) bound to serum transferrin: the iron contained in free haemoglobin is not detected.
Iron is not excreted in the urine, but is passed from the body in bile and faeces, and in menstrual blood. Iron deficiency causes an anaemia in which the number of red blood cells is normal, but the amount of haemoglobin in each cell is low. Greater than normal concentrations of serum iron occur in iron-overload disorders, such as haemochromatosis, and in acute iron poisoning. Iron concentrations may also be increased in acute hepatitis, lead poisoning, acute leukaemia, thalassaemia, or in women using oral contraception.
In clinical trials, measurement of serum iron can aid in the diagnosis of anaemia, iron deficiency, and haemochromatosis. Thus it is useful mainly as a screening test.

Method

Enzymatic reaction rate

Sample requirements

Serum or heparinised plasma. Iron is stable in serum and plasma for 3 weeks when stored at 2–8°C, and for 7 days when stored at 15–25°C.

Maximum turnaround time

24 h

Alternative test name

LDH, EC 1.1.1.27

Purpose and scope

Lactate dehydrogenase is a hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to pyruvate. There are 5 isoenzymes, LDH 1 to 5, which are widely distributed around the body. There are various causes of an elevated LDH and they include: artefactual owing to haemolysis in vitro or delayed separation of plasma from cells; diseases such as myocardial infarction, haemolytic anaemia, acute viral hepatitis, cirrhosis, and metastatic carcinoma of the liver, tumors of the lung or kidneys and skeletal muscle disease. In clinical trials of new medicines, a raised LDH is most likely due to artefact, or drug-induced haemolysis (with an associated decrease in haemoglobin).

Method

Enzymatic rate method based on the International Federation of Clinical Chemistry (IFCC) standard for enzyme determination

Sample requirements

Serum or heparinised plasma. Centrifuge the samples within two hours of collection. LDH is stable for 2 days at room temperature. Refrigeration or freezing of samples is not recommended.

Maximum turnaround time

24 h

Purpose and scope

Concentration of circulating leptin, an adipocyte hormone, reflects the amount of energy stored in adipose tissue, and is considered a marker of nutritional status. Leptin is bound to the soluble leptin receptor (sOB-R) in the circulation, which modulates steady-state leptin levels by protecting the hormone from degradation and clearance, and the sOB-R:leptin ratio may be used as a marker of bioavailable leptin.
In clinical pharmacology trials, leptin is used as a marker of the action of drugs that might affect appetite, adipocyte function, or body weight.

Method

Solid-phase, enzyme-linked, immunosorbent assay

Sample requirements

Serum. Store serum at 2–8°C for up to 24 h, or at –20°C for up to 6 months, before assay.

Maximum turnaround time

Batched

Purpose and scope

Measurement of circulating lipase activity is used mainly in the diagnosis of pancreatitis. Lipase is an enzyme produced in the acinar cells of the pancreas, and is responsible for the hydrolysis of water-insoluble long-chain fatty acid esters of glycerol.
Serum lipase may be increased in acute pancreatitis, acute episodes of chronic pancreatitis, and obstructive pancreatitis. Levels of up to 80 times the upper reference limit may be found in serious acute inflammation; on the other hand, complete absence of lipase can occur in congenital disorders. Lipase activity is reduced in chronic pancreatitis, owing to destruction of acinar cells. In acute upper quadrant abdominal syndrome, serum lipase activity may increase up to 5-fold. In clinical trials of new medicines, lipase serves as a marker for drug-induced pancreatitis.

Method

Enzymatic reaction based on the methodology of Panteghini

Sample requirements

Serum or heparinised plasma (lithium or sodium). Centrifuge the samples within 2 hours of collection. It is stable for 1 week at room temperature, or 3 weeks at 2–8ºC. Sample can be frozen at –20°C for up to 5 months for longer storage. Once thawed, do not refreeze.

Maximum turnaround time

24 h

Alternative test name

HDL-Cholesterol, HDL

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. Lipoproteins are classified according to their physical properties, including their hydrated densities, as: very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) or high-density lipoprotein (HDL).
HDL comprises 20–30% of total plasma cholesterol, and is capable of transporting cholesterol from the periphery to the liver (reverse cholesterol transfer pathway). Inert, disc-shaped, HDL particles from the liver are converted by the enzyme lecithin-cholesterol acyltransferase (LCAT) to an active, spherical form that transports cholesterol esters to the liver. The HDL particles bind to the liver cell via apoprotein E, releasing cholesterol for reuse by the liver.
HDL particles protect against atherosclerosis. Disorders of lipoprotein metabolism that lead to atherosclerosis may be related to decreased production of HDL-cholesterol (eg familial hypoalpha-lipoproteinemia), abnormal enzyme processing (eg Tangier’s disease), or defective cellular uptake of HDL. In clinical practice, HDL-cholesterol levels are measured to follow an individual’s risk of coronary heart disease (CHD), or response to treatment. In early clinical trials, HDL-cholesterol is used to screen for individuals with disorders of cholesterol metabolism, and to identify those at high risk of developing CHD.

Method

Timed-endpoint method

Sample requirements

Serum, heparinised plasma or 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 up to 3 months. Once thawed, do not refreeze.

Maximum turnaround time

24 h

Alternative test name

LDL cholesterol, LDL

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. Lipoproteins are classified, according to their physical properties, including their hydrated densities as: very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) or high-density lipoprotein (HDL).
LDL particles are the main carriers of cholesterol; they deliver it to both the liver and peripheral cells. The surface of an LDL particle contains embedded proteins called apoprotein B100 and apoprotein E. Those apoproteins are ligands for the LDL receptors on liver cells. After binding to the receptor, the LDL particle is internalised in the liver cell as a coated vesicle, which is degraded by cell liposomes, releasing the cholesterol.
The number of LDL receptors regulates the circulating LDL concentration. The circulating LDL concentration is also regulated by the activity of the rate- limiting enzyme in the cholesterol synthetic pathway (HMG Co-A). Cells regulate their cholesterol content by the following negative feedback mechanism: oversupply of free cholesterol leads to decreased rate of cholesterol synthesis by inhibiting HMG-CoA, and inhibiting synthesis of new LDL receptors.
However, LDL can also be taken up by non-receptor mediated mechanisms that are not regulated by the cell and so cannot be saturated. Those become significant as plasma LDL concentrations increase, as in familial hypercholesterolaemia. Scavenger cells, eg macrophages become engorged with LDL-cholesterol esters becoming ‘foam cells’ that act as precursors to atherosclerosis, a major cause of coronary heart disease.
Increased LDL-cholesterol, and decreased HDL-cholesterol, are associated with increased risk of coronary heart disease (CHD). More than half of all patients under 60 with confirmed CHD have a disorder of lipoprotein metabolism affecting apoprotein production, LDL receptor function, or enzyme function. In clinical practice, measurement of LDL is used to follow a patient’s response to cholesterol-lowering treatment. In early clinical trials, LDL-cholesterol is measured to screen for individuals with disorders of cholesterol metabolism who are at risk of CHD. Rarely, LDL-cholesterol is measured to assess the effect of novel lipid lowering drugs. A low HDL/LDL ratio is directly related to the risk of developing coronary artery disease (CAD). Elevated LDL cholesterol is the primary target of cholesterol-lowering therapy.

Method

Timed-endpoint method

Sample requirements

Serum or plasma (heparinised or EDTA). Centrifuge the samples within 2 h of collection.  It is stable for 8 h at room temperature or 5 days at 2–8ºC. Samples can be frozen at –80°C for longer storage. Once thawed, do not refreeze.

Maximum turnaround time

24 h

Alternative test name

LH

Purpose and scope

The pituitary gonadotrophins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), control the function and secretion of oestrogens (oestradiol being the most important ovarian oestrogen), progesterone, and testosterone. In addition, LH and FSH regulate the development of ovarian follicles in women, and FSH stimulates spermatogenesis in men. LH and FSH secretion is in turn regulated by the hypothalamic gonadotrophin releasing hormone (GnRH). In the woman, higher brain centres impose a 28-day menstrual cycle on the activity of GnRH.
In the pre-menopausal woman, the menstrual cycle has 3 phases: follicular, ovulation, and luteal. During the follicular phase, ovarian follicles are stimulated by FSH to develop: FSH first rises, then falls; LH remains low. Under the influence of LH and FSH, oestrogen concentrations rise steadily; progesterone remains low. At first there is negative feedback on the pituitary by oestrogen, inhibiting LH and FSH secretion. Later there is increased GnRH secretion, and increased LH sensitivity to GnRH (positive feedback), which leads to the mid-cycle surge of LH, inducing ovulation of the lead follicle. During the luteal phase, the follicle differentiates into a corpus luteum which secretes both progesterone and oestradiol. Those hormones then stimulate endometrial proliferation in preparation for implantation. If implantation does not occur, the corpus luteum regresses and the hormones decrease. If implantation and pregnancy do occur, the corpus luteum produces human chorionic gonadotrophin (HCG) which maintains the production of oestrogen and progesterone by the corpus luteum, until the placenta can make enough oestrogen and progesterone.
After the menopause, oestrogen concentrations decline, and, owing to the loss of the negative feedback, LH and FSH concentrations increase.
The interpretation of LH in women must make allowance for the phase of the menstrual cycle, and whether the woman is pre- or post-menopausal. LH surges during ovulation. After the menopause, LH rises as oestradiol concentrations fall.
In men, pulses of GnRH stimulate FSH and LH secretion from the pituitary. FSH stimulates the Sertoli cells in the seminiferous tubules of the testis to produce mature sperm. LH stimulates testosterone production from Leydig cells in the testis. Testosterone aids spermatogenesis, and is responsible for the male secondary sexual characteristics. It is also an anabolic hormone. Testosterone inhibits pituitary GnRH secretion.
LH secretion is different for the 2 sexes, but is required for normal sexual function. It occurs in pulses, with rapid fluctuations over the entire reference range. Values obtained in a single day from the same patient may therefore vary widely.
In clinical trials of new medicines, LH is not routinely measured. LH may be measured when the study medicine is expected to affect blood concentrations of LH. Also, LH, FSH and oestradiol are measured to confirm that a woman is post-menopausal.

Method

2-step immunoenzymatic ‘sandwich’ assay

Sample requirements

For analysis, use serum or plasma (heparin). Samples are stable for 8 h at room temperature, 48 h at 2–8°C and 6 months at –20°C.

Maximum turnaround time

24 h