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Laboratory Testing in Coagulation

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MLAB 1227- Coagulation Keri Brophy ... factors Detects inhibitors Screening Tests PT APTT Discussed in lab Fibrinogen Thrombin Time Thrombin Time Qualitative ... – PowerPoint PPT presentation

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Title: Laboratory Testing in Coagulation


1
MLAB 1227- CoagulationKeri Brophy-Martinez
  • Laboratory Testing in Coagulation

2
Lab Testing For Primary Hemostasis Disorders
  • Purpose
  • Evaluate platelet concentration and function
  • Tests
  • Bleeding time discussed in lab
  • PFA-100
  • Platelet aggregometry

3
Lab Testing For Secondary Hemostasis Disorders
  • Purpose
  • Evaluates coagulation factors
  • Detects inhibitors
  • Screening Tests
  • PT
  • APTT Discussed in lab
  • Fibrinogen
  • Thrombin Time

4
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

5
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

6
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7
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

8
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

9
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

10
Mixing Study
11
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

12
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.

13
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.

14
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.

15
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.

16
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.

17
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

18
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
19
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

20
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

21
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

22
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

23
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.

24
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.
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