HEMATOLOGY – Page 3

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.

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.

RETICULOCYTE COUNT TEST METHOD

1. Filter 2–3 drops of the stain into a small tube or vial.

2. Add about 4 drops of EDTA anticoagulated blood or capillary blood and mix well.
The amount of blood used is not critical. Use at least twice the volume of blood to stain if the patient is severely anaemic.

3. Incubate at room temperature for 20 minutes or 10–15 minutes at 35–37 C.

4. Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly spread thin films. Wave the slides back and forth to air-dry the films. Protect
the films from dust and insects until the count can be performed.

5. Count the reticulocytes microscopically. Use the 10 objective (with reduced condenser iris diaphragm) to check the distribution of the red cells. Select an area where the red cells can be seen individually, add a drop of immersion oil,
and examine using the oil immersion objective (open more the condenser iris diaphragm).

6. Count systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulocytes), noting the number that are reticulocytes.
Calculate the percentage of reticulocytes.

Appearance of reticulocytes

Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the form of small granules, distributed irregularly. Mature red cells stain pale green-blue.

Counting reticulocytes:

A convenient method of counting reticulocytes is to reduce the size of the microscope field by inserting in each eyepiece a circular piece of black (opaque) paper which has
a punched out hole of about 5 mm.
To calculate % of reticulocytes:
– Using a hand tally counter, count a total of 500 red cells, noting on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a white cell differential counter).
– Multiply the number of reticulocytes counted by 2.
– Divide the figure by 10 to obtain the percentage figure.

See also:

Reticulocytes count principle

SOURCES OF ERROR WHEN MEASURING ESR

● Using the wrong volume of blood to anticoagulant.

● Blood not sufficiently mixed with anticoagulant.

● Clots in the blood. Even the smallest fibrin clot in the sample will invalidate the test result.

● Air bubbles at the top of the column.

● Testing blood samples at the hottest time of the day, or leaving tests in direct sunlight. Temperatures over 25°C increase sedimentation.

● Using a pipette which is not clean or not dry.

● Pipette not positioned vertically. Even slight variations from the upright increase sedimentation.

● Not checking whether the ESR stand is level on the bench.

● Placing an ESR stand on the same bench as a centrifuge where vibration will interfere with sedimentation.

● Measuring the ESR when a patient is dehydrated.

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

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.