iii. Coagulation factors.
These act together in co-ordination to arrest bleeding. A breakdowns in the normal coagulation mechanism result in abnormal tendency to bleeding and may result from the defect in any one of these three components. Bleeding is especially liable to occur when one or more than one component is defective.
The various haemorrhagic disorders can result in uncontrolled haemorrhaging into joints, muscles and deep tissues with formation of haematomas. The laboratory investigations are carried out for patients who have a history of spontaneous bleeding after trauma or surgery or patients who have to undergo a major surgery.
The haemorrhagic disorders are either inherited or acquired.
Inherited Haemorrhagic Disorders:
These are transmitted as sex linked disorders and mainly observed among males, while females act as carriers. These disorders are mainly due to deficiencies of factors VIII and IX. Other inherited disorders of coagulation include factor XI deficiency which is transmitted as autosomal disorder and occur with equal frequency in males and females.
Acquired Haemorrhagic Disorders:
The commonest causes of acquired bleeding disorder includes:
1. Thrombocytopenias due to many reasons such as infections, malignant, drug induced immune and non-immune.
2. Deficiency of vitamin K.
3. Liver failure.
4. Therapy of heparin and coumarin group of drugs.
Screening Tests of Haemorrhagic Disorders:
These are non-specific tests designed to assess overall coagulation mechanism and are useful for the screening of patients who may have a bleeding disorders. Although these tests are simple, they lack sensitivity and it does not necessarily mean that a patient has a normal coagulation mechanism if the results of the tests are all normal. The following screening tests are recommended.
i. Bleeding time
ii. Whole blood coagulation time
iii. Clot retraction
iv. Prothrombin time
v. Platelet count
vi. Tourniquet test
Besides these, the routine haemorrhagic disorder tests include—Plasma recalcification time, Partial thromboplastin time, Activated partial thromboplastin time, Thrombin sulfate test and Fibrinogen determination are used to screen haemorrhagic disorders.
The duration of bleeding from a standard punctured wound of the skin is the bleeding time. It is the measure of function of platelet as well as integrity of the blood vessel wall.
A standard cut is made in the skin of patient and the length of time required to cease (to stop) the blood is noted. There are two methods for detecting the bleeding time:
i. Dukes method
ii. Ivy’s method
1. Dukes Method:
It requires sterile needle, filter paper, stop watch, spirit/alcohol.
Clean the ear lobe or fingertip with spirit by using a cotton swab. For ear lobe, a glass slide is placed behind the ear lobe to hold it firmly in place. This provides a firm site for incision. Puncture the ear lobe deeply, about 1 mm by using sterile lancet/needle. Start the stopwatch.
The blood should flow freely without pressing the ear lobe. Now remove the glass slide from ear lobe after pricking. The blood is allowed to drop on filter paper. The paper should be moved so that each drop will fall on fresh area. The bleeding of wound should be allowed without pressing.
When bleeding slows, the wound is touched gently with a fresh area of filter paper at 30 sec. interval. When blood stains disappear from the filter paper, stop the watch and note the time.
1. In case of children heel should be used.
2. In suspected cases of bleeding disorder, puncture the fingertip.
3. The area to be punctured should not be congested.
4. The size and depth of wound should be standard.
5. If bleeding continues for more than 15 min, it should be stopped by placing dry sterilized cotton and applying little pressure on the wound.
Normal value: 1 to 5 min.
2. Ivy’s Method:
Ivy’s method requires sphygmomanometer cuff, which is applied on the patient’s arm above the elbow. The puncture site is above the forearm. As soon as bleeding starts, touch the filter paper and start the stopwatch. When the blood drop on filter paper disappears, record the time.
Normal value: 5 to 11 min.
Prolonged bleeding time (BT) is generally found in thrombocytopenia (Pletlets below 50,000) in case of Vonwillebrand disease, the BT is high. It is also high in case of severe liver disease, leukemia, aplastic anaemia, etc.
BT can also be calculated by:
BT = 30.5 ? Platelet count / mm3 / 3,850
Whole Blood Coagulation Time:
The time required for blood to clot out of the blood vessel at 37°C, is known as clotting time. There are two different methods to determine clotting time:
1. Capillary method / Wright method
2. Vein punctures method / Lee-White method
Whole blood when removed from vascular system and exposed to foreign surface will form a solid clot. In Wright method, the blood is collected in capillary tube by finger puncture and in Lee-White method the blood is collected from vein in a test tube.
1. Capillary method/Wright method:
By using spirit, disinfect the tip of finger of patient and make about 1 mm deep cut with sterile lancet. Start the stopwatch, wipe-off the first drop of blood, and collect blood in capillary tube upto 2/3rd of its length.
After every 30 sec. break off about 1 cm of capillary to find out whether fibrin string /thread has formed. When fibrin string appears, stop the stopwatch and note the time.
The capillary method for clotting time can be advantageously combined with bleeding time test.
2. Vein puncture/Lee-White method:
Collect about 2 ml of blood in a syringe and dispense into a clean test tube. Start the stopwatch as soon as the first drop of blood is observed. Plug the tube with cotton and keep them into the water bath at 37°C. After every 30 sec. by tilting the tube find out whether the blood is clotted. When the blood has clotted, stop the watch and note the time.
Blood collected in a test tube should be above 1 ml. As less blood gives shortened clotting time. The water bath temperature should be 37°C ± 0.5°C. The venous puncture should be without trauma.
Vigorous agitation of tube is avoided to avoid air bubble in the tube.
Normal value (for both methods) = 5 to 10 min.
Severe deficiencies of any of the coagulation factor indicate prolongation of clotting time.
Prolonged clotting time is noted in a fibrinogenemia and hyper heparinema.
Lee-White method is more reliable than Wright method. Therefore Wright method should be used only when venous blood cannot be obtained.
A colt forms as the end product of blood coagulation. The clot under normal condition undergoes contraction, where serum is expressed from clot, and a clot becomes denser. This is called as clot retraction. Normal clot retraction starts within 30 sec. after the blood has clotted and is about 50% at the end of 1 hour.
Clean dry plane glass graduated centrifuge tube, water bath at 37°C, equipment for collecting the blood, timer, etc.
Take 5 ml blood by venous puncture, and transfer it to centrifuge tube. Incubate it at 37°C in vertical position. Record the degree of retraction after 1, 2 and 4 hours. If contraction is not apparent at the end of 1 hour loose the clot gently from the wall of the test tube.
The degree and rate of retraction should be noted. Also note the discoloration of serum. Measure the amount of serum expressed by the clot.
After 1 hour of blood collection, the clot should express 50% of serum.
Clot retraction is directly related to platelet count therefore, it is decreased in thrombocytopenia but normal in haemophilia.
It is requested in case of suspected haemorrhagic disorders.
Precaution should be taken at the time of removal of clot so that serum should not be haemolysed. Generally, clot is removed by using a hooked long needle. If the clot is small and serum is more, then, patient may have low haematocrit.
Patients with polycythemia also have poor clot retraction. Fragile clot represents low fibrinogen values.
Prothrombin time (PT) is the time required for clotting of citrate or oxalate plasma in a glass tube, after the addition of calcium and tissue thromboplastin.
Stage 1 is crossed by the addition of tissue thromboplastin. The time it takes for plasma to clot after addition of thromboplastin and calcium is a measure of substances in the stage 2 and Stage 3.
Out of these factors, the most important is prothrombin. Prothrombin time should be performed soon after blood is collected and plasma is obtained, i.e. within 1/2 hr. of blood collection.
Take 4.5 ml of patient’s blood in a clean test tube with 0.5 ml of oxalate or citrate anticoagulant. Mix well and centrifuge at 2000 to 3000 rpm for 10 min. to get plasma. In other test-tube take 0.1 ml of thromboplastin suspension and 0.15 ml calcium chloride (CaCl2) suspension.
Mix the tube, keeps both the tube at 37°C in water both. After 1 min, contents of second tube are mixed with 0.1 ml of plasma from the first tube, as soon as the contents are added, start the stopwatch and observe for plasma colt. If clot is observed, stop the stopwatch and note the time. This is the prothrombin time.
Normal value: 14 ± 2 seconds.
The commonest causes of pro-thrombin time prolongation are liver disorder, vitamin K deficiency, Haemolytic disease of newborn (HDN), etc.
Determination of platelet count is requested in the investigation of bleeding disorders. There are two methods by which we can get total platelet count. These are:
i. Direct method
ii. Indirect method
In this method, the blood is diluted with platelet diluting fluid and the cells are counted over a specified area under proper magnification.
Requirement Microscope, improved Neubauer chamber, RBC pipette, platelet diluting fluid.
i. Procaine hydrochloride – 3.0 gm.
ii. Sodium chloride – 10 gm.
iii. Distilled water – 100 ml.
Blood specimen EDTA anti-coagulated blood or capillary blood also can be used.
Mix the blood specimen carefully by using RBC pipette. Draw blood up to 0.5 marks. Wipe off the excess blood from outside of pipette. The diluting fluid is drawn upto 101 mark. This gives the blood dilution 1: 200. Mix the contents in the bulb thoroughly.
After 5 to 7 min. discard the first drop, and by using the second drop, charge the chamber. After charging keep the chamber undisturbed for 10 to 15 min. This will allow the settling down of cells. Count the cells in the central square of Nuberurs chamber, i.e. in all 25 squares.
No. of platelets / cu mm = No. of platelets counted ? Dilution / Volume of fluid
Where, Dilution = 200
Volume of fluid = Area ? depth
= 1 ? 0.1
= 0.1 cu mm
= o. of platelets ? 200
Platelets / cu mm = No. of platelets ? 2000
Dissolve 14 gm of Magnesium sulphate in distilled water and make up 100 ml.
Clean the fingertip and place a drop of 14% aqueous solution of magnesium sulphate on the skin. Puncture through this drop with little pressure and allow the blood to flow into the sulphate solution.
When the proportion is about 1 of blood to 5 of magnesium sulphate, mix thoroughly and make a thin smear. Clean the finger and make a RBC count. Stain the smear with Leishman’s ‘Blood Stain’. Cut a small square in a circular piece of paper and place it in the ocular of the microscope to reduce the size of the field.
Count the number of RBCs and the platelets in the field. Count 1000 RBCs. The number of platelets counted to 1000 RBCs is multiplied by the number of thousands of RBCs as determined by RBC count.
Platelets/cu mm = No. of Platelets ? RBC’s / 1000
Normal Value: 1.5 to 4 lakh/cu mm.
Increased platelet count is often associated with polycythaemia Vera, following splenectomy and in chronic myelogenous leukaemia.
This is called as thrombocytosis. Thrombocytopenia, i.e. decreased platelet count is often associated with prolonged bleeding and poor clot retraction, aplastic anaemia, megaloblastic anaemia, hypersplenesion, acute leukaemia and in immune thrombocytopenia.
Tourniquet test is one of the bleeding disorder tests, done by the physician and not by the technician. In this test, the blood pressure of the patient both the systolic as well as diastolic is measured.
The average is calculated, and a sphygmomanometer cuff is applied to the patients arm as in with average of blood pressure. Now, the hand is observed for internal capillary bleeding. This can be observed as red patches on the skin.
Purpura is a skin rash resulting from bleeding into skin from small blood vessels, i.e. capillaries. This results in individual purple spots of the rash called as petechiae. Purpura may be either due to defect in capillaries, called as non thrombocytopenic purpura or due to a deficiency of blood platelets, i.e. thrombocytopenic purpura.
Acute iodopathic thrombocytopenic purpura is a disease of children in which antibodies are produced that destroy the patient’s platelets. The child usually recovers without treatment. Schonlein-Henoch purpura is also seen in young children with unknown cause. It is associated with abdominal pain and kidney disturbance.