CLINICAL CHEMISTRY

HYPOGLYCAEMIA

A low blood glucose level is called hypoglycaemia. Persistent occurrences of hypoglycaemia with glucose levels less than 2.2 mmol/l accompanied by
symptoms such as fainting, fits, sweating, hunger, pallor, confusion, or violence, should be investigated. Causes of hypoglycaemia include severe malnutrition, kwashiorkor, severe liver disease, alcoholic excess, insulin secreting tumours, Addison’s disease, and certain drugs. Commonly, however, markedly
reduced blood glucose levels occur following the overtreatment of diabetes.

Neonatal hypoglycaemia

Newborn infants may suffer hypoglycaemia when blood glucose levels fall below 1.1 mmol/l. Infants particularly at risk are
underweight poorly nourished babies, twins, premature infants, and babies born of diabetic mothers. It is important to detect hypoglycaemia of the newborn because without treatment brain damage may
occur.

Malaria associated hypoglycaemia

In severe malaria, hypoglycaemia is a common finding and can increase mortality particularly in young children.
Hypoglycaemia can also occur in those being treated with quinine and quinidine.

False glucose values

A falsely high glucose level will result if a blood sample is collected from an arm receiving a glucose (dextrose) intravenous
(i.v.) infusion. A falsely low value may be obtained if the plasma is markedly icteric.

See also:

HYPERGLYCEMIA

A raised blood glucose level is called hyperglycaemia. When definite it is diagnostic of diabetes mellitus. In an adult with symptoms of diabetes mellitus, a random venous plasma glucose of 11.1 mmol/l or more on two occasions or a fasting value of 7.0 mmol/l or more on two
occasions is diagnostic.

Hyperglycemia occurs in pancreatic disease and some endocrine disorders such as thyrotoxicosis
Cushings syndrome.
Steroid therapy may also cause hyperglycaemia.
Transient hyperglycaemia often occurs following severe stress, e.g. after surgery, injury, shock, infections, or severe burns.

These are essentially forms of transient diabetes (e.g. “stress diabetes”, “steroid diabetes”). However, it is now recognized that a number of these patients
may have pre-existing type 2 diabetes, therefore it is important to check that hyperglycaemia does resolve when the intercurrent illness resolve.

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PREPARATION OF GLUCOSE CALIBRATION GRAPH

1. Take six tubes and label them ‘B’ (reagent blank) and 1 to 5. Pipette 1.5 ml of protein precipitant reagent into each tube. Note: The use of the protein precipitant reagent is required because it forms part of the buffer system and contains phenol which is needed for the colour reaction.

Add to each tube as follows:

Tube

B …………………….. 0.05 ml distilled water

1 ……………………. 0.05ml standard, 2.5 mmol/l

2 ……………………..0.05ml standard, 5 mmol/l

3 ……………………..0.05ml standard, 10 mmol/l

4 ………………………0.05ml standard, 20 mmol/l

5 ……………………..0.05ml standard, 25 mmol/l

3. Continue as described in steps 5 to 7 of the glucose method.

4. Take a sheet of graph paper and plot the absorbance of each standard (vertical axis) against its concentration in mmol/l (horizontal axis). A linear calibration should be obtained.

The useful working limit (linearity) of the glucose oxidase method is about 25 mmol/l.

Check the calibration graph by measuring a control serum. A new calibration graph should be prepared and checked against controls whenever stock reagents are renewed.

GLUCOSE

Glucose provides the energy for life processes. It is the main end product of carbohydrate digestion.

Oxidation of glucose by the glycolytic and tricarboxylic acid pathways provides the chemical energy needed for cellular activity.

When not required for the body’s immediate energy needs, glucose
is converted to glycogen and stored in the liver and muscles (glycogenesis).

When required to maintain the blood glucose level, liver glycogen is converted back to glucose (glycogenolysis).

Muscle glycogen provides the glucose for muscular activity. Excess glucose is oxidized to fatty acids and stored as fat in the tissues. If needed, glucose can also be formed from fats and protein (gluconeogenesis).

An increase in the breakdown of fats to provide energy, results in an increase in the
production of ketones.

Insulin is the most important hormone that regulates the amount of glucose in the blood, the rate at which glucose is
taken up by the tissues, and the conversion of glucose to glycogen. It is made and secreted by the beta-islet cells of the
pancreas. Only insulin is capable of reducing the concentration of glucose in the blood.

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PREPARATION OF GLUCOSE STANDARD SOLUTIONS

Stock glucose standard, 100 mmol/l

1. Weigh accurately 1.8 g of dry anhydrous glucose (analytical reagent grade).
Note: To ensure the glucose is dry, heat it in an open container in an oven at 60–80 °C for about 4 hours. Remove and close the container immediately. When cool, weigh the glucose.

2. Transfer the glucose to a 100 ml volumetric flask.
Half fill the flask with 1 g/l benzoic acid and mix until the glucose is fully dissolved. Make up to the 100 ml mark with the
benzoic acid reagent and mix well.
The glucose concentration in the flask is 100 mmol/l.

3. Transfer to a storage bottle and label. When stored at 2–8 °C the stock standard is stable for about 6 months. If stored frozen in tightly stoppered containers the stock standard is stable for at least 1 year.

Working Standards

1. Take five 100 ml volumetric flasks and number them 1 to 5. Pipette accurately into each flask as follows:

Flask Stock glucose, 100 mmol/l
1 ………………………………………. 2.5 ml
2 ………………………………………. 5.0 ml
3 ………………………………………. 10.0 ml
4 ……………………………………… 20.0 ml
5 ……………………………………… 25.0 ml

2. Make the contents of each flask up to the 100 ml mark with 1 g/l benzoic acid and mix well.
The concentration of glucose in each standard is as follows:

Flask
1 = 2.5 mmol/l
2 = 5.0 mmol/l
3 = 10.0 mmol/l
4 = 20.0 mmol/l
5 = 25.0 mmol/l

3. Transfer each solution to a storage bottle and label. Store at room temperature (20–28 °C).
The working standards are stable for about 2 months

TYPES OF JAUNDICE

Haemolytic jaundice

In haemolytic (prehepatic) jaundice, more bilirubin is produced than the liver can metabolize, e.g. in severe haemolysis
(breakdown of red cells). The excess bilirubin which builds up in the plasma is mostly of the unconjugated type and is therefore not found in the urine.

Hepatocellular jaundice

In hepatocellular (hepatic) jaundice, there is a build up of bilirubin in the plasma because it is not transported, conjugated, or excreted by the liver cells because they are damaged, e.g. in viral hepatitis. The excess bilirubin is usually of both the unconjugated and conjugated types with bilirubin being found in the urine.

Obstructive jaundice

In obstructive (post-hepatic) jaundice, bilirubin builds up in the plasma because its flow is obstructed in the small bile
channels or in the main bile duct. This can be caused by gallstones or a tumour obstructing or closing the biliary tract. The
excess bilirubin is mostly of the conjugated type and is therefore found in the urine. The term cholestasis is used to describe a failure of bile flow.

See also

Bilirubin test

THE METHOD OF PERFORMING A GLUCOSE TOLERANCE TEST

Prepare a GTT chart for the patient on which to record collection times and test results.

Collect a fasting venous blood sample into a bottle or tube containing fluoride-oxalate. Label the container ‘fasting blood’.

Give the patient 75 g of glucose (D-glucose monohydrate) in 250–300 ml water, to be drunk in 5 to 15 minutes. To reduce nausea a few drops of lemon juice may be added to the water.

Make a note of the time and enter on the GTT chart the time at which the next blood sample is to be collected, i.e. 2 hours after the glucose water has been drunk.

Instruct the patient to rest quietly and not to eat, drink, exercise, smoke, or leave the hospital during the test.

Inform the patient when the test will be completed.

Important: If the patient should feel faint, very nauseated or begin to perspire excessively, call a medical officer.

Collect the second blood sample at the correct time, labelling the container with the collection time.

Measure the glucose concentration in each of the blood specimens.

Enter the patient’s results on the GTT chart if the value of the control serum is acceptable.

See also:

Blood glucose test
Glucose colorimetry method

COLORIMETRIC GLUCOSE METHOD

COLORIMETRIC GLUCOSE METHOD
The glucose-oxidase enzymatic method is recommended because it is specific for glucose. A protein precipitation stage is included because this removes substances such as urate which may be present in blood samples in sufficient concentration
to interfere in the final stage of the reaction.

Principle of glucose oxidase-peroxidase
method

Glucose oxidase (GOD) catalyzes the oxidation of glucose to give hydrogen peroxide (H2O2) and gluconic acid. In the presence of the enzyme peroxidase (POD), the hydrogen peroxide is broken
down and the oxygen released reacts with 4- aminophenazone (4-aminoantipyrine) and phenol to give a pink colour.
The absorbance of the colour produced is measured in a colorimeter using a green filter 520 nm or in a spectrophotometer at
515 nm.

See also:

GTT TEST
BLOOD GLUCOSE TEST

SEROLOGY TESTS PART 2

Antistreptolysin O Test (ASOT)

This test is used to detect the presence of Antistreptolysin O (ASO) antibodies in the serum. It widely used to detect conditions resulting from streptococcal infection and conditions secondary to a streptococcal infection. It also assists in the diagnosis of rheumatic fever, glomerulonephritis, bacterial endocarditis and scarlet fever.

C-Reactive Protein

The protein rises during inflammation and tissue destruction. It thus tested to chart to he progress of Rheumatoid arthritis, acute rheumatic fever, widespread malignancy and bacterial infections.

Cold Agglutinins

This detects presence of antibodies called cold Agglutinins. It is performed by incubating the patient’s serum with erythrocytes as cold temperature. The agglutination confirms the presence of as antibodies. It used to diagnose Infectious mononucleosis, mycoplasma pneumonia, chronic parasitic infections and lymphoma.

ABO and Rh typing

It is done to prevent transfusion and transplant reactions and to identify problems such as hemolytic diseases of the Newborn.

Rh antibody Titre

Detects amounts of antibodies in the blood. The antibodies can occur in a pregnant woman who is Rh-negative and is carrying an Rh-positive foetus. Most frequently applied in detection of Rh incompatibility problem with a mother and her unborn child.

See also:

Serology tests part 1

FACTORS THAT AFFECT THE MEASUREMENT OF ENZYME ACTIVITY

Temperature

Reaction rate increases with temperature. Most enzymes show their optimal activity between 30 °C and 50 °C. Above 60 °C, enzyme denaturation occurs. The incubation temperatures in test methods
must be strictly followed.

Time

The substrate concentration falls with time as the product concentration increases. Timing is therefore important because substrate and product concentrations are changing all the time and it is one of
these that is being measured. An accurate timer must be used to measure the incubation time of an enzyme and its substrate.

Any increase or decrease in pH away from the optimum will cause a decrease in enzyme activity. A marked change in pH can lead to the denaturation of an enzyme. The pH of buffers and substrates used in enzyme tests must therefore be correct.

Light rays

Ultraviolet light tends to inhibit enzyme activity while blue or red light tends to increase it. Samples for enzyme analysis must therefore be protected from direct light.