BLEEDING TIME

(Duke's Method)
Principle
The bleeding time is the time it takes for a small standardized wound, introduced into the capillary bed of the finger or ear lobe,
to stop bleeding. It is dependent upon the elasticity of the skin and capillary vessels, the efficiency of the tissue fluids and the
mechanical and chemical action of the thrombocytes (blood platelets).
APPARATUS
Finger puncture material
Filter paper
Stop watch
TECHNIQUE
1. Make a small wound 3 mm deep in the lateral aspect of a fingertip or the lower portion of the ear lobe, using a
suitable lancet. The wound should be sufficiently deep to give a free flow of blood without squeezing.
2. Start the stop watch immediately after the puncture is made. Though the stop watch is started a few seconds after
puncturing the skin, very little error results in the bleeding time.
3. At intervals of one-half minute gently blot the blood from the wound with a piece of filter paper, being careful not to
touch the skin.
4. At the time when blood fails to appear on the filter paper, stop the stop watch.
5. The interval of time between the puncture and the cessation of bleeding is the bleeding time. The blood should fail to
appear on the filter paper in 1-3 minutes. All abnormal findings should be rechecked.

NORMAL
1-3 minutes

DISCUSSION
1. For this test to have diagnostic significance, it is necessary that a standardized wound be made. Too deep a wound
will prolong the bleeding time while a shallow wound will shorten the time necessary for hemostasis.
2. A single prolonged bleeding time is not significant due to the difficulty of producing a standardized wound. If a
prolonged bleeding time is encountered, additional determinations should be made.
3. In the event the puncture site is cold, there will be a constriction of the capillary vessels, resulting in a decreased
blood flow. In such cases, the area should be warmed to normal body temperature prior to performing the puncture.
4. The bleeding time depends primarily on extravascular and vascular factors and, to a lesser degree, on the factors of
coagulation. The chief factor controlling bleeding from a small cut is the constriction of the minute vessels following
injury. Accuracy in this test may be enhanced by blotting the drops of blood at shorter intervals of time as the drops
of blood become progressively smaller.
5. Thrombocytes play an important part in the formation of the hemostatic plug which seals off a wound. In
thrombocytopenic purpura there is a decrease in platelets resulting in a prolonged bleeding time due to a defective
platelet plug. An additional factor prolonging the bleeding time in this condition is a defect in capillary contraction.
6. In hemophilia the bleeding time is normal. This is explained by the fact that there are no vascular or extravascular
abnormalities. However, the test should not be performed on a known hemophiliac, for delayed oozing of blood is a
real hazard.
7. Do not use a needle for the puncture, since a round wound does not bleed well enough to produce a sensitive test.
8. Factors influencing proper performance of the bleeding time include skin temperature, circulation, area punctured,
thickness of skin and depth of puncture.

SOURCES OF ERROR
1. Squeezing the finger or ear lobe.
2. Improper puncture.
3. Low skin temperature.
4. Use of a needle for the puncture.
5. Improper depth of wound.
 BLEEDING TIME

(Ivy Method)

Principle

The bleeding time is the time it takes for a small standardized wound, introduced into the capillary bed of the finger or ear lobe,
to stop bleeding. It is dependent upon the elasticity of the skin and capillary vessels, the efficiency of the tissue fluids and the
mechanical and chemical action of the thrombocytes (blood platelets).
APPARATUS
Blood pressure cuff
Sterile, disposable blood lancet, capable of a puncture 5 mm wide and 1 mm deep
Stopwatch
Circular filter paper

TECHNIQUE

1. Cleanse the area 3 fingerwidths below the antecubital space with alcohol and allow to dry.
2. Place a blood pressure cuff on the arm above the elbow and inflate to 40 mm Hg.
3. Select a cleansed area of the forearm without superficial veins. Stretch the skin laterally and tautly between the
thumb and forefinger.
4. Start a stopwatch. Use the edge of a 4x4-inch filter paper to blot the blood through capillary action by gently touching
the drop every 30 seconds. Do not disturb the wound itself. Remove the blood pressure gauge when bleeding stops
and a clot has formed. Apply a sterile dressing when the test is completed.
5. Remember that the end point is reached when blood is no longer blotted from the forearm puncture. Report in
minutes and half minutes (eg, 5 minutes, 30 seconds).
NORMAL
1-7 minutes

INTERFERING FACTOR
1. Normal values for bleeding time vary when the puncture site is not of uniform depth and width.
2. Touching the puncture site during this test will break off fibrin particles and prolong the bleeding time.
3. Excessive alcohol consumption (as in alcoholic patients) may cause increased bleeding time.
4. Prolonged bleeding time can reflect ingestion of 10 g of aspirin as long as 5 days before the test.
5. Other drugs that may cause increased bleeding times include dextran, streptokinase-streptodornase (fibrinolytic
agents), mithramycin, pantothenyl alcohol.
6. Extreme hot or cold conditions can alter the results.
7. Edema of patient's hands or cyanotic hands will invalidate the test.

SOURCES OF ERROR
1. Squeezing the finger or ear lobe.
2. Improper puncture.
3. Low skin temperature.
4. Use of a needle for the puncture.
5. Improper depth of wound.
 COAGULATION TIME
(Lee-White Method)
Principle
The coagulation time is the interval required for venous blood, in the absence of all tissue factors, to clot under controlled
condition.
APPARATUS
Venipuncture material
Pyrex tubes (13x100 mm with 11 mm internal diameter)
Stop watch

REAGENT
Saline, physiological, 0.85 percent

TECHNIQUE
1. Rinse a syringe and three scrupulously clean pyrex test tubes of the size designated, with physiological saline.
2. Apply the tourniquet just prior to the venipuncture.
3. Using gentle traction on the syringe plunger, draw 4 ml of blood from a nontraumatic venipuncture. As soon as blood
first appears in the neck of the syringe, start the stop watch and release the tourniquet. If the vein is not entered
quickly and without trauma, repeat the venipuncture.
4. Remove the needle and introduce exactly 1 ml of blood into each of three test tubes and discard the last 1 ml which
contains tissue fluid. Allow the blood to run down the sides of the tubes to prevent agitation which will hasten
coagulation.
5. Immediately place the tubes in a 37°C water bath. (Coagulation time is delayed when the test is performed at room
temperature).
6. Wait 3 minutes, then tilt the first tube at intervals of one-half minute until it can be completely inverted without loss
of the blood.
7. Now invert the second tube at one-half minute intervals until it can be inverted without loss of the blood.
8. Repeat the process with the third tube. When the third tube can be inverted without loss of blood, stop the stop
watch.
9. The interval of time between the appearance of blood in the syringe and coagulation in the third tube is the
coagulation time.
NORMAL VALUES
37°C 5-10 minutes

DISCUSSION
1. This test is of value primarily as a screening procedure in an effort to determine anticoagulant activity and plasma
coagulation defects. Additional tests are needed to identify the specific defects.
2. A prolonged clotting time immediately indicates impaired coagulation but a normal clotting time does not exclude
many serious clotting defects.
3. The determination of the coagulation time is the simplest, and the most informative of all common laboratory
procedures routinely performed in blood evaluation. Values varying by as much as 40 percent may occur unless all
precautions in the procedure are carefully observed.
4. There are many factors which affect the laboratory determination of the coagulation time. Rough handling of the
blood, presence of tissue fluids (traumatic venipuncture), and narrow and or unclean tubes will tend to hasten the
coagulation time. Extreme temperatures or pH or the use of silicone tubes or paraffin tubes tend to prolong the
coagulation time.
5. Three tubes are used in this test since the blood in the first two tubes is subjected to agitation (tilting of the tubes)
which hastens the coagulation process. The third tube, which is subjected to the least amount of agita tion, gives the
most accurate coagulation time test results.
6. The method of measurement of the coagulation time by the use of capillary blood drawn into a capillary tube is
completely unreliable and should never be done,  since the sample contains a large amount of tissue fluid.
 Furthermore, the volume of blood in a capillary tube is difficult to control. A capillary tube technique using  venous
blood has recently been described as having a high degree of accuracy.
7. A 15-20 minute coagulation time is suggestive of hemorrhagic tendency. Should it be prolonged beyond 20 minutes, it
is indicative of hemorrhagic disease.
8. In hemophilia there is a prolonged coagulation time. This is due to a deficiency of a plasma thromboplastin substance
(AHG).
9. The coagulation time is normal in thrombocytopenic purpura. This is explained by the fact that only a small number of
thrombocytes need be present for normal coagulation to take place.
10. When heparin is administered as a therapeutic anticoagulant, its effect may be determined by the degree of
prolongation of the coagulation time.

SOURCES OF ERROR
1. Traumatic venipuncture.
2. Inadequately cleaned test tubes.
3. Prolonged application of the tourniquet.
4. The presence of bubbles in the syringe or test tubes.
5. Loosely fitting syringe and needle.
6. Test tubes of varying sizes.
7. Varying amounts of blood in the test tubes.
8. Variation in temperature or pH.
9. Unnecessary agitation of the sample.
 CLOT RETRACTION
(MacFarlane's Method)
Principle
This test evaluates the functions of thrombocytes. A measured amount of blood is allowed to clot around a wire hook in a
graduated centrifuge tube. After coagulation, the clot is removed and volume of serum measured. The volume of serum is
reported as a percentage of the total possible serum obtained by a hematocrit determination. This test measures the degree of
contraction of the clot in a specified period of time.
APPARATUS
Venipuncture material
Centrifuge tube, graduated
Cork with wire hook attached
Hematocrit tube
Test tube with Heller-Paul anticoagulant

TECHNIQUE
1. Draw 6 ml of venous blood. Place 4 ml in a clean dry graduated centrifuge tube. Place the remainder of the blood into
the anticoagulant tube and mix well.
2. Place the cork holding the wire hook in the mouth of the centrifuge tube so that the wire extends well beneath the
surface of the blood.
3. Place the centrifuge tube in a 37°C water bath until the clot forms. Allow the tube to remain in the water bath for an
additional hour.
4. Carefully free the clot from the sidesof the centrifuge tube. Allow the clot to drain well on the side of the tube then
remove it from the serum.
5. Centrifuge the serum-cell mixture at 2,000 rpm for 15 minutes.
6. Obtain the packed cell volume and the total volume from the graduations on the centrifuge tube. Subtract the cell
volume from the total volume to determine the volume of expressed serum.
7. Fill the Wintrobe hematocrit tube from the well-mixed anticoagulated specimen and centrifuge at 3,000 rpm for 30
minutes. Read the percent ofplasma.
8. Calculate the percent serum expressed according to the formula given in the calculations.
CALCULATIONS

Volume of expressed serum x 100

= percent serum expressed
Percent plasma ( hematocrit ) x total blood volume ∈centrifugetube
EXAMPLE:
Volume of expressed serum: 2.0 ml
Hematocrit: 55.0% plasma, 45% total cells
Total blood volume in centrifuge tube: 4.0 ml

2.0 x 100
=91 % serum expressed
0.55 x 4.0
DISCUSSION

1. This procedure is basically that of McFarlane but includes the correction for hematocrit and for red cells not trapped
in the clot. The percentage of plasma (derived from the hematocrit) serves as an index of the total fluid which could
be expressed from the clot. These refinements increase the accuracy of the procedure.
2. When the rate of clotting is unusually slow or imperfect, the method is unreliable, since incomplete coagulation
renders the clot nonadherent and easily displaced.
3. Positive correlation of platelet numbers and clot retraction is such that a decrease in clot retraction (expressed
serum) is a more reliable index of thrombocytopenia than the platelet count. A normal degree of clot retraction is
strong evidence that the thrombocyte count is normal.
 4. The clot retraction is normal in hemophilia since there is a normal number of platelets. However, the onset of
contraction is often delayed in blood samples from hemophiliac patients.
5. A screening method of clot retraction may be done by placing the blood sample, maintaining sterile technique, in a
dry test tube which is then incubated. The time that retraction begins (30-60 minutes) and the degree of retraction
(poor, partial, complete) in 24 hours is observed.
SOURCES OF ERROR
1. Dirty glassware
2. Maceration of clot during extraction.
3. Errors associated with performance of hematocrit.
4. Presence of residual anticoagulants in centrifuge tube.
5. Presence of plasmin (fibrinolysin) in specimen.
6. Inaccuracy in reading.