laboratory Coagulation Tests


Don't forget to go to Practical Haemostasis for the real deal.


Pre-Lab Variables:

  • Patient identification

  • Sampling technique – needle gauge, patient stress

  • Sample volume & tube used

  • Transport – temperature, agitation, delays reaching lab




Prothrombin Time


1.     PPP + (Tissue Factor + Phospholipid) at 37oC

2.     Add calcium and time to clot


International Normalised Ratio


(Patient PT / Reference PT) to the power of ISI

Or can be expressed as (PT Ratio) to the power of ISI


Activated Partial Thromboplastin Time


1.     PPP + Activator + Phospholipid at 37oC

2.     Add calcium and time to clot


Activators – Kaolin, Silica


Thrombin Time


1.     PPP + Thrombin at 37oC

2.     Time to clot


Thombin – human or bovine source


Clauss Fibrinogen


1.     Dilute PPP + Excess of Thrombin + Phospholipid at 37oC

2.     Add calcium and time to clot

3.     Compare time to calibration curves of reference plasma


Drug Monitoring


Haemoclot Thrombin Time


1.     Dilute PPP + Normal Plasma + Thrombin at 37oC

2.     Time to clot


Time is proportional to plasma concentration of DTI


Reptilase Time


1.     PPP + Reptilase at 37oC

2.     Time to clot


Reptilase cleaves fibrinopeptide A to fibrin. Like TT but not prolonged by LMWH/VKA/DTI


Ecarin Clotting Time


1.     Dilute PPP + Ecarin

2.     Time to clot


Ecarin generates meizothrombin. This then has to be cleaved to thrombin, which is the rate limiting step to clot formation. ECT is prolong by DTIs, but unaffected by Heparins


Activated Clotting Time


1.     Immediate use of whole blood + Surface Activator

2.     Time to clot


Used for POC monitoring of UFH in bypass/ECMO


Chromogenic Anti-Xa Assay


1.     Commercial plasms with known LMWH concentration + known amount of Xa

2.     PPP + Known amount of Xa

3.     Heparin binds to Antithrombin, binds to Xa —> Hep-AT-Xa complex

4.     Residual Xa measured by chromogenic assay using a Xa-specific substrate


Result, ie residual Xa activity, is inversely proportional to the amount of heparin in sample

Result is compared to a reference curve constructed using dilutions of the relevant heparin


Factor Assays


1-Stage PT-based Assay


1.     PPP + Commercial factor deficient plasma

2.     Use this mix to perform PT

3.     Compare result to control plasma dilutions


1-Stage APTT-based Assay


As for 1-Stage PT but with alternative reagents.


Certain F8 gene mutations, and recombinant FVIII concentrates, can cause discrepant results between 1-stage vs 2-stage/chromogenic assays.

  • Typically results in 2x higher FVIII:C with the 1-stage compared to 2-stage/chromogenic

  • In these situations, the 1-stage may give a result within the normal range

  • But the discrepancy can be the other way round

  • The bleeding phenotype usually correlates best with the 2-stage/chromogenic assay


2-Stage Factor VIII Assay


1.     Adsorb patient’s PPP to remove II, VII, IX and X

2.     Mix with activated serum (IX, X, XIa) + V + Phospholipid + Calcium

3.     Incuate at 37oC to produce Xa (patient’s FVIII level is the rate limiting step)

4.     Now mix 1st stage product with normal plasma and perform APTT

5.     Time to clot is proportional to the Xa that was produced in the 1st stage, which is turn was proportional to the FVIII:C in the patient sample.


Chromogenic Factor VIII Assay


1.     PPP + Reagent Cocktail (IXa, X, Thrombin, Phospholipid, Calcium)

2.     Incubate at 37oC to produce Xa

3.     Add chromogenic substrate and incubate

4.     Colour change occurs on cleavage of substrate by Xa —> Calculate Xa concentration


As with 2-stage APTT assay, the patient’s FVIII level is the limiting step in Xa production




PPP – Platelet Poor Plasma

DTI – Direct Thrombin Inhibitors

VKA – Vitamin K antagonists

POC – Point of care

UFH – Unfractionated heparin