MLS 12Aa: Hematology 2 (Laboratory)

MODULE 2: BLEEDING TIME AND CLOTTINFG TIME Additional Notes (Fortuno):

Concept Map
î After learning the different platelet
disorders, it is important to know what are
the tests that may aid in diagnosis of
qualitative platelet disorders.
î In this module, we will learn about
the two original tests for platelet function
which are the bleeding time and clotting
time.
î Even though these two tests are
considered obsolete these are still
encountered in the clinical setting for a
more rapid assumption of bleeding
tendencies as a part of preoperative
screening along with prothrombin time
and activated partial thromboplastin time
results.
. bleeding
REVIEWED TOPICS:
1. Mechanism of Hemostasis
2. Differences between Primary and Secondary Hemostasis
3. Steps/Sequence of Hemostatic Plug
BLEEDING TIME
Bleeding time and clotting time are being requested together in the laboratory
9 CTBT blood test
Bleeding Time Clotting Time
Duke Method Capillary Method
Ivy Method Drop/slide Method
Other methods: Lee-White Clotting Time Method /
9 Mielke Method (Template Whole Blood Clotting Time / Venus
bleeding time) Coagulation Time
9 Simplate Method
9 Surgicutt Method
Bleeding time vs clotting time
Bleeding Time Clotting Time
time it takes for a small standardized length of time required to form clot in
wound, introduced into the capillary vitro under standard condition
bed of the finger or earlobe, to stop
length of time that the bleeding stops length of time that the fibrin clot will
9 when the bleeding stops, it form
doesn’t mean that there is a
clot formation
9 maybe when the bleeding
stops, the platelets starts to
form the platelet plug
assesses the primary hemostasis assesses the secondary hemostasis
9 ability of the platelets to 9 formation and ability of the
form the platelet plug to clotting factor to form a clot
stop the bleeding initially 9 coagulation (clotting)
9 platelets and capillary factors and platelets
vessels (endothelial cells)
9 function and number of
platelets (quantitative and
qualitative)
do not wipe the first drop of blood wipe the first drop of blood

Additional Notes (Tinasas):

How does bleeding time differ from Platelet Function Test?
9 In Platelet Aggregometry criteria, platelet count must exceed 50,000 Bleeding Time Clotting Time
platelets/uL and patients must experience mucocutaneous bleeding. 9 platelets 9 platelets
9 Bleeding count is affected by platelet count (thrombocytopenia)platelet 9 endothelial cells 9 clotting factors
function and lastly the platelet morophology. 9 primary hemostasis 9 secondary hemostasis
9 In platelet aggregometry, the only issue is the platelet function only and 9 bleeding will stop 9 fibrin clot fill form
platelet count must exceed 50,000 platelets/uL. 9 platelet plug 9 fibrin clot

PADAYON, RMT QUITAG, BULQUERIN, DEALA 1

 MLS 12Aa: Hematology 2 (Laboratory)

DISCUSSION Differences between Duke and Ivy method
î In order to detect qualitative platelet abnormalities
o platelet function tests are designed for patients who are experiencing Duke Method Ivy Method
symptoms of mucocutaneous bleeding preferred
type of lancet used normal lancet feather lancet
î Platelet count is an essential laboratory test number of punctures 1 2
o to perform before the analysis of platelet function location of puncture site
rd th
3 or 4 finger forearm just below the
o since thrombocytopenia is the common cause of hemorrhage earlobe antecubital fossa
pressure not standardized standardized
î Platelet morphology is also noted in cases of Bernard-Soulier Syndrome uses a blood pressure cuff
o which reveals large gray platelets on the peripheral blood smear and (sphygmomanometer)
thrombocytopenia maintained at 40 mmhg
o only in the presence of bleeding symptoms and a platelet count that
clotting time can be clotting time cannot be
exceeds 50,000/uL is suggestive of qualitative platelet disorders
performed using performed using the specimen
the specimen
Principle:
î Bleeding time
o the time it takes for a small standardized wound, introduced into the Procedure: Duke Method
capillary bed of the finger or earlobe, to stop bleeding Materials needed:
î 70% alcohol
î This test both reflects platelet number and platelet functional integrity î stopwatch – for the time
î lancet
î It is dependent upon the î filter paper – to blot the blood
o elasticity of the skin and capillary vessels î cotton – for aftercare
o the efficiency of the tissue fluids
o the mechanical and chemical action of thrombocytes Procedure:
1 Disinfect the 3rd or 4th finger or earlobe (puncture site) using 70%
2 MAIN METHODS: alcohol.
1. Duke Method 2 After drying, use the sterile lancet to puncture the selected site.
• first describe by Duke in 1912 3 Start the timer as soon as the blood is seen.
• involves the use of a lancet to make an incision either on the finger or on 4 Blot the blood using filter paper every 30 seconds, be careful not to touch
the earlobe the skin and do not apply pressure on the puncture site. Let the blood
• a drop of blood is blotted on to the filter paper every 30 seconds until the flow on its own.
blood ceases to flow 5 Stop the timer at the time blood does not appear on the filter paper.
• this is not a recommended method because it is not precise or accurate 6 Perform after care to your patient.
• still preferred by the medical technologists because clotting time can be 7 Record the bleeding time.
performed – isa nalang ka puncture sa patient
Normal value: 1-5 minutes
2. Ivy Method
• was modified by Ivy in 1941
• involves the use of a blood pressure cuff inflated at 40 mmHg
• two separate cutes in the forearm are made and blood is blotted on to
the filter paper every after 30 seconds until the bleeding stops
• the length of time required for the bleeding to stop is recorded as the
bleeding time.
• patients with high blood pressure will falsely prolonged BT
• indi pwede masabay ang clotting time kay ma puncture ka pagid liwat
sa finger

PADAYON, RMT QUITAG, BULQUERIN, DEALA 2

 MLS 12Aa: Hematology 2 (Laboratory)

note: Procedure: Ivy Method
9 just open the lancet once you are ready to puncture Materials needed:
o do not expose to avoid contamination î 70% alcohol
î stopwatch – for the time
9 puncture on the sides of the fingertips and perpendicular to the lines î feather lancet
o to avoid the nerve endings of the patient î sphygmomanometer
o also, for it to become less painful for the patient î filter paper – to blot the blood
o to avoid the possibility that the lancet will slide into the nailbed î cotton – for aftercare

9 do not wipe the first drop of blood

o allow the blood to flow freely Procedure:
o start the timer immediately after you puncture 1 Disinfect the selected site in the forearm with 70% alcohol.
2 Position the sphygmomanometer in the upper arm and inflate it to
9 blot the blood in the filter paper after 30 seconds 40mmHg, maintaining the pressure until the test is done.
st
o 30 seconds – 1 blot 3 Make two separate cuts into the forearm usually 5-10 cm apart in quick
nd
o 1 min – 2 blot succession.
rd
o 1min and 30 secs – 3 blot 4 Start the stopwatch immediately and every 30 seconds, filter paper is
th
o 2 mins – 4 blood used to draw off the blood.
o continue until no blood appears on the filter paper 5 Record the time at which blood ceases to flow.
o based on the photo, every blot of the blood represents 30 seconds
Normal Value: 2 – 9 minutes
9 avoid touching the skin and applying pressure on the puncture site
o do not squeeze the puncture site unlike in capillary puncture note:
o in order not to disturb or dislodge the formation of platelet plug 9 puncture site -- forearm
o to avoid falsely prolonged BT o just below the antecubital fossa about 3-4 fingers
9 recording of results 9 make sure that there is no hair on the puncture site
o because it will be hard to blot the filter paper

9 choose a site where there is no vein

o to avoid falsely prolonged BT
o damo blood
o capillary blood is needed not venous blood

9 make 2 separate cuts in the forearm in quick succession

nd st
o puncture the 2 cut immediately after the 1 one
o not after 5 secs

9 make 2 separate cuts in the forearm

o to compare the results of the 2
o to make sure that the results are accurate
BT: 2mins, normal
9 maintain the pressure at 40mmHg for Ivy method

9 the sphygmomanometer maintains the constant pressure in the capillaries

o in order to standardize the procedure
o because the first factor that may affect the bleeding time is the
intercapillary pressure

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 MLS 12Aa: Hematology 2 (Laboratory)
FACTORS AFFECTING BLEEDING TIME:
î Intercapillary pressure
o eliminated upon standardization of the method in Ivy Method
o pressure should be maintained at 40 mmHg to maintain consistent in
their capillary
î Size and depth of the wound
o bleeding time can be affected by the size and the depth of the wound
o spring loaded
9 equipment that is used to control the depth of the wound
o depends on the medical technologist on how he/she punctures the site
9 kis-a na umol-umol si Medtech sa patients
9 so ang tendency dalumon or duuton ni Meddtech and lancet sa
wound
9 as a result, there is an increased blood flow in the injury leading
to prolonged bleeding time
î Efficiency of the tissue fluids in accelerating the clotting process
î Thickness and vascularity of the skin
o in heavily callous patients, there will be an increased wound depth to
reach the capillary bed
o use the other hand if the skin is thinner
î Chemical and mechanical actions of platelets
o since bleeding time tests for the functional integrity of the platelets
o bleeding time may also be affected by the number of platelets because
bleeding time also reflects the platelet number
î Ability of the blood vessels to constrict and retract
o because here in bleeding time we test for primary hemostasis
o components involved in primary hemostasis are the vasculature (blood
vessels) and platelets
PADAYON, RMT
note:
9 Duke method is more affected than Ivy method by the differences in
intercapillary pressure
o since the procedure in Duke method is not standardized as compared
o with Ivy method
9 there is a recommended puncture depending on age
o for children and adults, we have the recommended puncture which is
2.4mm – 3mm
o for infants the recommended puncture is 1.6 mm
o these depths depending on the age are recommended in order to hit the
capillary bed
9 avoid heavily calloused fingers or puncture sites
EXAMPLES OF DISEASE/CONDITIONS THAT MAY YIELD ABNORMAL
BLEEDING TIME RESULTS:
î Von-Willebrand’s Disease
o deficiency, absence or problem in the VWF function
o VWF is needed for platelet adhesion in primary hemostasis
o if the platelets fail to adhere in the subendothelial collagen or
endothelium, then there will be a problem in the formation of platelet
plug
o prolonged BT
î Bernard-Soulier Syndrome
o problem in GP Ib
o GB Ib is a receptor for VWF in the platelets
o bisan normal si VWF kag absent ang receptor, VWF will not bind into
the platelet thus platelets will not adhere into the endothelium
o there is a problem in platelet adhesion in primary hemostasis
o prolonged BT
î Glanzmann Thrombasthenia
o absence of GP IIa/IIIa of the platelet cell membrane
o GP IIa/IIIa is a receptor for fibrinogen
o in order for the platelets to attach or aggregate with each other,
fibrinogen should bind to the platelet cell membrane
o fibrinogen serves as a bridge to the platelet
o fibrinogen is needed for platelet aggregation in primary hemostasis
o prolonged BT
note:
9 these conditions are under the qualitative platelet defects and this may also
affect the bleeding time results
9 in addition: Scurvy and Ehlers-Danlos Syndrome
QUITAG, BULQUERIN, DEALA 4
 MLS 12Aa: Hematology 2 (Laboratory)

OTHER METHODS FOR BLEEDING TIME: Surgicutt Method
Mielke Method (Template bleeding time) î utilizes a slicing action using a surgical blade
î a modification of Ivy method î it is spring-loaded and when activated, the blade moves forward to the outside
î utilizes a template containing a standardized slit in place of a disposable lancet of the unit and sweeps across the opening of the bottom of the housing
î a blade is placed in a special handle î the blade automatically retracts into the housing after it has made a standardized
o such that when the template is placed on the forearm, the blade would cut
make an incision having a standardized depth and length

note:
9 we make a slit (slice) instead of just puncturing the skin
9 uses different kind of blades
9 uses a template

CLOTTING TIME

Principle:
î Clotting time of whole blood
o the length of time required to form clot in vitro under standard condition
o assesses secondary hemostasis

note: î In this test, whole blood will form a solid clot when exposed to a foreign surface
9 the illustration above is the instrument used in Mielke method such as glass tube/glass slide

Simplate Method î It is also the time interval from puncture to form fibrin strands
î contains a spring-loaded blade within a plastic case which holds a double blade
î when activated, the blade springs forward from the housing and makes a cut on î this test is used to diagnose and assess bleeding problems and monitor
the skin anticoagulant therapy

3 MAIN METHODS:
1. Capillary Method
2. Drop/Slide Method
3. Lee-White Clotting Time Method / Whole Blood Clotting Time / Venus Coagulation
Time

Capillary Method Drop/Slide Method Lee-White Method

uses only 1 specimen uses 3 drops uses 3 tubes
capillary tube glass slide
tests the first part of the tests the last part of the capillary puncture
capillary puncture
note: more tissue factor present less tissue factor present
9 the spring makes the method standardized
9 the depth of the wound is standardized by the spring extrinsic pathway intrinsic pathway
9 contains 2 lancets forming 2 punctures (no need to puncture twice)

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 MLS 12Aa: Hematology 2 (Laboratory)

Capillary Method note:
î applicable only to infants and patients receiving heparin therapy 9 use PPE
rd th
o small amount of blood is needed 9 prick on the 3 or 4 finger, heel or earlobe
o Heparin: anticoagulant which targets factor II and factor X that belong 9 wipe off first drop of blood
to common pathway o to eliminate contamination of tissue factors that may cause shortened
result
î evaluates the extrinsic pathway of coagulation 9 collect using non-anticoagulated capillary tube (blue tip)
9 set aside the portion of the capillary tube
î since capillary method tests for extrinsic pathway, the results of patients o once a fibrin strand forms, stop the timer and count the broken parts of
receiving heparin therapy will not be affected the capillary tube

note:
9 the first sample testing from capillary puncture—the sample is still full of tissue
factor

Materials Needed: **Fibrin Strand Bridging the two

î 70 % alcohol broken ends of the Capillary**
î Lancet
î Stopwatch
î Cotton
î Non-anticoagulated capillary tube (blue-tip)

Procedure:
1 Prepares the materials required for the test.
2 Identifies the patient prior to collection.
3 Explains the procedure.
4 Selects and warms the puncture site. SOURCES OF ERROR:
5 Cleanses the incision site. Allows alcohol to dry. î Failure to wipe off the first drop of blood
6 Puncture the site. o this may be contaminated by tissue factor through pricking
o and may yield to shortened clotting time
7 Wipes off the first drop of blood.
8 Starts the timer as soon as the second drop of blood appears.
î Underfilled capillary tube
9 Fills a plain capillary tube with blood.
o will not give you enough segments to cut
10 Breaks off a portion of the tube every 30 seconds. o length needed to cover the capillary tube is about ¾ to provide
11 Stops the timer as soon as fibrin strands are seen bridging the two adequate of sample
broken ends of the tube. o should avoid bubbles
12 Applies after care.
13 Record clotting time accurately. î Failure to keep track of time
14 Identifies and segregates wastes generated and disposes them o each portion we break represents 30 seconds and we should not
properly. dispose these immediately

Normal Value: 3-7 minutes î Skin thickness at the puncture site

o too deep
o too narrow
o resulting to erroneous results

cut/break the capillary tube every 30 seconds

note:
9 avoid cyanotic areas, edematous areas and sites na kapila na ma-puncture

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 MLS 12Aa: Hematology 2 (Laboratory)

Drop/Slide Method 9 make sure to turn off electric fan or aircon when doing this test
î used to evaluate the intrinsic pathway of coagulation which involves Factors o fans and aircon can contribute to easy drying of blood
VII, IX, X, XI and XII o fibrin strands are affected

Materials Needed: SOURCES OF ERROR:

î 70% alcohol î Failure to wipe off the first drop of blood.
î Lancet o this may be contaminated by tissue factor through pricking
î Stopwatch o may yield to shortened clotting time
î Cotton
î glass slides î Contact of the slide to the puncture site
o will cause shortened clotting time
Procedure: o blasts can activate clotting factors faster
1 Adhere to safety precaution by using PPE.
2 Prepare the materials required for the test. î Failure to keep track of time
3 Identify the patient prior to collection.
4 Explain the procedure. î Drying of the blood sample on the slide
5 Select and warm the puncture site. o turn off electric fans and aircon
6 Cleanses the incision site. Allow alcohol to dry. o if nag-dry ang blood sample, you have to recollect again
7 Punctures the site without applying pressure.
Lee-White Method
8 Wipes off the first drop of blood.
î also known as Whole Blood Clotting Time or Venus Coagulation Time
9 Starts the timer.
10 Obtain 3 drops of blood consecutively in a clean dry glass slide not
î first described in 1913
touching the skin. î the first laboratory procedure to assess coagulation in vitro
11 Using a lancet or needle, check fibrin thread formation starting at the î rough measure of all intrinsic clotting factors in the absence of tissue factors
last drop of blood every 30 seconds. Move to the second drop once î variations are wide and the test sensitivity is limited
the fibrin is seen on the last.
12 Stops the timer as soon as fibrin strand has formed on the first drop. î whole blood, when removed from the vascular system and exposed to a
13 Applies after care. foreign surface, will form a solid clot
14 Records clotting time accurately.
15 Identifies and segregates wastes generated and disposes them î within limits, the time required for the formation of the solid clot is a measure
properly. of the coagulation system

Normal value: 2- 6 minutes Principle:

check fibrin thread formation
only at number 3 every 30 î The time interval from the initiation of clotting to visible clot formation reflects
seconds the condition of coagulation mechanism.
î This is not used anymore in laboratory setting because the variations are wide
if fibrin thread is present in 3, and the test sensitivity is limited
1 2 3 check number 2 then 1
Materials Needed:
stop the timer if fibrin strand Venipuncture materials LWCT materials
formed in 1
70% alcohol Plain test tubes with label (Tube 1, Tube 2, Tube 3)
note: Tourniquet Water Bath set at 37°C
9 do not touch the skin while collecting blood 5 cc Syringe Timer/Stopwatch
o to avoid premature activation of contact factors Cotton & Plaster
o which can be activated through contact of negatively-charged surfaces
such as the glass slide

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 MLS 12Aa: Hematology 2 (Laboratory)

Procedure: LWCT 9 how would you know that the blood had already form a clot?
1 Adheres to the safety precautions by using PPE. o clot is formed when the blood doesn’t flow when you tilt the tube
2 Prepares the materials required for the test. o stop the timer when the blood clots in tube 3
3 Identifies the patient prior to collection.
4 Explains the procedure. FACTORS AFFECTING LEE-WHITE COAGULATION TIME
5 Label the glass tubes #1, #2 and #3 and place them in a water bath at 37 î Poor venipuncture technique
C o stitching – if there is an injury, clotting factors will be activated prior
6 Cleanses the puncture site. Allows the alcohol to dry. to blood draw
o repeated insertion of the needle will also activate clotting factors
7 Withdraws 4 ml of blood
8 Starts the timer as soon as blood enters the syringe (backflow).
î Bubbles entering the syringe
9 Delivers 1 ml of blood in each tube starting with tube 3, the tube 2,
o can disrupt the formation of the clot
and tube 1.
10 Discards the last ml of blood. î Diameter of the tube
9 First portion of blood is already contaminated by the tissue factors o If gamay ang diameter, mas close ang blood cells to each other so
and already came in contact with the needle that activates the tubes dasig sila mag-clot
in the water bath after each delivery. o tube serves as the activator of clotting factors
o thin tube will have a closer contact of the blood into the blood, clotting
After discarding the last ml of blood, return the tube in the water bath for factors will be activated thus shortened CT
incubation for 5 mins o use regular test tube
11 Checks for clot formation starting at tube 1 after exactly 5 minutes.
12 Applies after care. î Temperature
13 Records clotting time accurately. o the higher the temperature, the more active the clotting factors
14 Identifies and segregate wastes generated and disposes them properly. o shortened CT
o if you didn’t use water bath, clotting time will prolonged
Normal Value: 5 - 15 minutes o make sure to prewarm and the water bath is in 37°C before collecting
blood and placing the tubes
1 2 3
î Mixture of blood with tissue juice
o shortened CT

Sample Set-up of LWCT:

check clot formation only at tube 1 after exactly 5 mins

9 If wala pa nag-clot si tube 1, indi mag proceed with tube 2 kay we should
check for clot formation in tube 1 first
9 Ibalik sa water bath and wait for the next 30 seconds and check again in one
direction
9 If nag-clot na sa tube 1 proceed with tube 2 without stopping the timer and
check again for clot formation then proceed with tube 3 and if may clot na,
stop the timer

note:
9 in transferring the blood into the tube, don’t do it drop by drop
o this will disrupt clotting

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 MLS 12Aa: Hematology 2 (Laboratory)

SUMMARY o deficiency in clotting factors
o to know what specific clotting factors, do mixing studies
Prolonged Prolonged Normal
Normal BT
BT CT CT
Von Willebrand disease / /
Thrombocytopenia / /
Scurvy
/ /
Cushing syndrome
Vitamin A deficiency / /

note:
9 Von Willebrand factor
o performs two critical functions in primary hemostasis
o acts as a bridging molecule at sites of vascular injury for normal
platelet adhesion, and under high shear conditions, it promotes
platelet aggregation

9 Thrombocytopenia
o decreased platelet count
o affects quantitative and qualitative aspects of platelets

9 Scurvy
o lack of vitamin C
o problem in the production of collagen
o problem with blood vessels

9 Vitamin A deficiency
o hemophilia A
o the body doesn't make enough factor VIII, one of the substances the
body needs to form a clot

9 prolonged BT and CT
o problem with the platelets because platelets are common on both
o problem with platelet number and function
o confirm with platelet count if the problem is with the platelet number
or platelet function
§ if abnormal, the problem is with platelet number
§ if normal, the problem is with platelet function
§ proceed to the peripheral blood smear
9 Prolonged BT, normal CT
o problem with primary hemostasis
o specifically, in capillary vessels (endothelial cells)
o platelets are normal
o elasticity of the skin --- Scurvy (vitamin C deficiency)
o deficiency in cortisol --- Cushing syndrome

9 Prolonged CT, normal BT

o problem with secondary hemostasis

PADAYON, RMT QUITAG, BULQUERIN, DEALA 9