A Practical Guide to Haemostasis

Protamine Sulphate Neutralisation Test


The Protamine Sulphate Neutralisation Assay is an assay that has been used historically to calculate the amount of Protamine Sulphate required to correct the anticoagulation induced by Unfractionated Heparin [UFH]. However, it can also be used to calculate the concentration of heparin within a plasma sample.

It is not applicable to use with LMWHs as these do not, in general, prolong the Thrombin Time.

Principles & Method

The principles of these two tests are very similar.

Reagents 1. Increasing dilutions of Protamine Sulphate [0-500 µg/mL] are made in buffer.
2. Thrombin is diluted in buffer so that 0.1mL of thrombin clots 0.2mL of normal plasma at 37°C in 10s
3. Patient citrated Platelet Poor Plasma [PPP]
Test: In the assay, test plasma is incubated with buffer at 37°C and 0.1mL of Thrombin is added.  If the sample clots within 10s – no UFH is present and no further tests are necessary.

If the Thrombin time is prolonged, the buffer is replaced with Protamine Sulphate [500 µg/mL] and the test repeated.  The tests are repeated with increasingly dilute concentrations of Protamine Sulphate until a concentration is found which corrects the thrombin time.
For example if 30 µL of 200 µg/mL Protamine Sulphate corrects the Thrombin time but there is no correction when using 150 µg/mL Protamine sulphate, then 20 µg of Protamine Sulphate is sufficient to neutralise the heparin in 1 mL of plasma.

Assuming weight-for-weight neutralisation [i.e. 1µg of Protamine Sulphate neutralises 1µg of UFH] and 1mg of UFH = 100 IU – then in the above example, 20 µg of Protamine Sulphate neutralises 2 IU of UFH.
If we assume a blood volume of 75ml per Kg body weight then in an 80kg person with a Hct of 0.5:

20 x 75 x body weight x [1-Hct]/1000

= 20 x 75 x 80 x 0.5/1000  =  60

So 60mg of Protamine Sulphate will neutralise all the circulating UFH in this case.

It is relatively straightforward to see that if we know the amount of Protamine Sulphate we need to achieve neutralisation we can deduce the heparin concentration.