LABORATORY PROCEDURES – Page 2

THROMBIN TIME (TT) TEST

Thrombin time (TT) test
The TT test is sensitive to a deficiency of fibrinogen or inhibition of thrombin. It measures the formation of a fibrin clot by the action of thrombin on fibrinogen.

Principle of test
Thrombin is added to citrated plasma at 37°C. The time taken for the mixture to clot is measured and the appearance of the
clot noted.

Reagent
The use of a thrombin time test kit is recommended.
This provides the correct concentration of thrombin to use in the test, i.e. that which gives a clotting time of 12–15 seconds with pooled normal plasma.

Collect venous blood into citrate
anticoagulant and centrifuge to obtain platelet poor plasma as described for the APTT test. Perform the test with as little delay as possible.

See also:

Procedure for TT test

PROTHROMBIN TIME (PT) TEST

The PT is a screening test for the extrinsic clotting system, i.e. factor VII. It will also detect deficiencies of factors, prothrombin, V, X, and fibrinogen. It is mainly used to monitor patients receiving warfarin
anticoagulation.

Principle of the test

Plasma or capillary blood is added to a thromboplastin and calcium chloride reagent at 37°C and the time taken for a clot
to form is measured. The clotting time in seconds is converted to the International Normalized Ratio (INR), usually by reference to a table provided by the manufacturer of the reagent OR by the formula:
INR= (PT PATIENT/PT CONTROL)^ISI
ISI is provided by reagent manufacturer

Reagents

Thromboplastin calcium chloride combined reagent.
Several different thromboplastin calcium combined reagents are available depending on the source of the thromboplastin and whether the test is to be performed using plasma or capillary whole blood. Some manufacturers supply a thromboplastin calcium reagent that can be used with both capillary blood and plasma.

Capillary blood PT testing:

This avoids the need to collect venous blood when monitoring patients being treated with warfarin. It should not be used
however when a patient is anaemic or polycythaemic. Plasma should then be used.

See also

Prothrombin time test procedure

PROTHROMBIN TIME (PT) TEST PROCEDURE

Pipette 0.25 ml of the thromboplastin/calcium reagent into a small glass tube. Place in a 37°C water bath for 1–2 minutes.
Using a calibrated capillary or delivery pipette, add (0.05 ml) of capillary blood or plasma, 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
Convert the clotting time to the INR using the table provided by the manufacturer. Separate INR tables are provided for capillary blood and plasma.

The INR conversion table provided by the manufacturer is specific for The batch of thromboplastin supplied with it.

What is the normal range for Prothrombin time?

11 – 16 seconds

What are Causes of Prolonged PT?

Treatment with oral anticoagulant drugs (vitamin K antagonists) such as warfarin.
Liver disease
DIC
Haemolytic disease of the newborn
Rarely a deficiency of factor VII, X, V, or prothrombin.

HAEMOGLOBIN ELECTROPHORESIS

Haemoglobin electrophoresis is used to separate and identify the different haemoglobins by their migration within an electric field. Haemoglobin variants separate at different rates due to differences in their surface electrical charge as determined by their amino acid structure.

Alkaline cellulose acetate electrophoresis

Several techniques are available to separate haemoglobin variants by electrophoresis. For routine work, electrophoresis in an alkaline buffer at pH 8.4–8.6 using a cellulose acetate membrane is adequate.
This gives good separation of HbA, HbF, HbS, and HbC. On alkaline electrophoresis HbD and HbS have the same mobility and HbC, HbE and HbO also co-migrate. In specialist laboratories agarose gel
electrophoresis at an acid pH (6.0) can be used to separate these haemoglobins and also to provide a clear separation of HbF from HbS and HbC.

PREREQUISITES FOR MYCOLOGY SPECIMEN COLLECTION

Always consult the SOP manual of the diagnostic laboratory for the particular specimen and test.

Aim to collect sample in a sterile kit under aseptic conditions.

Ensure that you have the appropriate swab kit (alcohol swabs, gloves, transportation tube and media).

If it is a special or non-routine test, coordinate with the laboratory about specific requirements for collection and handling.

Document the following:

Patient identifiers (e.g. name, age, medical record number);
Sample identifiers (e.g. swab, smear, scrape, urine, blood);
Location and type (e.g. lower limb wound, nail scraping, and hair sample);
Date and time of collection;
Deviations from standard protocol during collection (e.g. not performed under aseptic conditions if large traumatic wound);
Relevant clinical information including recent and current antimicrobial therapy.

Ensure that adequate material (at least 2 ml of bodily fluids) is sent to the laboratory for proper yield. An inadequate specimen may lead to a false negative result.

Transport the sample within 2 hours and process promptly for optimum recovery of fungi.

If a delay is anticipated, refrigerate specimens at 4°C (exceptions: blood, bone marrow, CSF and sterile tissues should be stored at 35-37°C).

Ideally, collect specimens as soon as symptoms appear and whenever possible before antifungal therapy is initiated.

Staff must take all precautions to avoid inadvertent contamination of sample as well as for their own personal safety.

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

HbS SOLUBILITY TEST METHOD

Set up with the test, a negative control (HbAA) and a positive control using blood from a person with sickle cell trait (HbAS).

1. Pipette 2 ml of working reagent into a test tube approximately 13 × 77 mm.

2. Wash in (0.1 ml) of capillary blood or well mixed venous blood.
Note: When the haemoglobin is below 70 g/l (7 g/dl), use twice the volume of blood or if a venous blood sample, use plasma reduced blood (remove about half the plasma).

3. Mix well and filter through a small (5.5 cm diameter) No. 1 filter paper.

4. Note the colour of the solution (pale yellow, pink red, or dark red) and whether there is any red precipitate (insoluble reduced HbS) on the filter paper.

Findings

HbSS . . . . . . Clear pale yellow filtrate. Abundant red precipitate on filter paper

HbAS. . . . . . Clear pink filtrate. Small amount of red precipitate on filter paper
Same result will be obtained with HbSC and HbS with other Hb variant

HbAA (normal) . . . . . . . . Dark red fluid (soluble reduced Hb) with no precipitate on filter paper
Same result will be obtained with HbAC and HbAD

Reporting results

–‘Positive for sickle cell anaemia’ when result shows HbSS appearance.

– ‘Positive for sickle cell haemoglobin’ when result shows HbAS appearance.

– ‘Negative for HbS’ when result shows HbAA appearance.

See also:

HbS SOLUBILITY FILTRATION TEST

Value:

When reagents are available, this test should be performed in preference to the sickle cell slide test because it provides information about the different sickle cell disorders. In areas where both HbS
and HbD occur this test can be used to differentiate sickle cell anaemia (HbSS) from HbSD sickle cell disease.

Principle of the test

Blood is mixed in a phosphate buffer-saponin solution containing sodium dithionite and filtered. In its deoxygenated
form, HbS is insoluble. HbSS is indicated by a red precipitate on the filter paper with a pale yellow filtrate. Other forms of
haemoglobin are soluble when in a reduced state.

Reagents

● Phosphate buffer-saponin pH 7.1
Store at 2–8 C. Renew every 3 months or if it
becomes turbid.
● Sodium dithionite powder.

Making a working reagent:

– Measure 20 ml of buffer-saponin solution.
– Add 0.2 g sodium dithionite and mix gently until the chemical is dissolved.
Note: The working reagent is not stable. It can be used only on the day it is prepared.

See also:

HbS Solubility Test Procedure
Sickling test Procedure

SICKLING TEST METHOD

Negative Control: Deliver one drop of
blood from a person that does not have a sickle cell disorder on a slide marked ‘Neg Control’.
Add an equal volume of 2% sodium metabisulphite and mix. Cover with a cover glass. Exclude any air bubbles.

If a blood from a known sickle cell trait person is available, set up also a Positive Control.

3. Place the slides in a container (plastic box or petri dishes) with a damp piece of blotting paper or tissue in the bottom to prevent drying of the preparations. Close the container and leave at room temperature.

4. After 10–20 minutes, examine the patient’s preparation microscopically for sickle cells. Focus the cells first with the 10 objective and examine for sickling using the 40 objective.
Examine several parts of the preparation. Sickling often occurs quicker in one area than the other.

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