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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

FORMS OF DIABETES MELLITUS BASED ON WHO CLASSIFICATION

Type 1 diabetes : formerly known as insulin-dependent diabetes (IDDM):

Insulin treatment is required to sustain life. There is an absolute insulin deficiency due to the immune destruction of pancreatic beta-cells possibly triggered in genetically
susceptible persons by a viral infection (e.g. congenital rubella), consumption of cows milk early in life, or possibly by chemical toxins. The onset of type 1 diabetes is
abrupt with severe symptoms, often including ketosis.
Type 1 diabetes is the commonest form of diabetes among children and young adults in European countries but has a low prevalence in tropical countries.

Type 2 diabetes : formerly known as non-insulin dependent diabetes (NIDDM):

Individual or ethnic genetic factors lead to susceptibility.
There is some secretion of insulin but a decrease in insulin action (insulin resistance). Several factors are associated
with the development of type 2 diabetes in susceptible individuals. These include dietary changes, overnutrition
with increased intake of saturated fats and decreased intake of dietary fibre, obesity, physical inactivity, and ageing. There is often arterial hypertension and
dyslipidaemia. Some drugs and hormones can also cause glucose intolerance and diabetes.

Malnutrition-related diabetes mellitus (MRDM):

MRDM is a controversial entity, and current WHO classifications of diabetes does not include MRDM. It is also known as ‘tropical pancreatic diabetes’, and is seen in
localized areas throughout tropical countries. It is related to pancreatic damage and is characterized by young age of
onset and past or present malnutrition. Steatorrhoea may occur due to exocrine pancreatic deficiency. Pancreatic
calcification can occur in some cases. The cause is unknown; toxicity from the cyanide content of the root crop cassava has been suggested but not definitely
substantiated.

Gestational diabetes mellitus (GDM):

This is defined as diabetes first recognized in pregnancy. Glucose values often return to normal postpartum but glucose intolerance and type 2 diabetes may occur later in life.

Other forms of diabetes:

These rarer forms can be found with pancreatic disease, some hormonal diseases and genetic syndromes, abnormalities of insulin or its receptors, and as a result of treatment with certain drugs or exposure to certain chemical
toxins.

See also blood glucose for diabetes diagnosis

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

The APTT is a screening test of the intrinsic clotting system. It will detect the inhibition or deficiency of one or more of the following factors: prothrombin, V, VIII (antihaemophilic factor), IX, X, XI, XII and fibrinogen. The APTT is also used to monitor patients being treated with heparin.

Principle of test

Kaolin (surface activator) and platelet substitute (phospholipid) are incubated with citrated plasma at 37°C for the time
specified in the test method. Calcium chloride is added and the time taken for the mixture to clot is measured.

Reagents

Kaolin/platelet substitute mixture
Purchase from a reliable manufacturer in lyophilized form and reconstitute as instructed.

Calcium chloride, 0.025 mol/l (25 mM)
This is best obtained ready made unless the laboratory has facilities to make the reagent accurately and standardize it.

See also:

APTT TEST PROCEDURE

SOURCES OF ERROR ASSOCIATED WITH THE HbS SOLUBILITY

● Using a reagent that has deteriorated. If after adding blood to the working reagent, a pale orange colour develops, this indicates reagent deterioration. A fresh reagent must be prepared.

● Using blood with too few red cells. Either the volume of blood used should be increased or preferably the test performed on a plasma reduced blood sample.

● Testing the blood of a transfused patient. The test should be performed at a later date to obtain a reliable test result.

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

SOURCES OF ERROR WHEN PERFORMING APTT, PT & TT TESTS

● Difficulty in obtaining a venous sample, resulting in haemolysis or small clots in the sample.

● Delay in testing the plasma and leaving the sample at room temperature.

● Using tubes or pipettes which are not clean and dry or are contaminated with detergent. Whenever possible use disposable tubes.

● Not timing accurately the different stages of the test (a stop-watch must be used).

● Using unsatisfactory reagents (will be detected when testing control plasma).

● Not pipetting correct volumes of plasma and reagents.

● Reconstituting reagents and control plasma with contaminated deionized water.

THROMBOCYTOPENIA

It occurs when platelets fall below the normal range of 150–400×10^9 per litre of blood

REDUCED PRODUCTION OF PLATELETS

● Infections, e.g. typhoid, brucellosis
● Deficiency of folate or vitamin B12
● Aplastic anaemia
● Drugs (e.g. cytotoxic, quinine, aspirin), chemicals
(e.g. benzene), some herbal remedies, alcoholism
● Leukemias, lymphoma, myeloma, myelofibrosis, carcinoma
● Hereditary thrombocytopenia (rare condition).

INCREASED DESTRUCTION OR CONSUMPTION OF
PLATELETS

● Infections, e.g. acute falciparum malaria, dengue, trypanosomiasis, visceral leishmaniasis
● Disseminated intravascular coagulation (DIC)
● Hypersplenism
● Immune destruction of platelets, e.g. idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), other connective tissue disorders, chronic lymphatic leukaemia, lymphomas and HIV/AIDS. Also, exposure to drugs, e.g. quinine, mefloquine, penicillin, and some herbal remedies.

APTT MEASUREMENT USING PLATELET SUBSTITUTE MIXTURE

Test the control plasma and patient’s plasma in duplicate.

1. Pipette 0.2 ml of well-mixed kaolin/platelet substitute in a small glass tube.

2. Add 0.1 ml of plasma, mix, and incubate at 37° C for exactly 2 minutes (tilting the tube at intervals).

3. Add 0.1 ml 0.025 mol/l calcium chloride, mix and start the stop-watch. Hold the tube in the water bath and tilt the mixture back and forth, looking for clot formation. When a clot forms, stop the stop-watch and record the time.

4. Report the patient’s APTT (average of the duplicate tests) providing the APTT of the normal control plasma is satisfactory.

Reference APTT range

36–50 seconds

What causes prolonged APTT?

DIC (involving several clotting factors)
Deficiency of clotting factors: prothrombin, V, VIII, IX, X, XI or XII due to vitamin K deficiency, liver disease, heparin or warfarin anticoagulation, or less commonly an inherited coagulation disorder.

See also:

The Principle of APTT test

RETICULOCYTE COUNT

Value of test:

Reticulocytes are immature red cells
normally present in small numbers in the blood (up to 2%). Reticulocyte numbers increase when there is an increase in erythropoietic activity. A reticulocyte count assesses bone marrow activity, e.g. whether there is an effective erythropoietic response when there is a reduction in the number of red cells due to haemolysis or haemorrhage. A reticulocyte count is also of value in monitoring the erythropoietic response of an anaemic patient following treatment.

Principle of test
An isotonic solution of a supravital stain (i.e. one that stains living material) such as New methylene blue or brilliant cresyl blue is incubated with a few drops of blood. To detect ribosomal RNA in reticulocytes, the red cells must be stained while they are still living (not fixed). A thin preparation is made and the reticulocytes counted microscopically. Reticulocytes are recognized by the violet-blue stained granules of ribosomal RNA (reticulin) they contain.

The reticulocyte count is expressed as a percentage, or preferably in absolute numbers when an electronic analyzer RBC count is available.

See also:

Procedure for Reticulocytes count