CLINICAL BIOCHEMISTRY

THE IMPORTANCE OF LABORATORY TESTS

IN MEDICINE
 Laboratory tests of one kind or another are an essential part of medicine.

 Biochemical tests can be used for

1. screening for disease,
2. for confirmation (or otherwise) of a diagnosis made on clinical
examination,
3. for monitoring progression of a disease and the outcome of treatment.

 Blood and urine samples are most commonly used;

1. occasionally feces,
2. saliva, or
3. cerebrospinal fluid may be used.
 LABORATORY TESTS

 Advances in technology mean that many tests that were formerly carried
out only in specialist laboratories can now be performed at the bedside, in
the doctor’s office or veterinary practice, sometimes even at home by
patients themselves.

 Other tests are still conducted in hospital laboratories or by private clinical

chemistry laboratories, with samples sent in by the referring physician.

 Some tests that are less commonly requested and may be technically more
demanding are performed only in specialist centers.

 In addition, testing of samples from athletes (and race horses) for

performance-enhancing drugs and other banned substances is normally
carried out in only a limited number of specially licensed laboratories.
 CAUSES OF ABNORMALITIES IN LEVELS OF ANALYTES
MEASURED IN THE LABORATORY

 A great many different conditions can lead to abnormalities of the results of

laboratory tests;

 Tissue injury that results in damage to cell membranes and an increase in the
permeability of the plasma membrane leads to leakage of intracellular
material into the bloodstream (eg, leakage of creatine kinase MB into the
bloodstream following a myocardial infarction).

 In other cases, the synthesis of proteins and hormones is increased or

decreased (eg, C-reactive protein [CRP] in inflammatory states, or hormones
in endocrine disorders).

 Kidney and liver failure lead to the accumulation of a number of compounds

(eg, creatinine and ammonia respectively) in the blood, due to an inability of
the organ concerned to excrete or metabolize the compound.
 Common Causes for Abnormalities in
Blood Analytes With Selected Examples for Each

1. High serum and urine levels of human chorionic gonadotropin (hCG) in pregnancy;
2. high blood lactate - following strenuous exercise
3. Changes in fluid balance - Hypernatremia (high serum sodium) in patients who are
dehydrated , due to excessive sweating or vomiting
4. Changes in blood pH - Serum bicarbonate is low in metabolic acidosis (eg, diabetic
ketoacidosis) and high in metabolic alkalosis (eg, severe vomiting due to pyloric stenosis)
5. Changes in endocrine function - Serum TSH is low in primary hyperthyroidism and high
in primary hypothyroidism
6. Changes in nutritional status - Serum albumin and retinol binding protein are low in
protein-energy malnutrition
7. Cell injury or death (necrosis) - Serum creatine kinase MB is elevated in myocardial
infarction; serum pancreatic amylase is elevated in pancreatitis
 Common Causes for Abnormalities in
Blood Analytes With Selected Examples for Each

1. Acute or chronic inflammation (including infection) - C-reactive protein is

elevated in inflammation
2. Genetic diseases - Plasma phenylalanine is elevated in phenylketonuria; serum
ammonium is elevated in disorders of the urea cycle
3. Organ failure - Serum creatinine and urea are elevated in renal failure; serum
ammonium and bilirubin are elevated in liver failure
4. Trauma - Serum myoglobin may be elevated following muscle injury
5. Cancer - Various tumor markers are elevated in specific cancers—eg, α-
fetoprotein in hepatocellular cancer, prostate specific antigen in prostate cancer
6. Drugs - Drugs used in cancer chemotherapy increase serum uric acid
7. Poisons - Organophosphorus poisons decrease the activity of
butyrylcholinesterase in blood
8. Others - Stress increases serum cortisol and catecholamines
 THE REFERENCE RANGE

 For any compound that is measured (an analyte), there is a range of values around the average or
mean that can be considered to be normal.
 This is the result of biological variations between individuals.
 In addition, day-to-day or week-to-week variations can occur in the results for the same
individual.
 Therefore, the first step in establishing any new laboratory test, is to determine the range of
results in a population of healthy people.
 For some tests, this will also mean determining the normal ranges of analytes in people of
different ages.
1. The normal range of some analytes will differ between men and women,
2. and there may be differences between different ethnic groups to be considered as well.

 This range includes 95% of the target population, and is known as the reference range.
 Values outside the reference range are considered to be abnormal, meriting further
investigation.

 For some tests, the results from different laboratories will differ, usually because they use
different methods of measurement. Each laboratory establishes its own set of reference ranges
 ASSESSMENT OF A LABORATORY TEST

 The two terms: sensitivity and specificity

 The sensitivity of a test refers to the percentage of positive test results in patients with the
disease (“true positive”).
 The test for phenylketonuria is highly sensitive; a positive test is obtained in all who have
the disease (100% sensitivity).
 The carcinoembryonic antigen (CEA) test has lower sensitivity; only 72% of those with
carcinoma of the colon test positive when the disease is extensive, and only 20% with
early disease.

 The specificity of a test refers to the percentage of negative test results among people who
do not have the disease.
 The test for phenylketonuria is highly specific; 99.9% of normal individuals give a
negative result.
 Only 0.1% gives a false-positive result.
 In contrast, the CEA test for carcinoma of the colon has a variable specificity; about 3% of
nonsmoking individuals give a false-positive result (97% specificity), whereas 20% of
 The predictive value

 The predictive value of a positive test (positive predictive value) defines the percentage of
positive results that are true positives.
 Similarly, the predictive value of a negative test(negative predictive value) defines the
percentage of negative results that are true negatives.

 This is related to the prevalence of the disease.

 For example, in a group of patients in a urology ward, the prevalence of renal disease is higher
than in the general population.
 In this group, the serum concentration of creatinine will have a higher predictive value than in
the general population.
 An ideal diagnostic test is one that has 100% sensitivity, 100% specificity, and 100% predictive
value.
 However, this is not true for most, if not all, tests available nowadays.

 However, before ordering a test, it is important to attempt to determine whether the sensitivity,
specificity and predictive value of the test are adequate to provide useful information.
 The result obtained should influence diagnosis, therapy and prognostication or lead to a better
 SAMPLES FOR ANALYSIS

 The common samples for analysis are blood and urine.

 Blood is collected into tubes with or without an anticoagulant, depending on whether plasma
or serum is required for the estimation.
 Less commonly, samples of saliva, cerebrospinal fluid, or feces may be used.

 There is a difference between measurement of an analyte in a blood sample and in urine.

1. The concentration of an analyte in blood reflects levels at the time the sample was taken,
2. whereas a urine sample represents the cumulative excretion of the analyte over a period of
time.

 A further difference is that it is usual to report results of blood tests as amount of analyte (or
enzyme activity) per milliliter or liter of blood (or plasma or serum).
 Reporting the concentration of the analyte in urine in the same way is not useful, since urine
volume depends very largely on fluid intake.
 In some cases the patient is asked to provide a complete 24-hour urine sample; this is a tedious
procedure, and it is difficult to know whether there really has been a complete 24-hour
collection.
 Creatinine excretion is reasonably constant from day to day for any one individual, but
varies between individuals because it depends mainly on muscle mass,
 because creatinine is formed nonenzymically from creatine and creatine phosphate,
most of which is in skeletal muscle.
 Apart from measurement of blood gases, for which arterial samples are required, blood
samples are usually of venous blood.
 Blood glucose is often measured in capillary blood from a finger prick.
 Some analyses use whole blood; others require either serum or plasma.

 For a serum sample, the blood is allowed to clot, then the red cells and fibrin clot are removed
by centrifugation.
 For a plasma sample, the blood is collected into a tube containing an anticoagulant, and the
red cells are removed by centrifugation.
 The difference between serum and plasma is that plasma contains prothrombin and the other
clotting factors, including fibrinogen, while serum does not.

 Different anticoagulants are used for collection of plasma samples, depending on the assay to
be performed: citrate, EDTA or oxalate, all of which chelate calcium and so inhibit
coagulation.
 Heparin, which acts by activating antithrombin III, is another commonly used anticoagulant.
 For measurement of blood glucose, potassium fluoride is added, as an inhibitor of glycolysis by
 Enzymes in Clinical Chemistry

 Enzymes are important in clinical chemistry in three differentways:

1. to measure analytes in a sample,

2. to measure the activityof enzymes themselves in a sample, and
3. as a test of vitamin nutritional status.

 Using an enzyme to measure the concentration of an analyte confers a high degree of

specificity on the assay, since in general an enzyme will act on only a single substrate, or a
small range of closely related substrates, while a simple chemical reaction may well
respond to a variety of (possibly unrelated) analytes.

 When an enzyme is used to detect an analyte, the limiting factor in the assay must be the
analyte itself; the enzyme and other reagents must be present in excess.
 Enzymes in Clinical Chemistry

 When cells are damaged or die, their contents leak out into the bloodstream.
Measurement of enzymes in plasma can therefore be used to detect tissue
damage; information is obtained from the pattern of enzymes (and tissue-specific
isoenzymes) released.

 The increase in enzyme activity in plasma above the normal range often
indicates the degree of severity of tissue damage.

 If an enzyme has a vitamin-derived coenzyme that is essential for activity, then

measurement of the activity of the enzyme in red blood cells with and without
added coenzyme can be used as an index of vitamin nutritional status.

 This provides an indication of functional nutritional status, while measurement

of the vitamin and its derivatives commonly reflects recent intake rather than
physiological adequacy.
 Enzymes in Clinical Chemistry

 The underlying assumption is that red blood cells have to compete with
other tissues in the body for what may be a limited supply of the coenzyme.

 Therefore the extent to which the red cell enzyme is saturated with its
coenzyme will reflect the availability of the coenzyme over a period of time
corresponding to the half-life of red cells.

 Such an assay consists of incubating two samples of the red cell lysate:
 one has been preincubated with, and one without, addition of the
coenzyme; then substrate is added to both, and the activity of the enzyme is
measured.
 In the sample preincubated without addition of the coenzyme, only that
enzyme that had coenzyme bound (the holoenzyme) will be active.
 In the sample that was preincubated with the coenzyme, any apoenzyme (inactive
enzyme protein without bound coenzyme) will have been activated to the holoenzyme.
1. There is, therefore, always either no change in enzyme activity on addition of coenzyme,
indicating complete saturation of the enzyme with coenzyme,
2. or an increase in activity, reflecting the activation of the apoenzyme by added coenzyme.

 Reference ranges for the activation coefficient are established in the same way as for any
other test.
 Such enzyme activation assays are available for thiamin (vitamin B1, using red cell
transketolase), riboflavin (vitamin B2, using red cell glutathione reductase), and vitamin
B6(using one or the other of the red cell transaminases)
 Screening Neonates for Inborn Errors of Metabolism

 Many of the inborn errors of metabolism can lead to very severe

mental retardation if treatment is not initiated early enough.

 For conditions such as phenylketonuria and maple syrup urine disease,

dietary restriction of the amino acids that are not metabolized
normally (phenylalanine in phenylketonuria; the branched-chain
amino acids leucine, isoleucine, and valine in maple syrup urine
disease) is essential for management of the condition

 Therefore, it is usual in most developed countries to screen neonates

for such conditions.
 Screening Neonates for Inborn Errors of
Metabolism

 The concentration of the offending amino acid(s) is measured in a blood sample that is
normally taken a week after birth, when the enzymes that are affected in the disease
should have reached full expression.

 Most commonly, a capillary blood sample is taken by heel prick, and is blotted onto
absorbent paper to be sent to the laboratory for analysis.
 The first such screening test for an inborn error of metabolism was the Guthrie bacterial
inhibition test.
 A disk from the paper containing the blood sample is laid onto an agar plate that has been
seeded with a phenylalanine-requiring strain of Bacillus subtilis, together with a
competitive inhibitor of phenylalanine uptake into the bacteria (β-thienylalanine) at such
a concentration that it will compete with phenylalanine at levels normally found in
blood, so that the bacteria will not grow.
 Screening Neonates for Inborn Errors of
Metabolism

 If the concentration of phenylalanine is more than that usually found in

blood, it will be taken up by the bacteria more than the inhibitor, and the
bacteria will form visible colonies on the agar.

 In most centers, the bacterial inhibition test has been superseded by

chromatographic techniques that permit the detection of a variety of
abnormal metabolites, and hence the detection of a variety of different
inborn errors of metabolism.