HEMA Lab

• Bleeding Time


  The time it takes for a small standardized wound, introduced into the capillary bed of the finger or earlobe, to stop bleeding
• Bleeding Time


  • Reflects both platelet number and platelet functional integrity
  • Puncture sites: third & fourth finger or ring finger
  • Burned patients: earlobe
• Bleeding Time Results


  • Normal
  • Shortened (below normal range)
  • Prolonged (above normal range)
• Platelet Function Test


  Performed to assess the quality of platelets, usually done if patients have cutaneous bleeding
• Bleeding Time


  In vitro measurement of platelet adhesion and platelet aggregation, usually done before surgery but is now considered obsolete
• Duke Method


  1. Disinfect puncture site

  2. Use sterile lancet to puncture site

  3. Start timer when blood is seen

  4. Blot blood using filter paper every 30 seconds

  5. Stop timer when blood no longer appears on filter paper

  6. Perform aftercare

  7. Record bleeding time
• Duke Method


  First described by Duke in 1912, involves using a lancet to make an incision on the finger or earlobe
• Ivy Method


  1. Disinfect forearm site

  2. Position sphygmomanometer and inflate to 40mmHg

  3. Make two separate cuts into forearm

  4. Start stopwatch and use filter paper to draw off blood every 30 seconds

  5. Record time when blood ceases to flow
• Ivy Method


  Modified by Ivy in 1941, involves using a blood pressure cuff inflated at 40 mmHg and making two separate cuts in the forearm
• Factors affecting bleeding time

  • Intercapillary pressure
  • Size and depth of the wound
  • Efficiency of the tissue fluids in accelerating the clotting process
  • Thickness and vascularity of the skin
  • Chemical and mechanical actions of the platelets
  • Ability of the blood vessels to constrict and retract
• Conditions that may yield abnormal bleeding time results

  • Von-Willebrand's Disease
  • Bernard-Soulier Syndrome
  • Glanzmann Thrombasthenia
• Mielke Method (Template Bleeding Time)


  A modification of Ivy method that utilizes a template containing a standardized slit instead of a disposable lancet
• Simplate Method


  Contains a spring loaded blade within a plastic case which holds a double blade, making two incisions
• Surgicutt Method


  Utilizes a slicing action using a surgical blade that is spring-loaded and makes a standardized cut
• Capillary Fragility Test


  Evaluates the stability of capillaries under increased hypoxia and hydrostatic pressure, a positive test indicates weakness of the capillary walls or capillary permeability and fragility
• Capillary Fragility Test Procedure

  1. Examine for existing petechiae

  2. Place blood pressure cuff on upper arm and inflate to 80mmHg

  3. Maintain pressure for 5 minutes

  4. Remove cuff and wait 10 minutes

  5. Examine for presence of petechiae and count them
• Capillary Fragility Test Grading

  • 1+ = 0-10 petechiae
  • 2+ = 11-20 petechiae
  • 3+ = 21-50 petechiae
  • 4+ = 51 and over petechiae
• Clot Retraction Time


  Determines the length of time required for firm clot formation by both platelets and fibrinogen, measures the ability of the blood to retract and decrease clot size during formation of hemostatic plug
• Clot Retraction Time Procedure

  1. Withdraw 3mL venous blood and place in calibrated tube

  2. Incubate for 1 hour at 37°C

  3. Let stand at room temperature and inspect clot at 1, 2, 4, and 24 hours

  4. Note time of initial and final retraction

  5. Measure amount of serum expressed
• Clot Retraction Time Interpretation

  • 0: no serum extruded
  • +1: 5-10% serum extruded
  • +2: 10-20% serum extruded
  • +3: 20-35% serum extruded
  • +4: 35-50% serum extruded
• Normal clot retraction is 2-4 hours, poor is after 4 hours and within 24 hours, no retraction is after 24 hours
• Clot Retraction Time Computation

  % Clot retraction = (mL of serum / mL of whole blood) x 100
• Hirschboeck Method

  Makes use of castor oil, reference values: <15 minutes = thrombotic tendency, >45 minutes = hemorrhagic tendency
• Stefanini-Dameshek Method


  Makes use of a single test tube to observe clot retraction
• Macfarlane Method


  Places 5mL fresh blood in a calibrated tube with a glass rod, measures the amount of serum left after retraction
• Factors affecting clot retraction

  • Platelet number and function
  • Fibrinogen concentration and function
  • Packed Cell Volume/ Hematocrit/ RBC mass
  • Size, shape, and nature of the glass surface
  • Amount and handling of the blood sample
  • Temperature
• Conditions that affect clot retraction

  • Fibrinogen deficiency (congenital or acquired)
  • Thrombocytopenia (less than 100,000/uL)
  • Thrombasthenia
  • Polycythemia vera
• Conditions that affect clot retraction

  • Fibrinogen deficiency (congenital or acquired)
  • Thrombocytopenia (less than 100,000/uL)
  • Thrombasthenia
  • Polycythemia vera = Increased clot
  • Glanzmann thrombasthenia = platelet aggregation
  • DIC (Disseminated Intravascular Coagulation) = Excessive clotting and excessive fibrinolysis
  • hypofibrinogenemia = low fibrinogen
  • dysfibrinogenemia = nonfunctional/dysfunctional fibrinogen
• Red Cell Fall Out = RBC trapped in the clot = it will fall out if not hold properly
• Causes of excessive red cell fall out

  • Fibrinogen concentration is low or absent
  • A fibrinolysin may be present
  • Factor XIII deficiency
• Platelet adhesiveness test


  One of the functions of platelets in their participation in hemostasis, where they adhere to each other and to the walls of damaged blood vessels to form a hemostatic plug
• Platelet adhesiveness test procedure

  1. Perform 2 clean venipuncture at separate sites, with and without the use of the glass bead collecting system

  2. Perform a platelet count on both blood samples

  3. Calculate the percentage of platelet adhesiveness
• Platelet aggregometry


  Designed to detect qualitative (functional) platelet abnormalities in patients who are experiencing the symptoms of mucocutaneous bleeding
• Functional platelets


  • Adhere to subendothelial collagen
  • Aggregate with one another
  • Secrete contents of their a-granules and dense granules
• Platelet aggregometry using platelet-rich plasma

  1. The operator pipettes the PRP to instrument compatible cuvettes

  2. Drop in one clean plasticized stir bar per sample

  3. Place the cuvettes in incubation wells

  4. Allows the samples to warm to 37C for 5 mins

  5. The operator than transfers the first cuvette, containing specimen and stir bar, to the instrument's reaction well

  6. Start the stirring device and the recording computer

  7. The instrument directs focused light through the sample cuvette to a photodetector

  8. The operator pipettes an agonist (platelet activator) directly into the same to trigger aggregation
• Whole blood platelet aggregometry


  1. The operator pipettes aliquots of properly mixed whole blood suspension to cuvettes

  2. Adds equal volumes of physiologic saline

  3. The operator drops in one stir bar and places cuvette in 37 C incubation wells for 5 mins

  4. The operator transfers the first cuvette to a reaction well, pipettes an agonist directly into the specimen, and suspends a low-voltage cartridge-mounted disposable direct current electrodes in the mixture
• Platelet lumiaggregometry


  • The procedure differs little from that for conventional aggregometry and simplifies the diagnosis of platelet dysfunction
  • As ATP is released, it oxidizes a firefly-derived luciferin-luciferase reagent to generate cold chemiluminescence proportional to the ATP concentration
• Platelet lumiaggregometry procedure


  1. Add an ATP standard to the first sample and then add luciferin-luciferase and tests for full luminescence

  2. Add luciferin-luciferase and an agonist to the second sample; the instrument monitors for aggregation, and secretion simultaneously
• Platelet agonists (activating agents used in aggregometry)


  • List of agonists