Category Archives: Coagulation Tests

Mixing Studies


Prolongation of a coagulation test may be as a result of a deficiency in coagulation factors or because of the presence of in inhibitor of coagulation. Mixing studies are performed to differentiate between the two.

Principle of Mixing Studies

Inhibitors affecting activated partial prothrombin time (aPTT) are more common in clinical practice and the discussion that follows is in relation to aPTT. A mixing study with prothrombin time (PT) may be performed using the same principle. Prolongation of aPTT indicates decrease in the coagulation factors the test is dependent on. aPTT prolongs when the factor levels fall to less than 40% of normal.

When the test plasma is mixed with normal pooled plasma in a 1:1 ratio the factor levels are and average of that in the two samples. The coagulation factor levels in pooled plasma are close approximately 100%. Patients with severe coagulation factor deficiency typically have <1% of normal coagulation factors levels. When plasma from such patients is mixed with normal pooled plasma the factor levels in the plasma are will be 50%. For patients with a lesser degree of coagulation factor deficiency the coagulation factor levels in mixed plasma will be greater than 50%. As a normal aPTT needs 40-50% coagulation factor activity and mixing a factor deficient plasma with normal plasma  would result in normalization of a prolong aPTT even in the most severe form of coagulation factor deficiency.

Inhibitors of coagulation are antibodies directed against coagulation factors. Presence of the antibody is associated with diminished coagulation factor activity and a prolonged aPTT.  When the plasma from a patient carrying an inhibitor is mixed with equal amount of normal plasma the inhibitor present in the patients plasma inhibits the activity of the coagulation factors present in pooled plasma. The prolonged aPTT of patient’s plasma fails to correct because the activity of the mixture falls to below that needed for a normal aPTT. Some antibodies act with a lag period. The aPTT of these patients may correct immediately on mixing but will prolong after 2 hours of incubation.

 

Performance of the Mixing studies

Mixing studies involves performance of duplicate aPTT on the following samples

  • Tube 1: Pooled plasma – Plasma from at least 20 normal individuals
  • Tube 2: Patients plasma
  • Tube 3: Patients plasma and pooled plasma incubated separately for 2 hours at 37°C. The two are mixed and the mixture immediately tested.
  • Tube 4: Patients plasma and pooled plasma mixed and incubated for 2 hours at 37°C

 

 

Interpretation of Mixing Studies

Diagnosis
Normal Plasma (Tube 1)
Test Plasma (Tube 2)
Incubated separately and mixed (tube 3)
Mixed and Incubated (tube 4)
Normal Normal Normal Normal Normal
Immediate Inhibitor Normal Prolonged Prolonged Prolonged
Delayed inhibitor Normal Prolonged Minimally prolonged if at all Prolongrd
ISI Curve

Prothrombin Time


Prothrombin time was described by Quick in 1935. It is the time taken by re-calcified plasma to clot in the presence of tissue procoagulant extract known as thromboplastin. It asses the efficiency of the extrinsic coagulation system. The test depends on activation of factor X by factor VII by tissue factor.

The Reagents

1. Thromboplastin: Thromboplastin is a mixture of tissue procoagulants. Thromboplastins have different sources e.g. placenta, human brain, rabbit brain. Recombinant thromboplastins are available.

2. Calcium Chloride: 25mM calcium chloride.

Method

The details of the method are beyond the scope of this text. The outline is as follows. Thromboplastin is added to plasma that has been separated from blood collected in sodium citrate and allowed time to mix. To this mixture calcium chloride is added. The time taken for the plasma to clot is the prothrombin time. The end point (clotting) may be determined manually or using automated (optical or magnetic) methods.

Reporting Results

The results is reported as the number of seconds taken for the clot to form along with a normal value and as INR.

The Concept of International Normalized Ratio

Thromboplastin is a mixture of tissue procoagulants. The source of thromboplastin affects the test results. From the time Quick described the prothrombin time till the 1950s the cadaveric human brain was the source for thromboplastin. Each laboratory had to make it’s own thromboplastin.

In the 1950s commercially available thromboplastins became available. These were derived from animal tissue. Contamination by serum resulted in the presence of some coagulation factors in the commercially available thromboplastin. Oral anticoagulants reduce the levels of vitamin K dependent factors (II, VII, IX, X) prolonging the prothrombin time in proportion to the decrease in the levels of coagulation factors. Use of thromboplastins contaminated with coagulation factors made the test less sensitive to the decrease in coagulation factors in the test plasma. An important clinical consequence of the insensitivity of prothrombin time performed with use of animal thromboplastin was increased risk of bleeding in patients on oral anticoagulant therapy.

A survey of average warfarin dose showed that the average dose of warfarin was higher in North America where commercially manufactured thromboplastins were used compared to UK that used human thromboplastin. Anticoagulation monitored by human thromboplastin at PT ratio (Patient’s PT/Normal or control PT) of 1.5-2 was compared with anticoagulation monitored with animal thromboplastin and a target PT ratio of 2-2.5. The two were found to be equally effective. Also the patients monitored by PT preformed by using human thromboplastin had a 20% lower risk of bleeding.

These observations suggested the need for standardization of prothrombin time to adjust for variations in the reagents used. The concept of international normalized ratio was introduced by WHO in 1983 to fulfil this need.

International Normalized ratio (INR) is defined as

INR = (Patient’s PT/Mean normal PT)ISI

Mean normal PT is the geometric mean of prothrombin time of plasmas collected from at least 20 normal individuals. The group should include members of both sexes. The ISI is the international sensitivity index of the thromboplastin that is calculated as a part of caliberating of thromboplastin by comparing the thromboplastin against a the WHO reference thrombopastin (Internationla Referenmce Preperation, IRP).

Importance of International Sensitivity Index (ISI)

ISI CurveINR depends of the prothrombin ratio and ISI. The difference between prothrombin ratios at INR 2 and 3 for thromboplastin with ISI between 1 and 2 are plotted in the graph above. The INR is maintained between 2 and 3 fro most indications. With a thromboplastin with an ISI of 1 the difference between prothrombin ratio at INR 2 and 3 is 1. This reduces to just over 0.3 for thromboplastin of ISI 2. Thromboplastins with a low ISI give a wider prothrombin time range for a given INR range allowing a more accurate control of anticoagulation. Higher ISI thromboplastins also give a less precise measurement of INR with a high coefficient of variation. It is recommended that the ISI of the thromboplastin used for prothrombin time should be < 1.7 (preferably between 0.9-1.2).

Target INRs for Anticoagulant Therapy

There are many recommendations for target INR for anticoagulation. In general these recommend that therapeutic anticoagulation in most situations needs a target INT of 2.5. Patients at high risk e.g. those having thrombosis on oral anti coagulants or those having mechanical prosthetic valves with moderate to high thrombotic risk (see British Committee for Standardization in Haematology Oral Anticoagulant with Warfarin Guidelines – Fourth Edition and Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines)

Pre-Analytic Variables in Prothrombin Time

Sample collection is critical for prothrombin time. Faulty collection may result in activation of coagulation while collection. Sample for prothrombin time must be collected from a large vein with free flow of blood with a reasonably large calibre needle. The sample should not be a part of a large collection. If evacuated blood collection tubes are used then the prescribed order of draw must be followed (see the BD order of draw chart). If the sample is to be drawn from indwelling catheters 10-15ml blood is drawn and discarded before collection of sample for prothrombin time. Lipaemic plasma may interfere with the function of photo-electric measurements. Samples should not be obtained after meal. Samples should be collected in 3.2% buffered sodium citrate. Nine parts of blood should be mixed with 1 part of anticoagulant. It is not recommended toThe amount of blood in patients with very high or very low haematocrit may be calculates as follows

Amount of blood for every 0.5ml 3.2% sodium citrate =

[60/{100-haematocrit}] X 4.5

 

Related links in this site

  1. Bleeding Time
  2. Pharmacogenetically Guided Warfarin Therapy

 

Further Reading

  1. Poller L. International Normalized Ratio (INR): The First 20 Years. J Thromb Haemost 2004; 2: 849–60. – One of the most comprehensive articles discussing INR and it’s evolution I have come across