MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y.

2022-2023

LABORATORY EVALUATION OF PRIMARY HEMOSTASIS (PART 1)

INTRODUCTION
 Platelet function tests are designed to detect qualitative (functional) platelet abnormalities in
patients with mucocutaneous bleeding.
 Two procedures performed prior to platelet function tests:
 Platelet Count
 Peripheral Blood Film examination for platelet morphology
 If platelet count is normal, but the patient has suggestive bleeding history, an assessment of platelet
function should be conducted.
 Platelet Function Tests:
 Bleeding Time Test
 Tourniquet Test (Capillary Fragility Test)
 Clot Retraction
 Platelet Aggregometry
 Platelet Adhesiveness Test
 Von Willebrand Factor Activity Assays
 Heparin-Induced Thrombocytopenia Assays or Heparin Associated Thrombocytopenia Test (HATT
Test)

PLATELET COUNT: number of platelets in 1 liter (L) or 1 microliter (µL) of whole blood
 Reasons why platelets are hard to count:
 Platelets adhere to foreign surfaces.
 Platelets easily disintegrate.
 They are hard to differentiate from debris.
 Platelets are unevenly distributed in the blood because they tend to clump.

 Principle:
 Whole blood, with EDTA as the anticoagulant, is diluted with platelet diluting fluid in an RBC pipet
and counted using the hemocytometer. The platelets are counted in the 25 small squares in the
large center square (1 mm2) of the hemocytometer using bright-field microscopy.

 Specimen: Venous blood anticoagulated with EDTA

 Methods:
A. Indirect Method: platelets are counted in relation to 1,000 RBCs in the blood smear
B. Direct Methods: whole blood is diluted with platelet diluting fluid in an RBC pipette and counted
in a hemocytometer
 Light Microscopy Method
 Rees and Ecker’s
 Diluting Fluid (isotonic) composition:
 Sodium Citrate  prevent platelet aggregation
 Formaldehyde  preservative
 Brilliant Cresyl Blue  stain/dye

PREPARED BY: KAREN B. ROSETE, RMT 1

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

 Distilled Water
 Appearance of platelets: small | round/oval | highly refractile | Bluish
 Guy and Leake’s
 Diluting Fluid (isotonic) composition:
 Sodium oxalate  prevent platelet aggregation
 Formaldehyde  preservative
 Crystal Violet  stain/dye
 Distilled Water
 Appearance of platelets: highly refractile | Lilac
 Phase Microscopy Method
 Brecher-Cronkite Method
 Diluting Fluid used: 1% Ammonium oxalate (hypotonic)
 Unopette
 Diluting Fluid used: 1% Ammonium oxalate
 Others: Tocantin’s Method, Nygard’s Method, Walker and Sweeney’s
Method
C. Automated Methods

 Dilution:
 1:100 (most common)
 < 50 platelets counted on each side = 1:20
 > 500 platelets counted on each side = 1:200

 Calculation:

cells counted × dilution factor

Platelet count =
area counted (mm2) × depth

 Normal Values or Reference Range:

Parameter Conventional Units Standard International

(cells/mm or cells/µL)
3 (SI) Units (× 109/L)

Platelet count (adult) 150,000 – 450,000 150 – 450

 Sources of Error and Comments:

 Normal platelet: count 5 small RBC squares = area is 0.2 mm2
 Inadequate mixing or poor collection  platelet clumping 
false ↓ platelet count
 Short draw: excess anticoagulant  swelling of platelets 
fragmentation  false ↑ platelet count
 Skin puncture specimen is less desirable
 Platelet satellitosis
 Occurs in EDTA anticoagulated samples

PREPARED BY: KAREN B. ROSETE, RMT 2

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

 Adherence of platelets around neutrophils, producing a ring or satellite effect

 Causes pseudothrombocytopenia
 Solution: use sodium citrate as anticoagulant; multiply the obtained platelet count by
1.1 for accuracy

Platelet Estimate (Peripheral Blood Film Examination)

 Locate/select the ideal area of examination
 Using the 100× oil immersion objective, count the number of platelets in 10 consecutive fields,
and calculate the average number of platelets per field.
 To obtain the platelet estimate per µL of blood, multiply the average number of platelets per field
by 20,000.
 Compare the instrument platelet count with the platelet estimate from the blood film.
 In instances of significant anemia or erythrocytosis, use the following formula for the platelet estimate:
Average no. of platelets × total RBC count/ 200 RBCs
 If platelet count is normal, there must be:
 About 1 platelet per 10-30 red blood cells
 7-20 platelets per oil immersion field

BLEEDING TIME (BT)

Introduction:
 Screening test of primary hemostasis
 Screening test for detecting disorders of platelet function and von Willerand’s disease; evaluates vascular
integrity
 In vivo measurement platelet adhesion and aggregation on locally injured vascular subendothelium
 Directly affected by the platelet count and ability of platelets to form a plug

Methods:
 Duke Method
 Procedure:
 Earlobe or fingertip: punctured (3 mm deep) using a sterile lancet
 Start the timer as soon as the first drop of blood appears.
 Blot drop of blood with filter paper every 30 seconds (filter paper must not touch the
wound)
 Stop timer as soon as bleeding stops.
 N.V. = 2 to 4 minutes
 Not precise or accurate
 Ivy Method
 Refer to the procedure below
 Mielke Method (Template Bleeding Time)
 Modification of the Ivy Method
 A Bard-Parker or similar disposable blade is used, along with a rectangular polystyrene or
plastic template that contains a standardized slit.
 The blade is placed in a special handle containing a gauge in order to standardize the depth
and length of the incision.

PREPARED BY: KAREN B. ROSETE, RMT 3

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

IVY METHOD
Principle:
 A blood pressure cuff is placed on the patient’s arm above the elbow, inflated, and maintained at a constant
pressure throughout the procedure. One (or two) standardized incisions is (are) made on the volar surface
of the forearm. The length of time required for bleeding to stop is recorded as the bleeding time.

Equipment:
 Blood pressure cuff
 Bleeding time device (refer to the table below)
 Stopwatch
 Circular filter paper
 Alcohol prep pads
 Bandage
Bleeding Time Devices
Device No. of Length/Depth of Position of Age Example of
Incisions Incision (mm) Choice Normal Range
Simplate Pediatric 1 3.0 × 0.5 Perpend./Parallel Pediatric 1.6-6.8 minutes
Simplate 1 5.0 × 1.0 Parallel 2.3-9.5 minutes
Simplate II 2 5.0 × 1.0 Parallel 2.3-9.5 minutes
Surgicutt 1 5.0 × 1.0 Parallel >15 y/o 1.6-8.0 minutes
Surgicutt 2 2 5.0 × 1.0 Parallel >15 y/o 1.6-8.0 minutes
Surgicutt Newborn 1 2.5 × 0.5 Perpend. Newborn to 4 51-99 seconds
months
Surgicutt Jr. 1 3.5 × 1.0 Parallel 5 months to 15 1.3-9.0 minutes
y/o
Procedure:
1. Locate the area for the bleeding time.
 Standardized test site for the puncture: Volar surface of the forearm, 5 cm below the
fold of the elbow (or three finger-width below the bend of the elbow)
2. Cleanse the site with an alcohol sponge and allow to dry.
3. Place a blood pressure cuff on the patient’s arm above the elbow. Increase the pressure to 40 mm Hg
(or lower for newborns) and hold this exact pressure for the entire procedure.
4. Prepare the bleeding time device.
5. Activate the trigger and start the stopwatch. Remove the device approximately 1 second after making
the incision.
 The incision must be made and the bleeding time started within 30-60 seconds (within 30
seconds for infants) after the blood pressure cuff has been inflated.
6. Blot the blood from the puncture site on a clean section of circular filter paper every 30 seconds.
 The filter paper must not touch the wound at any time.
7. When bleeding ceases, stop the watch and release the blood pressure cuff.
8. Place a bandage over the puncture site.

Normal Values/Reference Range:

 3-8 minutes (Turgeon)
 2-8 minutes (Henry’s)
 2-9 minutes (Rodak’s)

PREPARED BY: KAREN B. ROSETE, RMT 4

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

 BT result between 8-11 minutes (not clinically significant)

Clinical Application:
 ↑ Bleeding Time
 Platelet count: <30,000 to 50,000/µL  BT increases up to more than 30 minutes
 Platelet count: >100,000/ µL  impaired platelet function or defect of subendothelial factor
 Dysfunctional platelets
 von Willebrand Disease
 following ingestion of aspirin
 aspirin-containing compounds
 anti-inflammatory drugs
 anticoagulants
 some antibiotics and certain other drugs
 Drug therapy: most common cause of prolonged bleeding time

OTHER METHODS:
Aspirin Tolerance Test
 Useful in distinguishing functionally abnormal platelets from normal platelets
 Bleeding time: measure before and after 2 hours of aspirin (650 mg) ingestion
 Bleeding time after aspirin ingestion is usually slightly longer

Copley and Lalitch Method

 Procedure:
 Clean the finger
 Make a puncture wound 6mm deep
 Immerse the wounded finger in a sterile NSS warmed at 37°C until bleeding stops
 the same procedure applies to Adelson-Crosby Method

MacFarlane’s Method
 Procedure:
 Same procedure with Adelson-Crosby method only that it uses the earlobe as the site of puncture

TOURNIQUET TEST (CAPILLARY FRAGILITY TEST/RUMPEL-LEEDE TEST/HESS TEST)

Introduction:
 Crude measure of capillary fragility
 Degree of thrombocytopenia correlates with tourniquet test
 Clinical diagnostic method to determine hemorrhagic tendency of a patient
 Normal individuals: none to very few petechiae formed during the test

Principle:
 An inflated blood pressure cuff on the upper arm is used to apply pressure to the capillaries for 5
minutes. The arm is then examined for petechiae.

Equipment:
 Blood pressure cuff
 Stethoscope

PREPARED BY: KAREN B. ROSETE, RMT 5

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

Procedure:
1. Examine the forearm, hand, and fingers to make certain no petechiae are present.
2. Apply a blood pressure cuff on the upper arm above the elbow, and take a blood pressure reading.
3. Inflate the blood pressure cuff to a point halfway between the systolic and diastolic pressures.
 Never exceed a pressure of 100 mm Hg. Maintain the pressure for 5 minutes.
4. Remove the blood pressure cuff and wait 5 to 10 minutes before proceeding.
5. Examine the forearm, hands, and fingers for petechiae.
 Disregard any petechiae within ½ inch of the blood pressure cuff.
6. The test results may be graded roughly as follows:
 1+ (0-10) a few petechiae on the anterior part of the forearm
 2+ (10-20) Many petechiae on the anterior part of the forearm
 3+ (20-50) Multiple petechiae over the whole arm and back of the hand
 4+ (>50) Confluent petechiae on the arm and back of the hand
 Note: Positive tourniquet test: presence of numerous petechiae (10 or more petechiae)

Clinical Application:
 Thrombocytopenia
 Decreased fibrinogen
 Vascular purpura

Normal Result:
 1+  0 to occasional petechiae present

CLOT RETRACTION TIME (CRT)

Introduction:
 When blood coagulation is complete, the clot normally undergoes retraction (serum is expressed from
the clot, and the it becomes denser)
 Originally used as a screening test for platelet function

Methods:
 Qualitative CRT
 Hirschboeck/Castor Oil
 Place castor oil in a red top tube (3/4)
 Add one drop of fresh blood into castor oil
 Observe for “dimpling” phenomenon
 N.V.: 15-45 minutes
 <15 minutes  thrombotic tendency
 >45 minutes  hemorrhagic tendency
 Quantitative CRT
 Stefanini Method
 Refer to the procedure below
 MacFarlane Method
 Specimen: 5 mL fresh WB
 Uses tube containing glass rod
 Temperature: 37°C
 Incubation time: 1 hour

PREPARED BY: KAREN B. ROSETE, RMT 6

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

 Computation: %CR = (amount of serum left in the tube/amount of blood used)

× 100
 N.V.: 44-67%

STEFANINI METHOD
Principle:
 Fresh whole clotted blood is placed in a 37°C water bath and inspected at 1, 2, 4, and 24 hours for the
presence of a retracted clot.

Specimen:
 3 mL of whole fresh blood, placed in a glass test tube

Equipment:
 Water bath, 37°C
 Glass test tubes

Procedure:
1. Obtain 3 mL of blood and dispense carefully into a glass test tube.
2. Place the test tube of blood in the 37°C water bath and allow the blood to clot.
3. As soon as the blood has clotted, inspect the clot at 1, 2, 4, and 24 hours for the formation of a retracted
clot.
 Normally, there is a small amount of red blood cell fallout during the clot retraction.
4. Results are reported as the length of time it took for the clotted blood to retract.

Results:
 Normal: clot retraction occurs at 2-4 hours
 Poor: clot retraction occurs after 4 hours and within 24 hours
 None: clot retraction occurs after 24 hours

Clinical Significance:
 Glanzmann’s thrombasthenia
 Thrombocytopenia (Platelet count: <100,000/µL)
 Clot retraction reflects the:
 Dysfibrinogenemia or Hypofibrinogenemia  formed
 Number and quality of platelets
clot is small; ↑ RBCs expressed from the clot (RBC
 Fibrinogen concentration
fallout)
 Fibrinolytic activity
 Paraproteinemias
 Packed red cell volume
 Disseminated Intravascular Coagulation  formed clot
is small and ragged; ↑ RBC fallout  Degree of clot retraction is:
 directly proportional to the
Note: number of platelets
 Clot retraction begins within 30 seconds after the blood  inversely proportional to
has clotted the hematocrit, RBC count
 At the end of 1 hour, clot retraction is observed with and fibrinogen level
most retraction occurring within the first 4 hours.
 Clot retraction should be complete within 4 hours.

PREPARED BY: KAREN B. ROSETE, RMT 7

 MT 64 | BSMLS-3A/3B/3C/3D/3F | S.Y. 2022-2023

MAIN REFERENCES:
 Keohane, E.M., Smith, L.J., Walenga, J.M. (2016). Rodak’s Hematology: Clinical Principles and Applications. (5th Ed.).
Missouri: Elsevier Saunders
 Keohane, E.M., Otto, C.N., Walenga, J.M. (2020). Rodak’s Hematology: Clinical Principles and Applications. (6th Ed.).
Singapore: Elsevier
 Brown, B. A. (1993). Hematology: Principles and Procedures. (6th Ed.). Philadelphia: Lippincott Williams & Wilkins
 Turgeon, M.L. (2012). Clinical Hematology: Theory and Procedures. (5th Ed.). Philadelphia: Lippincott Williams &
Wilkins

PREPARED BY: KAREN B. ROSETE, RMT 8