General Approach in Investigation of Haemostasis by elMMsd14


									    General Approach in
Investigation of Haemostasis

        Lecture 1: Introduction
     Preanalytical Variables including
1.   Sample Collection.
2.   Site Selection.
3.   Storage Requirements.
4.   Transportation of Specimen.
 Hemostasis is a complex interaction
  between vessels, platelets and
  coagulation proteins that, when working
  properly, stops bleeding while
  maintaining blood flow in the vessel.
 Specific tests are available to evaluate
  platelet function, coagulation proteins,
  natural occurring inhibitors and
Sample Collection
   Proper sample collection is of utmost importance for reliable
    test results to evaluate the bleeding patient, thrombosis or
    fibrinolysis (preanalytical phase)
   All these tests are influenced by sample collection, sample
    processing and sample storage.
   The laboratory will not evaluate samples that are hemolyzed,
    clotted, contain fibrin strands or improperly stored.
   Reference Laboratory Services will immediately notify the client
    of any problems with the sample.
   When blood is withdrawn from a vessel, changes begin to take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.
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 (1:9)

       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

* CLSI : Clinical and Laboratory Standards Institute
Sample Collection
   Other anticoagulants, including oxalate, heparin, and EDTA, are
   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
    neutralized more rapidly in citrate, and APTT tests are more
    sensitive to the presence of heparin.
Sample Collection : Samples with High
    According to the latest CLSI (formerly NCCLS) guideline on
     coagulation testing, it is important to adjust the sodium
     citrate volume when a patient’s hematocrit is greater than
     Examples of patients who may have elevated hematocrit
     values are newborns or people with polycythemia vera.
    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

    * National Committee for Clinical Laboratory Standards
X = (100–PCV)*vol./(595–PCV)
X= volume of sodium citrate
Vol =volume of whole blood drawn
PCV= patient’s hematocrit

Examples:                                 Citrate
Patients Hct= 60%, V= 5 mL         HCT
X=(100-60)*5 / (595-60)
 = 40*5 / 535 = 0.34 ml            0.20    0.70
                                   0.25    0.65
Patient Hct = 25%, V=5 ml
X=(100-25)*5 / (595-25)            0.30    0.61
  = 75*5 / 570 = 0.65 ml           0.55    0.39
                                   0.60    0.36
                                   0.65    0.31
                                   0.70    0.27
Site Selection
   Untraumatic venipuncture is required
     Traumatic venipunctures release tissue factor and initiate

   Fingersticks/Heelsticks are not allowed
   Indwelling IV line draws are discouraged
     Contain heparin & diluted blood

     Falsely increased results

   Order of Draw
   Evacuated tube system
     Blue top is 2nd

      If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red
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

   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
Storage Requirements
                              CENTRIFUGE TO                                  FREEZE
                                  PREPARE          REFRIGERATION            PLATELET-
                              PLATELET-FREE       (Or transport on ice)       FREE
                                   PLASMA                                    PLASMA
                                                                          If >24 hour delay
PT       24 hours                                 Do not refrigerate
                                                                               in testing
                                                                          If >24 hour delay
PTRX     24 hours                                 Do not refrigerate
                                                                               in testing
                                                                          If >4 hour delay in
PTT      4 hours
                             Within one hour of                           If >2 hour delay in
PTTRX    2 hours
                                 collection                                    testing
                                                                          If >4 hour delay in
TT       4 hours
OTHER                        Within one hour of                           Within one hour
         4 hours
ASSAYS                           collection                                   of collection
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 C for two weeks
       -70 C for six months
Transportation of Specimen
   Send specimen on ice OR deliver to lab ASAP

   Separate cells from plasma immediately via
Platelet Poor Plasma
   Platelet –Poor plasma (PPP)
     Platelet-Poor plasma is necessary for coagulation testing to

       prevent activation of platelets and release of PF4, a
       heparin inhibitor.
     The plasma platelet count must be < 10,000 /mm3.

     Specimen has been centrifuged for 15 minutes @ 2500 x g

     Why is PPP essential?

       1.  Contains platelet factor 4 (heparin neutralizer)
       2.  Contains phospholipids (affects lupus anticoagulant and
           factor assay testing)
       3.  Contains proteases (affect testing for vWF)
Platelets Poor Plasma preparation:
To prepare platelet-Poor plasma

   Centrifuge the blue top evacuated tubes (CLSI, formerly
    NCCLS recommendation is 1500 rpm for 15 minutes).

   Using a plastic pipette, immediately remove the top 2/3
    of the plasma to a plastic aliquot tube.
   Centrifuge this plasma sample and remove the top ¾ of
    the plasma to a second plastic aliquot tube with a fresh
    plastic pipette.

   Freeze the specimen within one hour of collection.
Platelets Rich Plasma (PRP)
   Platelet-Rich plasma (PRP)
     Used in platelet function studies

     200-300 x 10
                     9 /L

     Specimen must be centrifuged for 10 minutes @ 200 x

Common Collection Problems
Error             Consequence            Comment
Short draw        PT/PTT falsely         Anticoagulant to blood ratio exceeds
<2.7 mL           prolonged              1:9
Failure to mix    PT/PTT falsely         Blood clots form when anticoagulant
specimen after    prolonged              & blood do not mix
Excess vigorous   PT/PTT falsely         Hemolysis and platelet activation
mixing            shortened              cause start of cascade

Hemolysis         PT/PTT falsely         Reject specimen
Improper          PT/PTT falsely         Must follow storage requirements
storage: wrong    prolonged
temperature or
held too long
Chilling in       PT falsely shortened   Chilling to 4 oC activates factor VII.
refrigerator or
placing on ice
Common Collection Problems
Error                   Consequence                 Comment

Inadequate              PTT loses sensitivity for   Factor assays
centrifugation          lupus anticoagulants and    inaccurate
Prolonged tourniquet    Falsely elevates vWF,       Tourniquet causes
application             factor VIII                 venous stasis,
Drawing coagulation     PT/PTT falsely affected     Contamination
tube after to other
anticoagulant tubes
Probing the vein        PT/PTT falsely shortened Tissue thromboplastin is
                                                 released activating
Heparin contamination   PTT falsely prolonged       Heparin keeps the blood
from line draw                                      from clotting
Lipemia                 Test may not work           Photo-optical methods
Principles of Laboratory Analysis
    The more detailed investigations of coagulation proteins
     also require caution in their interpretation depending on
     the type of assay performed. These can be divided into
     three principal categories, as described in the following
1.   Immunological
2.   Assays Using Chromogenic Peptide Substrates
     (Amidolytic Assays)
3.   Coagulation Assays
4.   Other Assays
 Include         immuno-diffusion,   immuno-electrophoresis,
  radioimmunometric assays, latex agglutination tests, and tests
  using enzyme-linked immunosorbent assays (ELISA).

 Fundamentally, all these tests rely on the recognition of the
  protein in question by polyclonal or monoclonal antibodies.
  Polyclonal antibodies lack specificity but provide relatively high
  sensitivity, whereas monoclonal antibodies are highly specific
  but produce relatively low levels of antigen binding.
   latex agglutination kit: Latex microparticles are coated with
    antibodies specific for the antigen to be determined. When the latex
    suspension is mixed with plasma an antigen–antibody reaction takes
    place, leading to the agglutination of the latex microparticles.
   Agglutination leads to an increase in turbidity of the reaction
    medium, and this increase in turbidity is measured photometrically
    as an increase in absorbance.
   Usually the wavelength used for latex assays is 405 nm, although for
    some assays a wavelength of 540 or 800 nm is used. This type of
    assay is referred to as immuno- turbidimetric.
   Do not freeze latex particles because this will lead to irreversible
   An occasional problem with latex agglutination assays is
    interference from rheumatoid factor or paraproteins. These may
    cause agglutination and overestimation of the protein under assay.
Chromogenic Assay
   Chromogenic, or amidolytic, methodology is based on the use of a
    specific color-producing substance known as a chromophore.
   the chromophore normally used in the coagulation laboratory is
    para-nitroaniline (pNA), which has an optical absorbance peak at
    405 nm on a spectrophotometer.
Coagulation Assays
   Coagulation assays are functional bioassays and rely on
    comparison with a control or standard preparation with a known
    level of activity.
   In the one-stage system optimal amounts of all the clotting factors
    are present except the one to be determined, which should be as
    near to nil as possible.
   The best one-stage system is provided by a substrate plasma
    obtained either from a patient with severe congenital deficiency or
    artificially depleted by immuno-adsorption.
Coagulation Assays
   Coagulation techniques are also used in mixing tests to identify a
    missing factor in an emergency or to identify and estimate
    quantitatively an inhibitor or anticoagulant.
   The advantage of this type of assay is that it most closely
    approximates the activity in vivo of the factor in question.
    However, they can be technically more difficult to perform than
    the other types described earlier.
Other Assays
   Using snake venoms (The Taipan venom time employs a reagent
    isolated from the venom of the Taipan snake (Oxyuranus
    scutellatus) that directly activates prothrombin in the presence of
    phospholipid and calcium.)
   Aassay of ristocetin cofactor (used to diagnose von Willebrand
    disease )
   The clot solubility test for factor XIII.
    DNA analysis is becoming more useful and more prevalent in
    coagulation. However, this requires entirely different equipment and

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