Basis of Analysis of Body Fluids for Diagnostic, Prognostic and Monitoring Purposes
Underlying most human diseases is a change in the amount or function of one or more proteins that in turn triggers changes in cellular, tissue or organ function. The dysfunction is commonly characterised by a significant change in the biochemical profile of body fluids. The application of quantitative analytical biochemical tests to a large range of biological analytes in body fluids and tissues is a valuable aid to the diagnosis and management of the prevailing disease state.
Body fluids such as blood, cerebrospinal fluid and urine, in both healthy and dis eased states, contain a large number of inorganic ions and organic molecules. While some of these chemical species exert their normal biological function within that fluid, the majority of these take a passive role and are only transported by the fluid. The presence of this latter group of chemical species within the fluid is due to the fact that normal cellular secretory mechanisms and the temporal synthesis and turnover of individual cells and their organelles within the major organs of the body both result in the release of cell components into the surrounding extra-cellular fluid and eventually into the blood circulatory system. This, in turn, transports them to the main excretory organs, namely the liver, kidneys and lungs, so that these cell components and/or their degradation products are eventually excreted in faeces, urine, sweat and expired air. Examples of cell components in this category include enzymes, hormones, intermediary metabolites, small organic molecules and inorganic ions.
The concentration, amount or enzyme activity of a given cell component that can be detected in these fluids of a healthy individual at any point in time depends on many factors that can be classified into one of three categories, namely chemical characteristics of the component, endogenous factors characteristic of the individual and exogenous factors that are imposed on the individual. • Chemical characteristics: Some molecules are inherently unstable outside their normal cellular environment. For example, some enzymes are reliant on the presence of their substrate and/or coenzyme for stability and these may be absent or present only in low concentrations in the extra-cellular fluid. Molecules that can act as substrates of catabolic enzymes found in extra-cellular fluids, in particular blood, will also be quickly metabolised. Cell components that fall into these two categories therefore have a short half-life outside the cell and are normally present in low concentrations in fluids such as blood.
• Endogenous factors: These include age, gender, body mass and pregnancy. For example:
(a) Serum cholesterol concentrations are higher in men than pre-menopausal women, but the differences decrease post-menopause
(b) Serum alkaline phosphatase activity is higher in children than in adults and is raised in women during pregnancy
(c) Serum insulin and triglyceride concentrations are higher in obese individuals than in the lean
(d) Serum creatinine, a metabolic product of creatine important in muscle metabolism, is higher in individuals with a large muscle mass
(e) Serum sex hormone concentrations differ between males and females and change with age.
• Exogenous factors: These include time, exercise, food intake and stress. Several hormones are secreted in a time-related fashion. For example, cortisol and to a lesser extent thyroid-stimulating hormone ( TSH) and prolactin all show a diurnal rhythm in their secretion. In the case of cortisol, its secretion peaks around 9.00 am and declines during the day, reaching a trough between 11.00 pm and 5.00 am. The secretion of female sex hormones varies during a menstrual cycle and that of 25-hydroxycho lecalciferol ( vitamin D3 ) varies with the seasons, peaking during the late summer months. The concentrations of glucose, triglycerides and insulin in blood rise shortly after the intake of a meal. Stress, including that imposed by the process of taking a blood sample by puncturing a vein ( venipuncture), can stimulate the secretion of a number of hormones and neurotransmitters, including prolactin, cortisol, adrenocorticotropic hormone ( ACTH) and adrenaline.
The influence of these various factors on the extent of release of cell components into extra-cellular fluids inevitably means that even in healthy individuals there is a considerable intra-individual variation (i.e. variation from one occasion to another) in the value of any chosen test analyte of diagnostic importance and an even larger inter-individual variation (i.e. variation between individuals). More importantly, the superimposition of a disease state onto these causes of intra- and inter-individual variation will result in an even greater variability between test occasions.
Many clinical conditions compromise the integrity of cells located in the organs affected by the condition. This may result in the cells becoming more ‘leaky’ or, in more severe cases, actually dying ( necrosis) and releasing their contents into the surrounding extra-cellular fluid. In the vast majority of cases, the extent of release of specific cell components into the extra-cellular fluid, relative to the healthy reference range, will reflect the extent of organ damage and this relationship forms the basis of diagnostic clinical biochemistry. If the cause of the organ damage continues for a prolonged time and is essentially irreversible (i.e. the organ does not undergo self-repair), as is the case in cirrhosis of the liver, for example, then the mass of cells remaining to undergo necrosis will progressively decline so that eventually the release of cell components into the surrounding extra-cellular fluid will decrease, even though organ cells are continuing to be damaged. In such a case, the measured amounts will not reflect the extent of organ damage.
Clinical biochemical tests have been developed to complement in four main ways a provisional clinical diagnosis based on the patient’s medical history and clinical examination:
• To support or reject a provisional diagnosis by detecting and quantifying abnormal amounts of test analytes consistent with the diagnosis. For example, troponin T (a component of the contractile apparatus of cardiac muscle), creatine kinase (CK, spe cifically the CK-MB isoform) and aspartate transaminase all rise following a myocardial infarction (MI, heart attack) that results in cell death in some heart tissue. The pattern and degree of response can be used to support a diagnosis of MI. Tests can also help a differential diagnosis, for example in distinguishing the various forms of jaundice (yellowing of the skin due to the presence of the yellow pigment bilirubin, a metabolite of haem) by the measurement of alanine transaminase (ALT) and aspartate transaminase (AST) activities and by determining whether or not the bilirubin is con jugated with β-glucuronic acid.
• To monitor recovery following treatment by repeating the tests on a regular basis and monitoring the return of the test values to those within the reference range. Following hepatitis, for example, raised serum ALT returns to reference range values within 10–12 days. Similarly, the measurement of serum tumour markers such as CA125 can be used to follow recovery or recurrence after treatment for ovarian cancer.
• To screen for latent disease in apparently healthy individuals by testing for raised levels of key analytes. For example, measuring plasma glucose for diabetes mellitus and immunoreactive trypsinogen for newborn screening of cystic fibrosis. It is now common for serum cholesterol and other factors, for example weight and blood pressure, to be used as a measure of the risk of an individual developing heart disease. This approach is particularly important for individuals with a family history of the disease. A serum cholesterol <=5 mM is recommended and individuals with values greater than this should be counselled on the importance of a healthy diet and regular exercise and, if necessary, on the requirement for cholesterol-lowering ‘ statin’ drugs to be prescribed.
• To detect toxic side effects of treatment, for example in patients receiving hepatotoxic drugs, by undertaking regular liver function tests. An extension of this is therapeutic drug monitoring in which patients receiving drugs such as phenytoin and carbamazepine (both of which are used in the treatment of epilepsy) that have a low therapeutic index (ratio of the dose required to produce a toxic effect relative to the dose required to produce a therapeutic effect) are regularly monitored for drug levels and liver function to ensure that they are receiving effective and safe therapy.
Reference Ranges
In order for a biochemical test for a specific analyte to be routinely used as an aid to clinical diagnosis, it is essential that the test has the required performance indicators, especially sensitivity and specificity . Sensitivity expresses the proportion of patients with the disease who are correctly identified by the test:
Specificity expresses the proportion of patients without the disease who are correctly identified by the test:
Ideally, both of these indicators for a particular test should be 100%, but this is not always the case. Such a discrepancy most likely occurs in cases where the change in the amount of the test analyte in the clinical sample is small compared with the reference range values found in healthy individuals.
Whereas both of the above indicators express the performance of the test, it is equally important to be able to quantify the probability that the patient with a positive test has the disease in question. This is best achieved by the predictive power of the test. This expresses the proportion of patients with a positive test that are correctly diagnosed as disease positive:
A low positive predictive value means that there will be many more false positives that will need to be followed up with additional testing. A test with a high negative predictive value can be useful as it means that a negative result makes the disease unlikely.
The concept of predictive power can be illustrated by reference to fetal screening for Down’s syndrome and neural tube defects. Preliminary tests for these conditions in unborn children are based on the measurement of α-fetoprotein (AFP), human chorionic gonadotropin ( hCG) and unconjugated oestriol ( uE3) in the mother’s blood and an ultra-sound assessment of nuchal translucency thickness – this is a space at the back of the baby’s neck. The presence of Down’s syndrome results in an increased hCG, inhibin and nuchal translucency (NT) as well as decreased AFP and uE3 relative to the average in healthy pregnancies, whereas open neural tube defects tend to have elevated AFP. The results of the tests are used in conjunction with the gestational and maternal ages to calculate the risk of the baby suffering from these conditions. If the risk is high, further tests are undertaken, including the recovery of some fetal cells for genetic screening from the amniotic fluid surrounding the foetus in the womb by inserting a hollow needle into the womb (amniocentesis). The tests have a detection rate of 90% for Down’s syndrome and 85% for open neural tube defects. Accordingly, the performance indicators of the tests are not 100%, but they are sufficiently high to justify their routine use.
The correct interpretation of all biochemical test data is heavily dependent on the use of the correct reference range against which the test data are to be judged. As previously pointed out, the majority of biological analytes of diagnostic importance are subject to considerable inter- and intra-individual variation in healthy adults, and the analytical method chosen for a particular analyte assay will have its own precision, accuracy and selectivity that will influence the analytical results. In view of these biological and analytical factors, individual laboratories should ideally establish their own reference range for each test analyte using their chosen methodology and a statistically determined number (usually hundreds) of ‘healthy’ individuals. The recruitment of individuals to be included in reference range studies presents a considerable practical and ethical problem due to the difficulty of defining ‘normal’ and of using invasive procedures, such as venipuncture, to obtain the necessary biological samples. The establishment of reference ranges for children, especially neonates, is a particular problem. It is therefore usual to accept the manufacturer’s quoted ranges where available.
Reference ranges are most commonly expressed as the range that covers the mean ±1.96 standard deviations of the mean of the experimental population. This range covers 95% of the population. The majority of reference ranges are based on a normal distribution of individual values, but in some cases the experimental data are asymmetric and then often skewed to the upper limits. In such cases it is normal to use logarithmic data to establish the reference range; nevertheless, the range may overlap with values found in patients with the test disease state. Typical reference ranges are shown in Table 1.
Table1. Typical reference ranges for biochemical analytes (IU: international unit)
