Coagulopathy Testing

Outline the methods for assessing coagulation, platelet function and fibrinolysis

Coagulation Factors

All these tests measure how long it takes to make fibrin. They evaluate different parts of the coagulation cascade, which help localise where a coagulopathy may be occurring.

In these tests:

• Citrate is added to blood
  Binds calcium and prevents clotting.
• Sample is centrifuged
• Plasma decanted
• Calcium (to replace the calcium lost by binding to citrate) and a reagent is added
• Time taken to clot measured

Prothrombin Time/INR

The prothrombin time measures the extrinsic pathway. Tissue factor has to be added to the sample in order start clotting - this is why it is known as the extrinsic pathway as a substance extrinsic to the sample must be added. As the PT varies significantly between different labs, the INR is used to allow values to be compared.

Any disorder of the extrinsic or common pathways will prolong the PT, i.e. deficiency or inhibition of:

• Factor VII
• Factor X
• Factor II (prothrombin)
• Factor V
• Factor I (fibrinogen)

Although warfarin affects factors in all three pathways, its clinical effects are measured using INR. This is because:

• Factor VII has the shortest half-life of the clotting factors affected by warfarin
  Therefore so its levels will fall the quickest.
• Therefore a fall in Factor VII levels is the earliest indication of changes in coagulation status due to warfarin
• As factor VII is only in the extrinsic pathway, the PT/INR are the only tests which can evaluate its function

(Activated) Partial Thromboplastin Time

The partial thromboplastin time measures the intrinsic pathway, which begins produce fibrin when activated by the addition of phospholipid to the sample (phospholipid is contained in platelets, and so is not technically "extrinsic"). The activated partial thromboplastin time is the same test, except an activating agent is added to speed up the reaction.

Any disorder of the intrinsic or common pathways will prolong the APTT, i.e. deficiency or inhibition of:

• Factor XI
• Factor IX
• Factor VIII
• Factor X
• Factor V
• Factor II (prothrombin)
• Factor I (fibrinogen)

Heparin affects both sides of the pathway (IIa, IXa, Xa, XIa) however typically affects intrinsic factors more than extrinsic.

In addition, anti-phospholipid antibodies will also prolong the APTT by binding the added phospholipid.

Activated Clotting Time

Activated Clotting Time is used to for the dosing and reversal of heparin in cardiopulmonary bypass and other extracorporeal circuits.

Fresh whole blood is added to a tube with an activator (e.g. glass beads) to stimulate the intrinsic pathway. The time until clot formation is measured in seconds. Different activators will have different normal ranges, and target ranges for the circuit in use.

Platelet Function

Evaluate how well platelets aggregate in response to factors like ADP, collagen, arachidonic acid, and adrenaline (i.e., endogenous stimulators of platelet aggregation).

In this test, the aggregating agent is added to a tube of platelets, and the change in turbidity measured. Different patterns of response (or non-response) can be diagnostic of different platelet function disorders.

Point of Care Testing

Point of care coagulation testing:

• Involves testing of whole blood
  Traditional testing uses plasma only.
  • Therefore includes the cell-based model of coagulation
    May better represent actual clotting function compared with traditional coagulation factor testing.
• Provides information on all phases of clotting

Viscoelastic Methods

Include:

• Thromboelastography (TEG)
  Continuous measurement and display of viscoelastic properties of a blood sample from initial fibrin formation to clot retraction, and ultimately fibrinolysis. Involves:
  • A known volume (typically 0.36ml) of whole blood added to activators in two disposable cuvettes (cups) heated to 37°C
    • Contact activators (such as kaolin) are added to the blood to accelerate clotting
    • A heparinase cuvette is also commonly used so clotting function can be measured during full anticoagulation (e.g. CPB)
  • Pin attached to torsion wire immersed into blood Torsion on the pin is converted (by a transducer) into a TEG tracing.
  • Cuvette rotates through 4°45′ in alternate directions
    Each rotation takes 10s.
  • Pin initially remains stationary as it rotates through the un-clotted blood
    This is represented by a straight line on the tracing.
  • As blood clots, cup rotation exerts torque on the pin
  • The stronger the blood clot, the greater the torque exerted on the pin

• Rotational Thromboelastometry (ROTEM)
  Modified version of TEG:
  • A pin fixed to a steel axis is rotated in blood via movement of a spring
    The cuvette remains stationary.
  • Two samples are used:
    • Tissue factor is added to measure the extrinsic pathway (known as the ENTEM cuvette)
    • Contact activator is added to measure the intrinsic pathway (INTEM cuvette)
  • Impedance to rotation is detected by an optical system:
    • LED
    • Mirror on the steel axis
    • Electronic camera
  • Uses different reference ranges and nomenclature to TEG

Advantages and Disadvantages of TEG/ROTEM

Interpreting TEG/ROTEM

Note that reference ranges are not included here, and will vary depending on the:

• Technique (TEG/ROTEM) used
• Activator used
• Adjuvants added
  e.g. Citrated vs. recalcified samples.

References

1. Krafts K. Clot or Bleed: A Painless Guide for People Who Hate Coag. Pathology Student.
2. Activated Clotting Time - Practical Haemostasis.
3. Srivastava A, Kelleher A. Point-of-care coagulation testing. Contin Educ Anaesth Crit Care Pain. 2013;13(1):12-16.