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

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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

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Thrombin Time

• Qualitative
• Useful to detect parameters not detected by PT
  and aPTT such as heparin, presence of FDPs,
  presence of dys/hypofibrinogenemia
• Thrombin time is prolonged with these parameters
• Measures conversion of fibrinogen to fibrin
• Thrombin cleaves fibrinogen in undiluted plasma
• Normal reference range 10-16 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 Causes
• Factor Deficiency
• Circulating Inhibitors

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Factor Deficiency Evaluation

• First Considerations
• PT and/or APTT must be prolonged
• Patient history and symptoms must be considered
• Bleeding tendency important to note
• Reflex Testing (follow-up tests)
• Mixing Study
• Will determine whether a factor deficiency is
  present or a circulating inhibitor

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Mixing Study

• Also referred to as a circulating inhibitor
  screen
• 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
• Clotting times tend to increase with time and
  incubation due to the loss of labile factors, so
  it is important to compare the results of the
  patients diluted sample with the normal pooled
  plasma

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Mixing Study

• If the procedure corrects the prolonged PT or
  APTT, the defect is in the procoagulant family.
• Mixing study correctsFactor deficiency
• If the procedure does not correct the PT or APTT,
  the defect is due to an inhibitor
• Mixing study does NOT correctInhibitor

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

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Factor Assay Procedure

• 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.

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Factor Assay Procedure, contd.

• 3. The patient mixture results and the normal
  control mixture results are then compared to
  quantitate the factor level in the patient
  plasma.
• 4. A factor assay curve is the basis for plotting
  patient clotting times at various dilutions
• 5. 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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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 Procedure

• Based on the fact that if a specimen contains at
  least 50 of a normal level of factors, the PT or
  APTT will give a normal result.
• Normal plasma (containing 100 of all factors and
  giving a normal APTT) is added in equal
  proportion to the unknown plasma (which has
  already given us an abnormal APTT result).
• This 11 mixture we know contains at least 50 of
  all factors (because we added it) so we expect
  the APTT on this mixture to be normal.

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Inhibitor Screen Procedure, contd.

• If the APTT on the 11 mixture results in a
  corrected APTT (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 APTT on the 11 mixture does not correct
  the APTT, (the APTT 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 APTT, but no
  clinical symptoms or bleeding problems.
• The abnormal antibody reacts mildly in vivo with
  thrombotic events, but reacts stronger 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
  APTT on these patients. APTT results will then
  be normal

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Factor Deficiency vs. Circulating Inhibitor
Deficiency or Inhibitor Immediate PT or APTT after Mixing Study Mixing study following 2 hour incubation
Factor deficiency Correction Correction
Lupus-like anticoagulant No correction No correction
F-VIII inhibitor Correction No correction
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Anti Xa Assay

• Used to monitor LMWH
• Chromogenic assay
• Procedure
• Excess FXa is added to patient PPP
• Heparin/AT complexes in patient sample will bind
  FXa
• Chromogenic substrate is added and any unbound
  FXa cleaves the chromogen
• Produces a yellow color, read spectrophotometrical
  ly
• Results read off a standard curve

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Factor XIII

• Necessary for the formation of a stable fibrin
  clot
• Cross-linking by Factor XIII does not affect PT
  and APTT
• F-XIII deficiency test indicated if screening
  tests are NORMAL
• Patient exhibits delayed bleeding, poor wound
  healing, or bruising

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F-XIII Test

• Principle
• Based on the observation that the fibrin clot has
  increased solubility because of the lack of
  cross-linking of the fibrin polymer in the
  absence of F-XIII
• Patient PPP mixed with calcium chloride and
  allowed to clot for an hour at 37C.
• The clot is removed and placed in another tube
  containing urea
• If the clot dissolves in less than 24 hours,
  there is less than 2 F-XIII activity

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Lab Testing of the Fibrinolytic System

• D-Dimer --Discussed in lab
• FDP
• Detects fibrin/fibrinogen degradation products
• Requires a special collection tube that contains
  thrombin and a fibrinolytic inhibitor
• Patient serum is mixed with latex particles that
  detect FDPs and slide is observed for
  agglutination

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Activated Clotting Time ACT

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

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References

• http//labmed.yale.edu/education/cme/casestudies/6
  /7.aspx
• McKenzie, Shirlyn B., and J. Lynne. Williams.
  "Chapter 40." Clinical Laboratory Hematology. 2nd
  ed. Boston Pearson, 2010. Print.