Laboratory Testing in Coagulation

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MLAB 1227- CoagulationKeri Brophy-Martinez

• Laboratory Testing in Coagulation

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Lab Testing For Primary Hemostasis Disorders

• Purpose
• Evaluates platelet concentration and function
• Tests
• Bleeding time discussed in lab
• PFA-100
• Platelet aggregometry

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PFA-100 Platelet Function Assay

• In-vitro system that stimulates the in-vivo
  function of platelets in primary hemostasis
  adhesion, activation and aggregation
• Aids in detection of platelet dysfunction
• Whole blood is subjected to high shear and the
  closure time is measured

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Platelet Aggregation Studies

• Tests the platelets ability to respond and
  aggregate
• In the presence of in-vitro platelet aggregating
  agents, normal platelets will aggregate producing
  characteristic patterns of aggregation. Refer to
  pg. 787 in text
• Agents include epinephrin, collagen, ADP,
  ristocetin, arachidonic acid, and thrombin

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Lab Testing For Secondary Hemostasis Disorders

• Purpose
• Evaluates coagulation factors
• Detects inhibitors
• Screening Tests
• PT
• APTT Discussed
  in lab
• Fibrinogen
• Thrombin Time
• Qualitative
• Measures conversion of fibrinogen to fibrin
• Thrombin cleaves fibrinogen
• Normal ref range lt20 SECONDS

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Lab Testing For Secondary Hemostasis Disorders

• If a screening test is prolonged, further testing
  must be performed to locate the specific cause of
  the abnormality
• 2 Possible Sources
• Factor Deficiency
• Circulating Inhibitors

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Factor Deficiency Evaluation

• First step
• PT and/or APTT must be prolonged
• Patient history and symptoms must be considered
• Second Step
• Mixing Study
• Will determine whether a factor deficiency is
  present or a circulating inhibitor

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Mixing Study

• Principle
• Patient plasma is diluted with normal pooled
  plasma to demonstrate factor levels
• The normal plasma provides the missing factor in
  the patient plasma
• 50 activity is generally ample to produce a
  normal PT or APTT

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Mixing Study

• If the procedure corrects the prolonged PT or
  APTT, the defect is in the procoagulant family.
  In other words a FACTOR ISSUSE
• If the procedure does NOT correct the PT or APTT,
  the defect is due to an INHIBITOR

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Factor Assays

• Determines type of factor deficiency and activity
• Targets either
• PT Factors VII, X,V and II
• APTT Factors XII, XI,IX AND VIII

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Factor Assays

• Principle
• Ability of the patients plasma to correct a
  prolonged PT or APTT of a known factor deficient
  plasma
• Normal activity range is 50-150 or 50 factor
  activity

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Factor Assays

• How is it done?
• Commercially prepared Factor deficient plasmas
  are used that contain 100 of all factors except
  the one in question. A series of these plasmas
  is usually used which contain various levels of
  the factor. For example 0, 10, 20, 30, 40,
  50, etc.
• As a control to compare results to, normal plasma
  (containing 100 of all factors) is added to the
  commercially prepared factor deficient plasma in
  the same way.
• The patient mixture results and the normal
  control mixture results are then compared to
  quantitate the factor level in the patient
  plasma.
• A factor assay curve is the basis for plotting
  patient clotting times at various dilutions
• Results of the patient are expressed as a percent
  of the normal plasma. A patient with a normal
  factor level should be 50-150 of the normal
  control plasma.

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Russell's Viper Venom Test (Stypven Time)

• This is a mixing study used to differentiate
  deficiencies of Factors VII and X
• If the patient lacks Factor VII, Viper Venom
  treated plasma added to the patient's plasma will
  correct the PT
• In Factor X deficiency, the PT is not corrected

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Inhibitor Screens

• When a PT or PTT is prolonged it must first be
  determined if the defect is due to a true factor
  deficiency or if it may be due to an abnormal
  circulating inhibitor (autoantibody to a factor).
  An inhibitor screen will rule out one or the
  other.

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Inhibitor Screen Example

• Based on the fact that if a specimen contains at
  least 50 of a normal level of factors, the PT or
  PTT will give a normal result.
• Normal plasma (containing 100 of all factors and
  giving a normal PTT) is added in equal proportion
  to the unknown plasma (which has already given us
  an abnormal PTT result).
• This 11 mixture we know contains at least 50 of
  all factors (because we added it) so we expect
  the PTT on this mixture to be normal.
• If the PTT on the 11 mixture does result in a
  corrected PTT (the patient sample was abnormal
  before but is normal now that we added normal
  plasma to it), then this indicates a factor
  deficiency was present in the original patient
  sample. The problem is not an autoantibody.
• If the PTT on the 11 mixture does not correct
  the PTT, (the PTT is still abnormal even after
  normal plasma was added), then this indicates
  there is an autoantibody present. This antibody
  is not only binding the patient's factors, but
  the factors in the normal plasma that was added
  to the mixture as well.

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Lupus Inhibitor Screen

• Lupus inhibitor should be suspected in a patient
  with markedly prolonged PT and PTT, but no
  clinical symptoms or bleeding problems.
• The abnormal antibody does not react in vivo
  (except a mild thrombotic tendency) but does
  react in vitro by binding to the phospholipids in
  the reagents used for coag testing. Commercially
  prepared reagents are available that do not
  contain phospholipids and should be used to
  perform the PTT on these patients. PTT results
  will then be normal

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Factor VIII Inhibitor

• Quantitate by preparing various dilutions of
  patient plasma with normal pooled plasma with a
  known amount of factor VIII activity
• If factor VIII inhibitor is present, it will
  inactivate factor VIII

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Lab Testing of the Fibrinolytic System

• FDP
• D-Dimer Discussed in lab
• Clot Lysis
• Detects increased fibrinolytic activity
• Increased fibrinolytic activity found in DIC,
  liver disease, surgery, malignancies and women on
  oral contraceptives
• The clot used in the clot retraction procedure
  should be kept at 37oC and examined at the end of
  8, 24, 48 and 72 hours for clot lysis. Normally
  there is no clot lysis before 72 hours. If the
  clot that was initially formed becomes fluid in
  less than 72 hours, abnormal clot lysis is
  present. The time at which lysis was observed is
  reported as the clot lysis time. If no lysis
  occurred, the results are reported as no clot
  lysis after 48 or 72 hours. (NOTE The clot
  should remain intact at least 48 hours.)

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Historical Tests

• In other words, might see on Registry

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Lee White Whole Blood Coagulation Time

• Uses whole blood
• Measures all stages of coagulation in the
  intrinsic system
• Screening test
• Principle
• The time interval from the initiation of the
  coagulation to visible clot formation reflects
  the condition of the clotting mechanism
• NOTE This test is outmoded and is no longer
  performed in laboratories.

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Clot Retraction

• Evaluates primary hemostasis
• Measures platelet retraction function
• Principle
• A function of platelets is to reduce the size of
  the fibrin clot. When a normal clot forms from
  whole blood, the clot retracts at 50 of original
  volume, expelling serum
• Thrombosthenin, released by the platelets, is
  responsible for clot retraction. In addition,
  the number of platelets present also affects the
  clot retraction
• Clot retraction begins within 30 seconds after
  the blood has clotted. At the end of one (1)
  hour, there should be appreciable clot
  retraction, and almost complete retraction by the
  end of four (4) hours. Clot retraction should be
  complete within 24 hours. An abnormal clot
  retraction time is found in Glanzmann's
  thrombasthenia

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Tourniquet Test

• Aka Rumpele-Leede or Capillary Fragility
• Principle
• Measures the ability of small blood vessels to
  retain rbcs under conditions of stress or trauma
• Platelet number and function also important
• No longer performed

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Activated Clotting Time ACT

• Developed to monitor coagulation status and
  heparinization in immediate need situations. The
  ACT uses tubes containing a negatively charged
  particulate activator of coagulation, such as
  kaolin, celite. When whole blood is drawn into
  the tube, the contact system is activated and
  clotting occurs. This assay is very sensitive to
  heparin, but is also affected by platelets.

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On the Horizon
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Thrombelastography TEG

• Non-invasive instrument designed to analyze a
  whole blood sample to assess a patients clinical
  hemostatic condition
• Primarily used during surgical procedures
• Principle
• Monitors hemostasis in its entirety
• Clot initiation through clot lysis
• Net effect of all hemostatic components
  interacting together
• Activated blood maximizes thrombin generation and
  platelet activation in vitro
• Demonstrates hemostatic potential of a blood
  sample at a given point

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TEG
Normal TEG
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TEG

• Advantages
• Differentiates surgical from pathological
  bleeding
• Reduces the use of unnecessary blood products