Introduction to
Advanced Clinical
Biochemistry
2024-2025
Assist. Professor
Dr. Salm K. Mohammed
Ph.D. of Clinical Biochemistry
Requesting and interpreting tests
Biochemical tests are crucial to modern
medicine. Most biochemical tests are carried
out on blood using plasma or serum, but urine,
cerebrospinal fluid (CSF), faeces, kidney stones,
pleural fluid, etc. are sometimes required.
Plasma is obtained by collecting blood into an
anticoagulant and separating the fluid, plasma
phase from the blood cells by centrifugation.
Serum is the corresponding fluid phase when
blood is allowed to clot. For many (but not all)
biochemical tests on blood, it makes little
difference whether plasma or serum is used.
There are many hundreds of tests available in
clinical biochemistry but a core of common
tests makes up the majority of tests requested
in clinical biochemistry. These core tests are
typically available over a 24 h period. Tests are
sometimes brought together in profiles,
especially when a group of tests provides
better understanding of a problem than a
single test (e.g. the liver function test profile).
Many of the other more specialist tests are
restricted to larger laboratories or specialist
centres offering a national or regional service.
In dealing with the large number of
routine test requests, the modern clinical
biochemistry laboratory depends heavily
on automated instrumentation linked to a
laboratory computing system. Test results
are assigned to electronic patient files
that allow maintenance of a cumulative
patient record. Increasingly, test requests
can be electronically booked at the ward,
clinic or in General Practice via a terminal
linked to the main laboratory computer.
Equally, the test results can be displayed on
computer screens at distant locations, even
negating the need for issuing printed reports.
In this first semester, we set out some of the
principles of requesting tests and of the
interpretation of results.
The effects of analytical errors and of
physiological factors, as well as of disease, on
test results are stressed.
Biochemical testing in differential diagnosis
and in screening is discussed.
The use of clinical biochemistry tests:
Biochemical tests are most often discretionary,
meaning that the test is requested for defined
diagnostic purposes. Tests may also be requested to
screen for a disease, without there being any specific
indication of its presence in the individual, or to
assess the risk of a particular disease or disease
prognosis in the individual:
1. Why do I request this test?
2. What will I look for in the result?
3. If I find what I am looking for, will it affect my
diagnosis?
4. How will this investigation affect my management
of the patient? Will this investigation ultimately
benefit the patient?
Discretionary testing is the more common
reason for biochemical tests to be requested.
The main reasons for this type of testing are
summarised in the Table 1.
Tests may also be used to help evaluate the
future risk of disease (e.g. total cholesterol
and HDL- cholesterol levels contribute to
assessment of an individual's risk of
cardiovascular disease) or in disease
prognosis (e.g. biochemical tests to asses
prognosis in acute pancreatitis or liver failure).
Table 1 Test selection for the purposes of discretionary testing.
Category Example
To confirm adiagnosis Serum [free T4] and [thyroid-stimulating hormone,
(TSH)] in suspected hyperthyroidism
To aid differential diagnosis To distinguish between different forms of jaundice
To refine a diagnosis Use of adrenocorticotrophic hormone (ACTH) to
localize Cushing’s syndrome
To assess the severity of disease Serum [creatinine] or [urea] in renal disease
To monitor progress Plasma [glucose] and serum [K+] to follow
treatment of patients with diabetic ketoacidosis
(DKA)
To detect complications or side Alanine aminotransferase (ALT) measurements in
effects patients treated with hepatotoxic drugs
To monitor therapy Serum drug concentrations in patients treated with
antiepileptic drugs
Screening may take several forms:
In well-population screening a spectrum of tests is carried
out on individuals from an apparently healthy population in
an attempt to detect pre-symptomatic or early disease. It is
easy to miss significant abnormalities in the 'flood' of data
coming from the laboratory, even when the abnormalities
are 'flagged' in some way. For these and other reasons, the
value of well- population screening has been called into
question and certainly should only be initiated under
certain specific circumstances that are listed in Table 2.
In case-finding screening programmers appropriate tests
are carried out on a population sample known to be at high
risk of a particular disease. These are inherently more
selective and yield a higher proportion of useful results in
Table 3.
Table 2 Requirements for well- population
screening:
The disease is common or life-threatening
The tests are sensitive and specific
The tests are readily applied and acceptable to
the population to be screened
Clinical, laboratory and other facilities are
available for follow-up
Economics of screening have been clarified
and the implications accepted
Table 3 Examples of tests used in case finding programmers:
Programmers to detect diseases in Chemical investigations
Neonates PKU Serum [phenylalanine]
Hypothyroidism Serum [TSH]
Adolescents and young adults
Substance abuse Drug screen
Pregnancy Diabetes mellitus Plasma and urine [glucose]
Open neural tube defect (NTD) in the Maternal serum [α-fetoprotein]
foetus
Industry Industrial exposure to lead Blood [lead]
Industrial exposure to pesticides Serum cholinesterase activity
Elderly Malnutrition Serum vitamin D levels
Thyroid dysfunction Serum [TSH] and [thyroxine]
Point of care testing (POCT)
These are tests conducted close to the patient in the
emergency department or an outpatient or general
practitioner surgery, for example. The instrumentation used
is typically small and fits on a desk or may even be
handheld. This approach can be helpful where there is a
need to obtain a result quickly (e.g. blood gas results in the
emergency department in a breathless patient) or where a
result can be used to make a real-time clinical management
decision (e.g. whether to adjust someone's statin dose on
the basis of a cholesterol result). A further attraction is the
immediate feedback of clinical information to the patient.
POCT can be used to monitor illness by the individual
patient and help identify if a change in treatment is needed
(e.g. blood glucose monitoring in a diabetic patient).
The UK government, in outlining the future of the National
Health Service, has indicated a desire to move laboratory
testing from the hospital laboratory into the community
setting. High street pharmacies have also taken up these
opportunities.
There is also an increasing number of urine test sticks that
are sold for home use (e.g. pregnancy and ovulation
testing by measuring human chorionic gonadotrophin
(hCG) and luteinising hormone (LH), respectively). Table 4
shows examples of POCT tests in common use.
The introduction of POCT methodology requires attention
to cost, ease of use, staff training, quality, health and
safety as well as need. The advantages and disadvantages
of POCT are summarised in Table 5.
Table 4 Example of POCT that are in common use .
Common POCT in blood Common POCT in urine
Blood gases Glucose
Glucose Ketones
Urea and creatinine Red cells/haemoglobin
Na, K and Ca Bilirubin
Bilirubin Urobilinogen
Salicylate pH
Paracetamol Protein
Alcohol hCG
Troponin Drugs of abuse
Table 5 Advantage and disadvantages of point of
care testing (POCT).
Advantages Disadvantages
Rapid results on More expensive than centralised
acutely ill patients tests
Allows more Wide staff training may be
frequent monitoring needed
Immediate patient Nontrained users may have
feedback access with potential for errors
Available 24h if Calibration and quality control
required may be less robust
Health and Safety may be less
well monitored
Results less often integrated into
patient electronic record
Interpretation of Clinical Biochemistry tests
Most reports issued by clinical biochemistry laboratories
contain numerical measures of concentration or activity,
expressed in the appropriate units. Typically, the result is
interpreted in relation to a reference range for the analyte in
question.
This section discusses the interpretation of laboratory
results and the factors that may cause them to vary, under
the following main headings:
1. Analytical factors These cause errors in measurement.
2. Biological and pathological factors Both these sets of
factors affect the concentrations of analytes in blood, urine
and other fluids sent for analysis.
Serial results in the same patient
Doctors often have to interpret two or more sets of results
for the same analysis or group of analyses performed on
different occasions on the same patient. An important
question is whether an analytical change is due mainly to
laboratory imprecision or to a true change in the patient's
clinical condition. Without explaining on the statistical
aspects of this, the following rule may be applied: if the
results for analyses performed on specimens collected on
different occasions, but under otherwise identical
conditions, differ by more than 2.8 times the analytical SD
then there is a chance of over 95% that a genuine change in
concentration of the substance has occurred.
Biological causes of variation
As well as analytical variation, test results also
show biological variation in both health and
disease. Key questions are:
• How do results vary in health?
• How do results vary in disease?
The concentrations of all analytes in blood vary
with time due to diverse physiological factors
within the individual.
There are also differences between individuals.
Within-individual variation
The following may be important causes of within- individual
variation:
1. Diet: Variations in diet can affect the results of many tests,
including serum [triglyceride], the response to glucose
tolerance tests and urinary calcium excretion.
2. Time of day: Several plasma constituents show diurnal
variation (variation with the time of day), or a sleep/wake
cycle. Examples include iron, adrenocorticotrophic hormone
(ACTH) and cortisol concentrations.
3. Posture: Proteins and all protein-bound constituents of
plasma show significant differences in concentration
between blood collected from upright individuals and blood
from recumbent individuals. Examples include serum calcium,
cholesterol, cortisol and total thyroxine concentrations.
4. Muscular exercise: Recent exercise, especially if vigorous or
unaccustomed, may increase serum creatine kinase (CK) activity and
blood [lactate], and lower blood [pyruvate]
5. Menstrual cycle: Several substances show variation with the phase
of the cycle. Examples include serum [iron], and the serum
concentrations of the pituitary gonadotrophins, ovarian steroids and
their metabolites, as well as the amounts of these hormones and
their metabolites excreted in the urine.
6. Drugs: These can have marked effects on chemical results.
Attention should be drawn particularly to the many effects of
oestrogen-containing oral contraceptives on serum constituents.
Even after allowing for known physiological factors that may affect
plasma constituents and for analytical imprecision, there is still
considerable residual individual variation (Table 6). The magnitude of
this variation depends on the analyte, but it may be large and must
be taken into account when interpreting successive values from a
patient.
Table 6 Residual individual variation of some serum
constituents (expressed as the approximated day-to-day,
within-individual coefficient of variation). CV =
coefficient of variation.
Serum CV(%) Serum CV(%)
constituent constituent
Sodium 1 ALT activity 25
Calcium 1-2 AST activity 25
Potassium 5 Iron 25
Urea 10
Between-individual variation
Differences between individuals can affect the
concentrations of analytes in the blood. The following are
the main examples:
1. Age: Examples include serum [phosphate] and alkaline
phosphatase (ALP) activity, and serum and urinary
concentrations of the gonadotrophins and sex hormones.
2. Gender: Examples include serum creatinine, iron, urate
and urea concentrations and y-glutamyltransferase (GGT)
activity, and serum and urinary concentrations of the sex
hormones.
3. Race: Racial differences have been described for serum
[cholesterol] and [protein]. It may be difficult to distinguish
racial from environmental factors, such as diet.
Reference ranges
When looking at results, we need to compare each result
with a set of results from a particular defined (or
reference) population. This reference range is determined,
in practice, by measuring a set of reference values from a
sample of that population, usually of healthy individuals.
The nature of the reference population should be given
whenever reference ranges are quoted, although a
healthy population is usually assumed. Even age-matched
and gender-matched reference ranges are often difficult
to obtain, since fairly large numbers of individuals are
needed. In practice, blood donors are very often selected
as the most readily available reference population.