laboratory Coagulation Tests

Intro

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

 

Basics

 

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

 

Glossary

 

PPP – Platelet Poor Plasma

DTI – Direct Thrombin Inhibitors

VKA – Vitamin K antagonists

POC – Point of care

UFH – Unfractionated heparin