MECHANIZATION AND

AUTOMATION IN
CLINICAL
BIOCHEMISTRY
CLINICAL
BIOCHEMICAL
ANALYSES OF
PROTEINS, PLASMA
PROTEIN SPECTRUM.

Introduction to Clinical
biochemistry
CLINICAL BIOCHEMISTRY (also known as clinical
chemistry or chemical pathology) is the laboratory
service absolutely essential for medical practice or
branch of laboratory medicine in which chemical and
biochemical methods are applied to the study of disease.
The results of the biochemical investigations carried out
in a clinical chemistry laboratory will help the clinicians
to determine the diseases (diagnosis) and for follow-up of
the treatment/recovery from the illness (prognosis).

Introduction to Clinical
biochemistry

Introduction to Clinical
biochemistry
The use of biochemical tests:
Biochemical investigations are involved in every branch of
clinical medicine.
The results of biochemical tests may be of use in:
diagnosis and in the monitoring of treatment.
screening for disease or in assesing the prognosis.
reseach into the biochemical basis of disease
clinical trials of new drugs
Biochemical investigations hold the key for the diagnosis and
prognosis of diabetes mellitus, jaundice, myocardial infarction,
gout, pancreatitis, rickets, cancers, acid-base imbalance etc.
Successful medical practice is unimaginable without the service
of clinical biochemistry laboratory.

Introduction to Clinical
biochemistry
In general, biochemical tests can be broadly divided into two
groups:
In discretionary or selective requesting, the tests are carried
out on the basis of an individual patient's clinical situation. The case
for discretionary requesting has been put admirably (Asher, 1954):
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?
5. Will this investigation ultimately benefit the patient?
In contrast, screening tests are used to search for disease without
there being any necessary clinical indication that disease is present.

Introduction to Clinical
biochemistry

Test selection for the purposes of discretionary

Category
Example
testing
To confirm a diagnosis
Plasma (free T ) and
4

To aid differential diagnosis

To refine a diagnosis
To asses the severity of
disease
To monitor progress
To detect complications or
side effects
To monitor therapy

(thyroid-stimulating
hormone, TSH) in suspected
hyperthyroidism
To distinguish between
different forms of jaundice
Use of ACTH to localize
Cushing's syndrome
Plasma (creatinine) or (urea)
in renal disease
Plasma (glucose) to follow of
patients with diabetes
mellitus
ALT measurements in
patients treated with
hepatotoxic drug
Plasma drug concentration in
patients treated with
antiepileptic drugs

Introduction to Clinical
biochemistry
Table 1.4 Examples of tests used in case-finding
Programmes to detect
Chemical investigations
programmes. diseases in
Neonates:
PKA (phenylketonuria)
Hypothyroidism
Adolescents and young
adults:
Substance abuse

Serum [phenylalanine]

Serum [TSH] and/or

[thyroxine]

Drug screen

Pregnancy:
Diabetes mellitus in the
mother

Plasma and urine [glucose]

Open neural tube defect

(NTD) in the foetus
Industry:

Industrial exposure to lead

Maternal serum [afetoprotein]

Blood [lead]

Introduction to Clinical
biochemistry
ADVANTAGES OF SCREENING
First, an uncommon or unexpected disease may be
found and created. Second, the early requesting of a
battery of tests might be expected to expedite
management of the patient. Most studies have not
Unexpected abnormal test results
shown this Disease
to be so.
Hyperparathyroidism

Raised plasma calcium

Hypothyroidism

Raised plasma TSH and/or a low T4

Diabetes mellitus

High random plasma glucose

Renal tract disease

Raised plasma creatinine or urea

Liver disease

Increased plasma ALT, AST

Introduction to Clinical
biochemistry
DISADVANTAGES OF SCREENING
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.
Most of the abnormalities detected will be of little or no significance, yet
may need additional time-consuming and often expensive tests to clarify
their importance (or lack of it).
In other instances, to simplify requesting, a wide range of tests are routinely
requested on all patients in a particular category, for example, admission
screening on all those admitted through the Accident and Emergency (A&E)
Department. Mention should also be made of batteries of tests which are
generally requested on a discretionary basis but where the test group
collectively provides information about an organ system (e.g. tests for liver
disease) or a physiological state (e.g. water and electrolyte status). Many
laboratories analyse and report these functional or organ-related groups.
For example, a 'liver function test' group might consist of plasma bilirubin,
alanine aminotransferase (ALT), alkaline phosphatase (ALP), fglutamyltransferase (GGT) and albumin measurements.

Introduction to Clinical
biochemistry

Introduction to Clinical
biochemistry
Clinical biochemical tests comprise over of all hospital laboratory
investigations.

Core biochemistry: Most biochemistry laboratories provide the "core analyses",
commonly requested tests which are of value in many patients, on a frequent basis.
Core biochemical tests:
Sodium, potassium, chloride and bicarbonate
Urea and creatinine
Calcium and phosphate
Total protein and albumin
Bilirubin and alkaline phosphatase
Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST)
Glucose
Amylase.

Introduction to Clinical
biochemistry
Specialized tests:
Not every laboratory is equiped to carry out all possible biochemistry
requests.
Large departments may act as reference centres where less commonly
asked for tests are performed.
Specialized tests:
Hormones
Specific proteins
Trace elements
Vitamins
Drugs
Lipids and lipoproteins
DNA analyses

Introduction to Clinical
biochemistry
The emergency lab:
All clinical biochemistry laboratories provide facilities for
urgent tests. An urgent test is designated as one on
which the clinician is likely to take immediate action.
The main reason for asking for an analysis to be
performed on an urgent basis is that immediate
treatment depends on the result.

Introduction to Clinical
biochemistry
TYPES OF LABORATORY TESTS:
The biochemical investigations (on blood/ plasma/serum) carried out in
the clinical biochemistry laboratory may be grouped into different types.
1. Discretionary or on-off tests : Most common clinical biochemistry
tests that are designed to answer specific questions, e.g., does the
patient have increased blood urea/glucose concentration? Normally,
these tests are useful to support the diagnosis.
2. Biochemical profiles : These tests are based on the fact that more
useful information on the patients disease status can be obtained by
analysing zor more constituents rather than one e.g., plasma
electrolytes (Na+, K+, Cl-, bicarbonate, urea); liver function tests
(serum bilirubin, ALT, AST).
3. Dynamic function tests : These tests are designed to measure the
body's response to external stimulus e.g., oral glucose tolerance test (to
assess glucose homeostasis) : bromosulphthfein test (to assess liver
function).

Introduction to Clinical
biochemistry
4. Screening tests : These tests are commonly employed
to identify the inborn errors of metabolism, and to check
the entery of toxic agents (pesticides, lead, mercury) into
the body.
5. Metabolic work-up tests : The programmed intensive
investigations carried out to identify the endocrinological
disorders come under this category.
The term emergency tests is frequently used in the
clinical laboratory. It refers to the tests to be performed
immediately to help the clinician for proper treatment of
the patient e.g., blood glucose, urea, serum electrolytes.

Introduction to Clinical
biochemistry
There are over 400 different tests which may be carried
out in clinical biochemistry laboratories. They vary from
the very simple, such as the measurement of sodium, to
the highly complex, such as DNA analysis, screening for
drugs, or differentiation of lipoprotein variants. Many
high volume tests are done on large automated
machines. Less frequently performed tests may be
conveniently carried out by using commercially
prepared reagents packaged in "kit" form. Some
analyses are carried out manually.

Introduction to Clinical
biochemistry
Specimen collection:
The biological fluids employed in the clinical
biochemistry laboratory include blood, urine, saliva,
sputum, faeces, tissue and cells, cerebrospinal
fluid, peritoneal fluid, synovial fluid, pleural fluid,
stones.
Among these, blood (directly or in the form of plasma or
serum) is frequently used for the investigations in the
clinical biochemistry laboratory.

Introduction to Clinical
biochemistry
Identification of patients and specimens
The correct patient must be appropriately identified on the
specimen and request form, as follows:
1. Patient identification data (PID). This usually comprises
name plus unique number.
2. Test request information. This includes relevant clinical
details (including any risk of infection hazard), the tests to be
performed and where the report is to be sent.
3. Collection of specimens. In the correct tube and the
appropriate preservative.
4. Matching of specimens to requests. Each specimen
must be easily and unequivocally matched to the
corresponding request for investigations.

Introduction to Clinical
biochemistry
Some commoner causes of errors arising from use
Error
of
theof laboratory.
Crossover
addressograph labels
between patients
Timing error

Sample collection tube error

Sample taken from close to the site

of an intravenous infusion

Analytical error

Consequence
This can lead to two patients each with the other's set of results. Labels between
patients
Where the patient is assigned a completely wrong set of results, it
is important to investigate the problem in case there is a second patient with a
corresponding wrong set of results
There are many examples where timing is important but not considered. Sending in
a blood sample too early after the administration of a drug can lead to misleadingly
high values in therapeutic monitoring. Interpretation of some tests (e.g. cortisol) is
critically dependent on the time of day when the blood was sampled
For some tests the nature of the collection tube is critical which is why the
Biochemistry Laboratory specifies this detail. For example, using a plasma tube
with lithium-heparin as the anticoagulant invalidates this sample tube for
measurement of a therapeutic lithium level! Serum electrophoresis requires a
serum sample; otherwise, the fibrinogen interferes with the detection of any
monoclonal bands. Topping up a biochemistry tube with a haematology (potassiumethylenediamine tetraacetic acid (EDTA) sample) will lead to high potassium and
low calcium values in the biochemistry sample
The blood sample will be diluted so that all the tests will be correspondingly site of
an intravenous (IV) infusion
low with the exception of those tests which might
be affected by the composition of the infusion fluid itself. For example, using
normal saline as the infusing fluid would lead to a lowering of all test results but
with sodium and chloride results which are likely to be raised
Although comparatively rare, these do inevitably happen from time to time and any
result which is unexpected should lead the requesting clinician to discuss the
matter further with the Laboratory. Transcription errors within the Laboratory are
increasingly less common because of the electronic download of results to the
Laboratory computer as a source of the printout or results on the VDU. Most errors
generated within the Laboratory occur at the Reception as a result of mislabelling
of samples within the Laboratory

Introduction to Clinical
biochemistry
COLLECTION OF BLOOD:
Venous blood is most commonly used for a majority of
biochemical investigations. It can be drawn from any
prominent vein (usually from a vein on the front of the elbow).
Capillary blood (<0.2 ml) obtained from a finger or thumb, is
less frequently employed.
Arterial blood (usually drawn under local anesthesia) is used
for blood gas determinations.

Precautions for blood collection : Use of sterile (preferably

disposable) needles and syringes, cleaning of patients skin,
blood collection in clean and dry vials/tubes are some of the
important precautions.

Introduction to Clinical
biochemistry
Biochemical investigations can be performed on 4 types
of blood specimens whole blood, plasma, serum and red
blood cells. The selection of the specimen depends on the
parameter to be estimated.
1. Whole blood (usually mixed with an anticoagulant) is used
for the estimation of hemoglobin, carboxyhemoglobin, pH,
glucose, urea, non-protein nitrogen, pyruvate, lactate, ammonia
etc. (Note : for glucose determination, plasma is prefered in
recent years).
2. Plasma, obtained by centrifuging the whole blood collected
with an anticoagulant, is employed for the parameters
fibrinogen, glucose, bicarbonate, chloride, ascorbic acid etc.

Introduction to Clinical
biochemistry
3. Serum is the supernatant fluid that can be collected
after centrifuging the clotted blood. It is the most
frequently used specimen in the clinical biochemistry
laboratory. The parameters estimated in serum include
proteins (albumin/globulins), creatinine, bilirubin,
cholesterol, uric acid, electroylets (Na+, K+, Cl-), enzymes
(ALT, AST, LDH, CK, ALP, ACP, amylase, lipase) and
vitamins.
4. Red blood cells are employed for the determination
of abnormal hemoglobins, glucose 6-phosphate
dehydrogenase, pyruvate kinase etc.

Introduction to Clinical
biochemistry

Introduction to Clinical
biochemistry
Collection and preservation of blood specimens
Lack of thought before collecting specimens or carelessness in collection may
adversely affect the interpretation or impair the validity of the tests carried out
on the specimens. Some factors to consider include the following:
1. Diet Dietary constituents may alter the concentrations of analytes in blood
significantly (e.g. plasma [glucose] and [triglyceride] are affected by
carbohydrate and fat-containing meals, respectively).
2. Drugs Many drugs influence the chemical composition of blood. Such
effects of drug treatment, for example, antiepileptic drugs, have to be taken
into account when interpreting test results. Details of relevant drug treatment
must be given when requesting chemical analyses, especially when
toxicological investigations are to be performed.
3. Diurnal variation. The concentrations of many substances in blood vary
considerably at different times of day (e.g. cortisol). Specimens for these
analyses must be collected at the times specified by the laboratory, as there
may be no reference ranges relating to their concentrations in blood at other
times

Introduction to Clinical
biochemistry
Care when collection blood specimens
The posture of the patient, the choice of skin-cleansing agent and
the selection of a suitable vien (or other source) are the principal
factors to consider before proceeding to collect each specimen:
1. The skin must be clean over the site for collecting the blood
specimen. However, it must be remembered that alconol and
methylated spirits can cause haemolysis, and that their use is
clearly to be avoided if blood [ethanol] is to be determined.
2. Limbs into which intravenous infusions are being given
must not be selected as the site of venepuncture unless particular
care is taken. The needle or cannula must first be thoroughly
flushed out with blood to avoid dilution of the specimen with
infusion fluid.

Introduction to Clinical
biochemistry
3. Venepuncture technique should be standardised as far as
possible to enable closer comparison of successive results on patients.
4. Venous blood specimens should be obtained with minimal
stasis Prolonged stasis can markedly raise the concentrations of plasma
proteins and other non-diffusible substances (e.g. protein-bound
substances). It is advisable to release the tourniquet before withdrawing
the sample of blood.
5. Posture should be standardised if possible When a patient's
posture changes from lying to standing, there may be an increase of as
much as 13% in the concentration of plasma proteins or protein-bound
constituents, due to redistribution of fluid in the extracellular space.
6. Haemolysis should be avoided, since it renders specimens
unsuitable for plasma K+, magnesium and many protein and enzyme
activity measurements.
7. Infection hazard High-risk specimens require special care in
collection, and this danger must be clearly indicated on the request form.

Introduction to Clinical
biochemistry
Care of blood specimens after collection
Blood specimens should be transported to the laboratory as
soon as possible after collection. Special arrangements are
needed for some specimens (e.g. for acid-base
measurements, or unstable hormones) because of their lack
of stability. Most other analytes are stable for at least 3 h in
whole blood, or longer if plasma or serum is first separated
from the cells. As a rule, whole blood specimens for chemical
analysis must not be stored in a refrigerator, since ionic
pumps that maintain electrolyte gradients across the cell
membrane are inactive at low temperatures. Conversely,
separated serum or plasma is best refrigerated, to minimize
chemical changes or bacterial growth.

Introduction to Clinical
biochemistry
Several changes occur in whole blood specimens following
collection. The commoner and more important changes that occur
prior to the separation of plasma or serum from the cells are:
1. Glucose is converted to lactate: this process is inhibited by
fluoride;
2. Several substances pass through the erythrocyte membrane, or
may be added in significant amounts to plasma as a result of red
cell destruction insufficient to cause detectable haemolysis.
Examples include K+ and lactate dehydrogenase;
3. Loss of CO2 occurs, since the Pco2, of blood is much higher than
in air;
4. Plasma [phosphate] increases due to hydrolysis of organic ester
phosphates in the red cells;
5. Labile plasma enzymes lose their activity.