Coagulation Screening Test - PowerPoint PPT Presentation

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Coagulation Screening Test

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Mr. Mohammed A. Jabar PT: INR Values INR = International Normalized Ratio. MNP = Mean Normal Plasma. INR = (PT / MNP)ISI An INR of 1.0 means that the patient PT is ... – PowerPoint PPT presentation

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Title: Coagulation Screening Test


1
Coagulation Screening Test
  • Mr. Mohammed A. Jabar

2
PREANALYTICAL VARIABLES INCLUDING SAMPLE
COLLECTION
  • Many misleading results in blood coagulation
    arise not from errors in testing but from
    carelessness in the preanalytical phase. Ideally
    the results of blood tests should accurately
    reflect the values in vivo.
  • When blood is withdrawn from a vessel, changes
    begin to take place in the components of blood
    coagulation. Some occur almost immediately, such
    as platelet activation and the initiation of the
    clotting mechanism dependent on surface contact.

3
Sample Collection
  • Anticoagulant of choice
  • 3.8 or 3.2 Sodium Citrate
  • 3.2 Preferred as the standard measure due to
    stability and closeness to the plasma osmolality
  • Anticoagulant/blood ratio is critical (19)
  • Exact amount of blood must be drawn. No short
    draws are acceptable, this will falsely increase
    results due to presence of too much anticoagulant
  • CLSI guideline is /- 10 of fill line
  • Purpose of the anticoagulant is to bind or
    chelate calcium to prevent clotting of specimen

4
Sample Collection
  • Other anticoagulants, including oxalate, heparin,
    and EDTA, are unacceptable.
  • The labile factors (factors V and VIII) are
    unstable in oxalate, whereas heparin and EDTA
    directly inhibit the coagulation process and
    interfere with end-point determinations.
  • Additional benefits of trisodium citrate are that
    the calcium ion is neutralised more rapidly in
    citrate, and APTT tests are more sensitive to the
    presence of heparin.

5
Sample Collection
  • Sidenote Samples with High hematocrits
  • NCCLS recommends adjusting anticoagulant ratio
    for patients with hematocrits exceeding 55
  • High hematocrits may cause falsely prolonged test
    results due to an over- anticoagulated sample
  • Formula correction achieves a 40 hematocrit

6
Correction Formula High Hematocrits
  • (1.85 x 10 -3) (100-Hct)V C
  • Where
  • C volume of sodium citrate
  • Vvolume of whole blood drawn
  • Hct patients hematocrit
  • Example
  • Patients Hct 63
  • V 5 mL
  • (1.85 x 10 -3) (100-63)5 C
  • (1.85 x 10 -3) (37)5 0.34 mL

Haematocrit Citrate (ml)
0.20 0.70
0.25 0.65
0.30 0.61
0.55 0.39
0.60 0.36
0.65 0.31
0.70 0.27
7
Site Selection
  • Untraumatic venipuncture is required
  • Traumatic venipunctures release tissue factor and
    initiate coagulation
  • Fingersticks/Heelsticks are not allowed
  • Indwelling IV line draws are discouraged
  • Contain heparin
  • Falsely increased results
  • Order of Draw
  • Evacuated tube system
  • Blue top is 1st or 2nd tube.
  • If 2nd tube drawn, 1st top must be anticoagulant
    free (i.e. red top)

8
Storage Requirements
  • Prothrombin Time PT
  • Uncentrifuged or centrifuged with plasma
    remaining on top of cells in unopened tube kept
    at 2-4 oC or 18-24 oC must be tested within 24
    hours of collection
  • Activated Partial Thrombin Time APTT
  • Uncentrifuged or centrifuged with plasma
    remaining on top of cells in unopened tube kept
    at 2-4 oC or 18-24 oC must be tested within 4
    hours of collection

9
Storage Requirements
  • Other Assays
  • Fibrinogen, Thrombin Time, Factor Assays
  • Centrifuged with plasma remaining on top of cells
    in unopened tube kept at 2-4 oC or 18-24 oC must
    be tested within 4 hours of collection

10
Storage Requirements
  • Other general notes
  • Perform coagulation tests ASAP
  • Specimen may deteriorate rapidly (especially
    factors V and VIII)
  • If the testing is not completed within specified
    times, plasma should be removed from the cells
    and placed in a frost free freezer
  • - 20 oC for two weeks
  • -70 oC for six months

11
Transportation of Specimen
  • Send specimen on ice OR deliver to lab ASAP
  • Separate cells from plasma immediately via
    centrifugation

12
Common Collection Problems
Error Consequence Comment
Short draw lt2.7 mL PT/PTT falsely prolonged Anticoagulant to blood ratio exceeds 19
Failure to mix specimen after collection PT/PTT falsely prolonged Blood clots form when anticoagulant blood do not mix
Excess vigorous mixing PT/PTT falsely shortened Hemolysis and platelet activation cause start of cascade
Hemolysis PT/PTT falsely shortened Reject specimen
Improper storage wrong temperature or held too long PT/PTT falsely prolonged Must follow storage requirements
Chilling in refrigerator or placing on ice PT falsely shortened Chilling to 4 oC activates factor VII.
Inadequate centrifugation PTT loses sensitivity for lupus anticoagulants and heparin. Factor assays inaccurate Desire platelet poor plasma
Prolonged tourniquet application Falsely elevates vWF, factor VIII Tourniquet causes venous stasis,
13
Common Collection Problems
Error Consequence Comment
Drawing coagulation tube PRIOR to other anticoagulant tubes PT/PTT falsely affected Contamination
Probing the vein PT/PTT falsely shortened Tissue thromboplastin is released activating coagulation
Heparin contamination from line draw PTT falsely prolonged Heparin keeps the blood from clotting
Lipemia Test may not work Photo-optical methods affected

14
Terms
  • Platelet Poor plasma (PPP)
  • lt10 x 10 9 /L
  • Specimen has been centrifuged for 15 minutes _at_
    2500 x g
  • Why is PPP essential?
  • Contains platelet factor 4(heparin neutralizer)
  • Contains phospholipid (affects lupus
    anticoagulant and factor assay testing)
  • Contains proteases (affect testing for vWF)

15
Terms
  • Platelet-Rich plasma(PRP)
  • Used in platelet function studies
  • 200-300 x 10 9 /L
  • Specimen has been centrifuged for 10 minutes _at_
    200 x g

16
Protime /Prothrombin(PT)
  • The prothrombin time is therefore the time
    required for the plasma to clot after an excess
    of thromboplastin and an optimal concentration of
    calcium have been added.
  • The PT measures functional activity of the
    extrinsic and common pathways.
  • The PT evaluates patients suspected of having an
    inherited or acquired deficiency in these
    pathways.

17
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18
PT Reagent
  • Calcium ions and Thromboplastin from brain tissue
    (Rabbit).
  • Thromboplastin (Tissue Factor) protein-lipid
    complex found in tissues outside blood vessels.
  • Thromboplastins were originally tissue extracts
    obtained from different species and different
    organs containing tissue factor and phospholipid.
    Because of the potential hazard of viral and
    other infections from handling human brain, it
    should no longer be used as a source of
    thromboplastin. The majority of animal
    thromboplastins now in use are extracts of rabbit
    brain or lung.

19
PT Reagent
  • The introduction of recombinant thromboplastins
    has resulted in a move away from rabbit brain
    thromboplastin. They are manufactured using
    recombinant human tissue factor produced in
    Escherichia coli and synthetic phospholipids,
    which do not contain any other clotting factors
    such as prothrombin, factor VII, and factor X.
  • Therefore they are highly sensitive to factor
    deficiencies and oral anticoagulanttreated
    patient plasma samples and have an International
    Sensitivity Index (ISI) close to 1.

20
PT Reagent Calibration
  • Reagents are calibrated against standard PT
    reagent established by the WHO.
  • ISI International Sensitivity Index.
  • ISI is assigned by the manufacturer for each lot
    of reagent using reference material from WHO.
  • The lower the ISI the more sensitive the Reagent
  • ISI of 1.8 to 2.4 Low sensitivity
  • ISI of 1.4 to 1.8 Average sensitivity
  • ISI 1.0 to 1.4 High Sensitivity

21
Principle
  • Principle
  • The procedure uses a tissue thromboplastin
    reagent with CaCl (Calcium Chloride) to provide a
    one step procedure for evaluating plasma
    clotting.
  • This test was devised on the assumption that when
    an optimal amount of calcium and an excess of
    thromboplastin are added to decalcified plasma,
    the rate of coagulation depends on the
    concentration of prothrombin in the plasma.

22
Principle
  • The normal values for the prothrombin time range
    from 10.0 to 13.0 seconds.
  • An elevated prothrombin time may indicate the
    presence of
  • Vitamin K deficiency,
  • DIC,
  • liver disease,
  • Presence of FSPs

23
Principle
  • a deficiency in one or more of the following
    factors
  • Factor I (Fibrinogen)
  • Factor II (Prothrombin)
  • Factor V (Proaccelerin, Labile Factor)
  • Factor VII (Proconvertin, Stable Factor)
  • Factor X (Stuart-Prower Factor)
  • Factor XIII (Fibrin Stabilizing Factor)
  • In addition, inhibitors can cause prolonged PTs.

24
When is it ordered?
  • Used to monitor oral anticoagulant therapy
    (Warfarin / Coumadin).
  • When a patient who is not taking anti-coagulant
    drugs has signs or symptoms of a bleeding
    disorder
  • When a patient is to undergo an invasive medical
    procedure, such as surgery, to ensure normal
    clotting ability.

25
Clot formation can be detected by
  1. Manual or Using, semiautomated or automated
    devices.
  2. Electromechanical methods.
  3. Optical Method.
  4. Electrochemical Method.

26
Electromechanical methods
  • Impedance, Steel Ball
  • The sample cuvette rotates and a steel ball
    remains stationary in a magnetic field until the
    formation of fibrin strands around the ball
    produces movement. This is detected by a change
    in the magnetic field, and the coagulation time
    is recorded.
  • A steel ball rotates under the influence of a
    magnet until the formation of fibrin strands
    around the ball stops it rotating. This is
    detected by a sensor, and the coagulation time is
    recorded

27
Photo Optical
  • Scattered Light Detection for Clotting Assays
    (660 nm)
  • The turbidity during the formation of a fibrin
    clot is measured as an increase in scattered
    light intensity when exposed to light at a
    wavelength of 660 nm.
  • Transmitted Light Detection for Chromogenic
    Assays (405 nm, 575 nm, 800 nm)
  • Colour production leads to a change in light
    absorbance, which is detected as a change in
    transmitted light. Over time the change in
    absorbance per minute is calculated (? OD/min).
    Various wavelengths can be used such as 405 nm,
    575 nm, and 800 nm.
  • Transmitted Light Detection for Immunoassays (405
    nm, 575 nm, 800 nm)

28
Electrochemical
  • INRatio Meter (Hemosense) Near Patient Testing
    Devices
  • The INRatio single-use test strip is made of
    laminated layers of transparent plastic. Each
    test strip has a sample well where blood is
    applied, three channels through which the blood
    sample flows to reach the testing areas, reagents
    to start the coagulation process, and electrodes
    that interface with the INRatio meter. The device
    detects a change in electrical resistance when
    blood clots.

29
Procedure
  • Reconstitute tissue thromboplastin according to
    instructions. Label the thromboplastin with the
    time, date and initials. The thromboplastin
    reagent is stabile for 7 days after
    reconstitution. Allow to sit 10-15 minutes,
    then invert gently several times. Mix well prior
    to pipetting any of this reagent at any step in
    this procedure.
  • Pipet 1-2 mls, using a plastic pipet, of the
    tissue thromboplastin-calcium chloride reagent (
    PT reagent) into a glass test tube and place in
    a 37 water bath incubator.

30
Procedure
  • Pipet 100 µL or .1 mL of normal control, Patient
    PPP into each of the test tubes.
  • Allow at least one (1) minute for the control to
    reach 37C.
  • Pipet 200 µL or .2 mLof PT reagent into the tube
    containing the control. Start the stop watch
    simultaneously.
  • Mix the tube and leave in the water bath for a
    minimum of 7-8 seconds. Then remove, wipe the
    exterior, tilt back and forth gently until a
    visible clot is formed. As the clot forms, the
    mixture will gelatinize and may turn cloudy.

31
Procedure
  • Stop the stop watch immediately when the clot
    begins to form and record the time in seconds.
    Carry out 1 significant figure passed the decimal
    point. For example, if your result is 12.23
    seconds, report as 12.2 seconds.
  • Repeat the procedure for the second run of normal
    control. Record the time.

32
Procedure
  • If the results from run 1 and run 2 are within
    1 second from each other, average the two
    results and report with appropriate units. For
    manual PT, results should match within 1.0 second
    (if result is less than 20 seconds). Results
    over 20 seconds should match within 2.0 seconds.
  • If results are not within required limits, a
    third run should be performed and average the two
    that match within acceptable limits. Be sure and
    cross out any values you are not using for the
    final calculation. Include measurement unit of
    seconds on report sheet.

33
Interpretation of Result
  • The Mean Normal Plasma is used to evaluate
    routine result.
  • The INR is not used to evaluate ROUTINE PT
    results.
  • The International Normalization Ratio (INR) value
    is only used on patients who are on oral
    anticoagulant therapy such as Coumadin. Because
    these patients must be continuously monitored and
    their dosage adjusted accordingly, it is very
    important to standardize the reported PT results.
    Standardization ensures uniformity and safety
    when a traveling patient is treated in different
    geographical areas.

34
PT INR Values
  • INR International Normalized Ratio.
  • MNP Mean Normal Plasma.
  • INR (PT / MNP)ISI
  • An INR of 1.0 means that the patient PT is
    normal.
  • An INR greater than 1.0 means the clotting time
    is elevated.

35
Expected PT Values
  • Mean Normal Plasma 10 to 14 seconds.
  • Mean Normal Plasma value varies with PT
    sensitivity.
  • A high sensitivity (Low ISI) PT will give a high
    normal PT value (13 to 15 seconds).
  • Oral anticoagulant monitoring Target INR of 2.0
    to 3.0.
  • INR of greater than 5 or 5.5 unacceptable high
    risk of bleeding.

36
Sources of Error
  • Associated with specimen
  • Inappropropriate ratio of anticoagulant to blood
  • Failure to correct citrate volume if hematocrit gt
    55
  • Clotted, hemolyzed or lipemic samples
  • Lack of PPP
  • Delay in testing or processing
  • Inappropriate storage

37
Sources of Error
  • Associated with storage
  • Incorrect preparation of reagents
  • Failure to properly store reagents
  • Use of reagents beyond reconstituted stability
    time or expiration date
  • Contaminated reagents

38
Sources of Error
  • Associated with procedure
  • Incorrect temperature
  • Incorrect incubation times
  • Incorrect volumes of sample, reagents or both
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