CLINICAL

BIOCHEMISTRY
Introduction
• Area of pathology concerned with analysis of bodily fluids.
• Blood, Urine
• Analysis of the soluble noncellular components of bodily fluid.
wind
• Vary from small inorganic compounds such as salts and ions to larger
organic compounds such as lipids and steroids and even drugs to
macromolecules such as albumin, enzymes and protein hormones
.
• Samples are collected in tubes appropriate to the test required.
Analytical methods
• The biochemical tests used in a clinical chemistry laboratory are designed to
accurately quantify these specific components.
• In order to reduce variation in laboratory measurement to an absolute
minimum, standard operating procedures (SOPs) and rigid quality control
testing regimes are enforced in all tests conducted.
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 Sample collection
• The principal biological fluid examined in clinical chemistry is blood serum as
distinct from blood plasma which still contains clotting factors including
fibrinogen.
• Urine is also collected for analysis and is usually a 24 h collection in a plastic
container supplied from clinical chemistry and containing an antibacterial
agent. Early morning urine (EMU) is often collected as it is considered to be
more concentrated whilst midstream urine (MSU) is often collected for
microbiological purposes.
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Blood

• In general blood, if not sampled by medical staff on the ward or the accident
and emergency department, will be sampled at a clinical/haematology blood
clinic collected by a qualified phlebotomist and many bio- medical scientists
are also phlebotomy trained.

• Samples are collected in tubes appropriate to the test required. For most
analytes to be measured by immu- noassay serum is the optimal fluid and
the blood is simply allowed to clot in a plain tube thereby allowing the
cellular components fibrinogen and clotting factors
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 Ø Hemolysin is the phenomenon when red
blood cells are destructed, hemoglobin and other
intracellular components are released into the
plasma. The delivery of hemoglobin causes the
serum or plasma to appear from pale red to
cherry red color.
Ø Reason: due to a mistake in the technique of
taking venous blood.
Ø Consequences: increases Potassium,
Phosphate, ... leading to incorrect results.
Hemolysic blood tube
 Principle:
Centrifugal force is generated when a mixture
is rotated in a centrifuge at a high speed.
When centrifuging a mixture of many
substances in solution, centrifugal force will
separate substances into different types to
create a separate layer.
At the end of the process, the original mixture
will be devided into separate components.
Function: Separate different substances in a
mixture into distinct layers for examination.
Centrifuge Rotofix 32A
 Principle:
The blood sample is drawn for analysing, then pumped to a
system of 3 electrodes: K+, Na+, Cl- (or Li+).
The Na+ electrode is a glass tube, which is made of a material
with a very high sensitivity to Na+ ions. K+ electrode is a plastic
tube, this tube contains Valinomycin which selectes all K+ ions
in the solution flowing through it. Similarly, the Cl- (or Li+)
electrode also contains substances which is sensitive to Cl- (or
Li+) ions. The potential of each electrode is compared with a
standard and stable potential produced by the Ag/AgCl
reference electrode. The relationship of potential difference is
determined based on the Nernst equation.

Electrolytes panel XL 921

To measure ions concentration in patient sample, the reference
measurement method is used. First, the machine measures the potential
when sample is drawn through a system of 3 electrodes. Next, a standard
solution with known concentration of ions is pumped through the
electrodes. The potential difference between the two measurements is
proportional to the concentration of the corresponding ion. Since the
potential difference is measured and the concentration of the standard ions
is known, the instrument can calculate the concentration of the ions in the
patient sample.
Functions:
Calculate the concentration of K+, Na+, Cl- in patient samples.
Normal value:
Na: 133 – 147 mmol/l fewer
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 Principle:
Based on the principle of colorimetric method.
The solution is inserted into the cuvette. A light
source with white light passes through the filter
to obtain a wavelength suitable for the solution.
The optical detector collects the intensity of
light passing through the cuvette containing the
solution and converts it into an electrical signal,
so that the result can be calculated and
displayed on the screen.
Functions:
Determine the concentration of Glucose, Ure,
Creatinin, Cholesterol, Triglyceride, HDL,
LDL, Acid urid, Billirubin Total, Billirubin
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Direct, Billirbin Indirect.
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Clinical assessments
The molecular structural categorization of bodily fluid components may
influence the biochemical tests used in their detection and quantification
Clinical chemistry is subdivided more along clinical/biological functions
relevant to the practice of medicine:
• Urea and electrolytes
• Gastro-intestinal markers
• Renal function markers
• Bone assessment
• Heart disease and lipid disorders
• Liver function markers
• Endocrine assessment
• Reproductive endocrinology
• Therapeutic drug monitoring and cancer biomarkers.
Urea and
electrolytes (U and
Es)
• Increased serum potassium level is observed in metabolic renal
tubular acidosis, shock or circulatory failure.
• Low serum potassium values are observed due to low intake of
dietary potassium over a period of time or increased loss through
kidney, vomiting or diarrhea
• Increased secretion of adrenal steroids or some diuretics may also
promote the loss of potassium
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Metabolism and gastrointestinal markers
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• Renal factors involve mainly damage to the glomeruli.

• Postrenal factors may be prostatic hypertrophy, calculi
blocking the ureters or neoplasms compressing the
ureters.
• The serum creatinine concentration is monitored closely
after a renal transplantation because a rising
concentration, even though small, may be an indication of
graft rejection.
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• Glucose is a reducing monosaccharide that serves as the principal
fuel of all the tissues.
• Diabetes mellitus the blood glucose levels are very high.
• Some patients with very high blood glucose levels may develop
metabolic acidosis and ketosis caused by the increased fat
metabolism, the alternate source for energy.
• Hyperglycaemia is also noted in gestational diabetes of pregnancy
and may be found in pancreatic disease, pituitary and adrenal
disorders.
• A decreased level of blood glucose, hypoglycaemia is often associ-
ated with starvation, hyperinsulinaemia and in those who are taking
high insulin dose for therapy and hypoadrenalism.
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Bilirubin too many protein →
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• Bilirubin is formed from the haem fragment of hae moglobin
released by aged or damaged red blood cells. Liver, spleen
and bone marrow are the sites of bilirubin production.
• Bilirubin formed in spleen and bone marrow is transported to
the liver. In the liver it is converted into bilirubin conjugates
and diglucuronides.
• Any liver disease affects the above systems, and hence
bilirubin accumulates in serum leading to jaundice.
Transaminases
• The two transaminases of diagnostic importance are serum
glutamic oxaloacetate transaminase (SGO) also called
aspartate amino transferase or AST, and serum glutamic
pyruvate transaminase (SGPT) also called alanine amino
transferase or ALT.
• While AST is found in every tissue of the body, including red
blood cells, and is particularly high in the cardiac muscle, ALT
is present at moderately high concentration in liver but is low in
cardiac, skeletal muscle and other tissues.
• Both AST and ALT measurements are useful in the diagnosis
and monitoring of patients with hepatocellular disease.
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Renal function
Implications of abnormal results
Abnormal results (lower-than-normal creatinine clearance) may indicate:
•. acute tubular necrosis;
•. bladder outlet obstruction;
•. congestive heart failure;
•. dehydration;
•. glomerulonephritis;
•. renal ischemia (blood deficiency);
•. renal outflow obstruction (usually must affect both kidneys to reduce the
creatinine clearance);
•. shock;
•. acute renal failure;
•. chronic renal failure;
•. end-stage renal disease. feddmay disease
Liver function tests
 Liver function tests
• Alanine amino-transferase (ALT) --- a marker of hepatitis
• A spartate aminotransferase(AST)---a marker of liverdamage, as is
albumin.
• Direct bilirubin and total bilirubin are markers for infant jaundice, whilst
alkaline phosphatase (ALP) is often elevated when the bile duct is
blocked.
• Gamma- glutamyltransferase (GGT). Isolated elevation of GGT level
may be induced by alcohol and aromatic medications, usually with no
actual liver disease.
• In determining the pathology of liver disorders the pattern of change in
the various biomarkers indicates the nature of disease.
Heart disease and lipid disorder tests
Lipid disorders, high blood cholesterol and triglycerides, increase the
risk for atherosclerosis, and thus for heart disease, stroke and
hypertension. There are three main serum forms of cholesterol
which are measured:
•. total cholesterol;
•. high-density lipoprotein (HDL) cholesterol; and
•. low-density lipoprotein (LDL) cholesterol.
•Along with triglycerides and Apolipoproteins (Apo A-1, etc.),
cholesterols are usually measured as a panel of tests.
•The laboratory test for HDL or LDL measures how much cholesterol
is present in high-density or low-density lipoprotein fractions, not the
actual
Several genetic disorders lead to abnormal levels of
cholesterol and triglycerides:
•. Familial combined hyperlipidemia
•. Familial dysbetalipoproteinemia
•. Familial hypercholesterolemia
•. Familial hypertriglyceridemia.
Abnormal cholesterol and triglyceride levels are more often caused by:
• . being overweight or obese (metabolic syndrome);
• . medications, including birth control pills, oestrogen,
corticosteroids, diuretics, beta blockers, and
• antidepressants;
• . pre -existing diseases, for example diabetes, hypo-
• thyroidism, Cushing’s syndrome, polycystic ovary
• syndrome and kidney disease;
• . excessive alcohol consumption;
• . diets that are high in saturated fats (red meat, egg
• yolks and high-fat dairy products) and transfatty
• acids (found in commercial processed foods);
• . lack of exercise and sedentary lifestyle;
• . smoking (depresses HDL levels).
Markers of myocardial infarction
Troponin T --- developed, Troponin T being spe- cific to
the cytoskeletal filaments found in heart muscle cells.
Troponin I and T are now the main stay markers of
myocardial infarction:
. Blood creatine kinase (CK or CPK) levels: male, 38---
174 units/L; female, 96---140 units/L;
. MBCK level: normally less than 5% of total CK;
. Troponin I and Troponin T: normally undetectable very
high in MI, but slight to moderate elevation during any
damage episode to heart tissue such as
inflammation --- myocarditis.
KIT FOR DIAGNOSIS (ABOTT)
Pancreatic function
Amylase

• In acute pancreatitis, amylase in the blood increases

often to four to six times higher than the highest
reference value (upper limit of normal). The increase
occurs within 12h of injury to the pancreas and
generally remains elevated until the cause is
successfully treated.
• Amylase values will return to normal in a few days. In
chronic pancreatitis, amylase levels initially will be
moderately elevated but often decrease over time with
progressive pancreatic damage.
Diagrammatic representations of proinsulin and its processing
to active insulin and c-peptide
Amylase

• A urine amylase test may also be ordered. Typically, its

level will mirror blood amylase concentrations, but both
the rise and fall occur later.
• Amylase levels may also be significantly increased in
patients with pancreatic duct obstruction, cancer of the
pancreas, and gallbladder obstruction or infection.
• Measured by colorimetric assay the normal range is 23-
--85 U/L.
Insulin and c-peptide

A significant use of c-peptide assay is in forensics: self

abuse with injected insulin or indeed injected insulin
induced coma death can be determined by the absence
of c-peptide in the serum.
Bone disease
assessment
Newer markers of bone metabolism have been discovered and
are moving from research to routine clinical assessment,
primarily in the assessment of primary bone cancers (Ewings
sarcoma and oesteo-sarcoma) and bone metastasis (from
primarily breast and prostate cancers).
Demonstrating the structure of
procollagen positions of N-terminal
(PINP) and C-terminal (PICP)
propedtides (a), their cleavage
when laid down as large complex
fibril units of extracellular collagen
fibres (b) and the structure and
position of C and N terminal
telopeptides which join the
collagen fibres together, but are
released during bone metabolism
(60% as free telopeptides) and
further degraded (40%) to
pyridinoline and deoxy- (c)
pyridinoline
Endocrinological assessments
• Endocrinological assessments cover a very broad
group of medical disciplines and disorders
• Neuroendocrinology to reproduction in which markers
of the hypothalamic-pituitary adrenal axis
• The pituitary thyroid axis and the pituitary gonadol axis
are equally important.
• The endocrine functioning of the other endocrine
glands such as the parathyroids are also important to
specialists in metabolic disorders.
 Thyroid function tests

• The hypothalamus senses low circulating levels of thyroid hormones ---

Triiodothyronine (T3)
• Thyroxine (T4) and responds by releasing thyrotropin releasing hormone
(TRH).
• Pituitary to produce thyroid stimulating hormone (TSH).
• The TSH stimulates the thyroid gland to produce thyroid hormones T3 and
T4 until levels in the blood return to normal.
• Thyroid hormone exerts negative feedback control over the hypothalamus
as well as the anterior pituitary thus controlling the release of both TRH
from the hypothalamus and TSH from the anterior pituitary gland.
Thyroid function tests

Ø The thyroid hormones T3 and T4 affect cells’ metabolism by

increasing metabolic rates. In clinical chemistry usually only
TSH and T4 levels are measured by specific immunoassay to
assess thyroid pathology.
Ø However, T3 has to be measured to confirm T3 toxicosis; given
the nonpeptide biochemical nature of T3 and T4:
•. TSH normal range: 0.3---3.5 mLU/L;
•. free T4 normal range: 10---25 pmol/L;
•. free T3 normal range: 3.5---7.5 pmol/L.
The hypothalamic pituitary thyroid axis
The hypothalamic--- pituitary---adrenal (HPA) axis
corticotropin-releasing hormone (CRH)
adrenocortico- tropic hormone (ACTH)
Addison’s disease --- ACTH stimulation test --- in normal individuals 30---60
min after being injected with an ACTH anolog drug plasma cortisol should nearly
double from 300---600 nmol/L to 500--- 1200 nmols/L but in Addison’s disease
cortisol levels will essentially remain unchanged.
Cushing’s syndrome --- dexamethasone suppression test --- in normal
individuals after administration of dexamethasone plasma cortisol will rapidly
drop from 300---600 nmols/L to a reference range in the order of undetectable to
$75 nmols/L.
Adrenal insufficiency --- ACTH stimulation test --- a response intermediate
between normal and full- blown Addison’s disease.
Diabetes insipidus --- major cause is deficiency in vasopressin or target tissue
insensitivity to vasopres- sin; often diagnosis is excessive urination and mon-
itoring a high plasma osmolarity and low urinary osmolarity.
Stress --- elevated basal cortisol.
In the control of
spermatogenesis,
GnRH is the key
neuropeptide acting
on gonadotrophs of
the anterior pituitary,
which in turn produce
LH and FSH. The
testicular gonadol
steroid testosterone
and protein hormone
inhibin B products,
feed back to
negatively
downregulate
Hypothalamic GnRH
and pituitary FSH
secretion
Changes in
Pregnancy
Cancer biomarkers
• Antigen or protein, which is secreted by the tumour itself
or by the surrounding tissues in response to the tumour
• Determined in the serum samples of the patient
 Classification of cancer biomarkers

• Organic and inorganic compounds and metabolites, peptide

hormones, monoclonal defined cancer antigens and
oncofetal antigens.
• Expression of proteins to be associated with highly
malignant tumours
Overview of biochemical cancer marker types and the nature of the tumours
with which they are associated
Examples of
major
monoclonal
antibody defined
cancer antigens
in clinical use.
Oncofetal antigens

• PLAP
• AFP
• hCG
References
• Gaw, A. et al. (2008) Clinical Biochemistry, 4th edn, Churchill
Livingstone.
• Hughes, J. and Jefferson, J.A. (2008) Clinical Chemistry Made Easy, 1st
edn, Churchill Livingstone.