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CH 2021 L19

Marc Mate
 Composition of Blood
 Collection and Preservation of
 Clinical Analyses: Common
 Immunoassays
Whole blood can generally be broken down into;
Plasma: which contains the serum and fibrinogen,
Cellular elements: which contain the erythrocytes,
leukocytes, and platelets.
Plasma is the
liquid portion of
circulating blood
Serum: the portion of plasma
remaining after coagulation of
blood, during which process the
plasma protein fibrinogen is
converted to fibrin and remains
behind in the clot
The majority of clinical analyses
are performed on whole blood,
plasma, or serum, and most of
these use serum.
Adapted from J. S. Annino, Clinical Chemistry. 3rd ed. Boston: Little, Brown and Company, 1960.
Blood and urine samples are often collected after the patient
has fasted for a period of time (e.g., overnight), particularly for
cholesterol or glucose analysis
 One study indicates that an average breakfast has no
significant effect on the concentration of the blood content
of carbon dioxide, chloride, sodium, potassium, calcium,
urea nitrogen, uric acid, creatinine, total protein, and
 Serum phosphorus is slightly depressed at 45 min after the
meal then stabilizes later
When serum is required for
the analysis, the blood is
collected in a clean and dry
tube to prevent contamination
and hemolysis.
Hemolysis is the destruction
of red cells, with the
liberation of hemoglobin and
other cell constituents into the
surrounding fluid (serum or
If plasma or whole blood is required for analysis, then
the blood is collected in a tube containing an
 A widely used anticoagulant is potassium oxalate ,
about 1 mg per mL blood.
 Oxalate precipitates blood calcium; the calcium is
required in the clotting process.
In CO2 analysis, the sample is kept anaerobically by
adding mineral oil to the collection tube
 A preservative is added to the sample, usually along
with an anticoagulant.
 NaF is widely used as a preservative for samples to be
analyzed for glucose.
 This is an enzyme inhibitor that prevents the
enzymatic break down of glucose, or glycolysis.
 One milligram sodium fluoride per milliliter blood is
 Since it also inhibits other enzymes, including urease,
sodium fluoride should not be added to samples to be
analyzed for enzymes or for urea based on urease
 The major serum electrolytes like
sodium, potassium, calcium,
magnesium, chloride, and bicarbonate
(CO2) are easily determined
 Blood glucose and blood urea
nitrogen (BUN) are the two most
frequently performed clinical tests
 The enzymatic determination of
glucose and BUN are the most
common methods
 Blood samples for glucose analysis should
not be left for a long time.
 Both red cells and leukocytes contain
glycolytic enzymes.
 Glucose will be consumed and the
concentration of glucose in a sample of
whole blood will decline with time.
 The rate of loss is generally said to be
approximately 5% per hour, but may be as
rapid as 40% in 3 hours.
 Consumption of glucose in whole blood
samples can be prevented by adding
sodium fluoride to the specimen to inhibit
the glycolytic enzymes
 Rapid separation of the sample or
cooling will also prevent glycolysis
and will allow the sample to be used
for other determinations.
 Unhemolyzed samples that have
been separated within 30 minutes of
drawing are generally considered
 Rapid cooling of the sample
followed by centrifugation is even
more effective in preventing
 Glucose concentration may be determined in whole
blood, plasma, or serum samples.
 If whole blood is used, the concentration will be lower
than if plasma or serum is used.
 This is due to the greater water content of the cellular
 Under usual circumstances, the concentration of
glucose in whole blood is about 15% lower than in
plasma or serum
 Blood glucose levels are measured by a procedure based
upon the enzyme glucose oxidase.
 The enzyme is very specific for only D-glucose, and will
not be subject to interferences from other molecules in
the blood.
 The enzyme glucose oxidase catalyzes the oxidation of
beta-D-glucose to D-gluconic acid
 Diatomic oxygen from the air is the oxidizing agent
 At the same time the oxygen in the presence of water is
converted to hydrogen peroxide
 The hydrogen peroxide reacts with
a second color producing chemical
(o-Toluidine or 2-methylaniline)
which reacts with the hydrogen
peroxide using an enzyme called
peroxidase to produce a color
forming chemical.
 The concentration of the glucose
can be related to the intensity of
color produced. The more the
intensity, the higher the
concentration of glucose.
 A simple color chart can be used to
"read" the concentration of the
 A blood urea nitrogen (BUN) test measures the amount
of nitrogen in your blood that comes from the waste
product urea.
 Urea is made in the liver and passed out of your body in
the urine.
 A BUN test is done to see how well your kidneys are
working. Normal human adult blood should contain 7
to 20 mg/dL (2.5 to 7.1 mmol/L) of urea nitrogen.
 If your kidneys are not able to remove urea from the
blood normally, your BUN level rises.
 Heart failure, dehydration, or a diet high in protein can
also make your BUN level higher.
 The enzyme Urease quantitatively converts urea to
ammonia and carbon dioxide.
 The amount of urea is calculated from a determination of
either the ammonia or the carbon dioxide produced,
usually the former.
 This can be done by reacting the ammonia with a color
Immunoassays are bioanalytical methods in which the
quantification of the analyte depends on the reaction of
an antigen (analyte) and an antibody.
 Immunoassays have been widely used in many important
areas of pharmaceutical analysis such as diagnosis of
diseases, therapeutic drug monitoring, clinical
pharmacokinetics and in drug discovery.
 An immunoassay capitalizes on the specificity of the
antibody-antigen binding found naturally in the immune
 The assay can be used to identify the presence of
pathogens in a clinical sample, or it can be used to measure
the amount of a target biomolecule.
 An antigen (e.g., a hormone) is a
(foreign) substance capable of
producing antibody formation in
the body
 It is able to react with (bind to)
that antibody.
 An antigen is always a large
molecule such as a protein.
 An antibody (immunoglobulin) is
a protein endowed with the
capacity to recognize, by
stereospecific association, a
substance foreign to the organism
it has invaded, for example,
bacteria and viruses.
 The antibody will specifically react with an antigen to form
an antigen-antibody complex.
 It is produced in the organism (where it will remain present
for some time) only after the organism has had at least one
exposure to the intruder (through vaccination, either
spontaneous or artificial).
 An antibody is produced for use in immunoassay by injecting
the antigen into an animal species to which it is foreign and
recovering the serum that contains the resultant antibody
 Immunoassay techniques generally involve a
competitive reaction between an analyte antigen and
a standard antigen that has been tagged (labelled), for
limited binding sites on the antibody to that antigen.
 The label may be a radioactive tracer, an enzyme, or a
RIA Principle: “Radio labelled antigen (Ag*) competes
with the unlabelled antigens for the binding site of the
fixed amount of antibody (Ab).”
0 ng – 100%
1 ng – 90%
The proportion of Ab-Ag* and
Ab-Ag at equilibrium will be
equal to the original
proportion of Ag* to Ag
 The initial reaction vessel contains antibody solution
(antiserum), labeled antigen, and the serum sample
that may contain unlabeled (natural) antigen (the
substance to be determined).
 Upon incubation, the antibody (Ab) will form an
antigen-antibody immunocomplex (Ag-Ab).
 In the absence of unlabeled antigen, a certain
fraction of the labeled antigen (Ag*) is bound (as
 Increasing amounts of unlabeled antigen (Ag) are
added, the limited binding sites of the antibody are
progressively saturated and the antibody can bind
less of the radiolabeled antigen.
 Following incubation, the bound antigens are
separated from the unbound (free) antigens, and the
labeled portion (radioactivity, fluorescence, etc.) is
measured to determine the percent bound of the
labeled antigen.
 The antibody solution is initially diluted so that, in the
absence of unlabeled standard or unknown antigen,
about 50% of the tracer dose of Ag* is bound.
 A diminished binding of labeled antigen when sample
is added indicates the presence of unlabeled antigen.
 A calibration curve is prepared using antigen
standards of known concentration by plotting either
the percent bound labeled antigen or the ratio of
percent bound to free (B/F) as a function of the
unlabeled antigen concentration.
 From this, the unknown antigen concentration can be