Coagulation‎ > ‎

Laboratory testing

Laboratory testing of clotting

Laboratory testing – clotting


Sample preparation

§        Minimise activation and venous occlusion, low surface contact plastic tubes

§        Plasma quality - lipaemia, jaundice, haemolysis, clotted

§        Anticoagulant tri-sodium citrate (106-129 mmol/L)

§        Time sensitive FV and FVIII labile (<4h)

§        Centrifugation (2000g x 15min) – double for APLS

§        Storage (4h at 4oC v 1 month at -20 and 6 months at -70)



First line investigations

Prothrombin time

§        Measures the extrinsic and common pathways (sensitive to V, VII and X less so to II and fibrinogen)

§        Tissue thromboplastin added to citrated plasma - triggered with CaCl2 – time for clot formation = PT

§        Thromboplastin now mainly recombinant human tissue factor (previously derived from various animal components – rabbit lung e/g/).  Combined with phospholipids

§        INR = (PT/MNPT)ISI

§        ISI = international sensitivity index – slope of the curve



§        Measures the intrinsic and common pathway (VIII, IX, XI)

§        Activator (kaolin) + phospholipid added to citrated plasma (activates XIIa which then converts XI to XIa in the absence of Ca) - remainder of cascade (IX to IXa etc) triggered with CaCl2

§        Causes of prolongation include:

§        DIC
Liver disease

§        Massive transfusion

§        Heparin

§        Inhibitors

§        Coagulation factor deficiency (except VII)


Thrombin time

§        Add thrombin to citrated plasma + OBS

§        Can be difficult to standardise due to instability of thrombin

§        Addition of Ca can unmask dysfibrinogenaemia

§        Causes of prolongation include:

§        Hypofibrinogenaemia

§        Raised FDPs (as in DIC)

§        Heparin (nearly always the cause of extreme prolongation)

§        Dysfibrinogenaemia

§        Hypoalbuminaemia / paraproteins



Many different ways of assaying

1. Clauss technique

§        Functional assay

§        Plasma is diluted to give low levels of any inhibitors or heparin

§        Then a strong thrombin solution is used so that the test is independent of the concentration of thrombin in the solution

§        Recommended for routine laboratory use

§        Causes of low levels

§        Inherited dysfibrinogenaemia (in which the physical amount of fibrinogen will be normal)

§        Heparin should not affect the assay unless it is at very high concentrations

2. Physicochemical estimation

§        Clot weight estimation / immunological measure

§        Fibrinogen (Dry clot weight) measurement

§        Fibrinogen is converted into fibrin with thrombin and calcium and the resulting clot is weighed. 

§        The plasma is placed in a tube with calcium and thombin and a stirrer which is used to pick the clot out of the tube after 15min.  This is then washed and dried out in an oven and weighed.

§        Will be discrepant from clauss fibrinogen in dysfibrinogenaemia


3. PT-derived fibrinogen

§        Automated analysers will give a derived estimate of fibrinogen

§        Tends to give higher estimates than the clauss method and will be inaccurate in many disease states and in anticoagulated patients

§        Does not measure fibrinogen quality


Calibration and quality control


Calibration (testing accuracy)

§   WHO and national standards are available for a number of coagulation factors

§   Can alternatively use a pool of normal subjects as the normal value (suggested minimum 20 people)

§   Need to repeat the assay at least 4 times to minimise errors and each sample must be tested in duplicate


Controls (testing precision)

§   Abnormal controls with values of approximately the upper and lower normal limits should be used to test precision

§   Variability is common – due to dilution errors, differences in reagents, failure to take time trends into account and differences in experience between operators. (Coefficient of variation of 15-20%).


Second line investigations


PT and APTT mixing studies

§        Perform PT or APTT on a mixture of patient and normal plasma

§        Caveats

§        Factor VIII inhibitors may require incubation for 2 hours or longer

§        Some Lupus anticoagulants are relatively weak and may only be apparent with 25:75 mixing studies


TT correction studies

§   Toludine blue and protamine sulphate can be use to investigate a prolonged TT

§   Protamine neutralises heparin and FDPs, touidine blue only neutralises heparin




Normal plasma


Toludine blue






Dysfibrinogenamia of liver disease





High FDP concentration






Reptilase time

§   Snake venom used instead of thrombin but same assay as for TT (not affected by heparin)

§   Will remain prolonged in raised FDPs, abnormal or reduced fibrinogen or hypoalbuminaemia


Factor Assays

One step assays

§   Test and control plasma (with 100% of the clotting factor) are both assayed in a range of dilutions in plasma deficient of the factor being investigated (but nothing else). 

§   PT (factor II, V, VII and X) or APTT (factors VIII, IX, XI, prekallikrein and HMWK) performed depending on the factor being assayed.

§   Results plotted on double log paper to produce semi-linear relationships and the level of factor deficiency can be calculated by comparing the clotting time with the known factor levels in the dilutions of test plasma.


Factor VIII assays

One step as above


§   Haemophilia A (<30)

§   Carriers of haemophilia

§   vWD

§   Congenital deficiency of V and VIII

§   DIC

§   Acquired haemophilia

§   (APLS)


§   Can be confounded by preactivation of VIII which occurs in some more mild forms of haemophilia and this results in a spuriously high FVIII level.

§   Relies on FVIII not being activated

§   Eg. Qualitative defect of FVIII molecule making it unstable leading to rapid dissociation of the activated molecule and sub-optimal factor X activation


Two step assays (Chromogenic Factor VIII assay)

§   Technique

1.      Incubate test sample with purified FIXa and FX to generate Xa – which is dependent on the amount of FVIII in the test sample (all FVIII is activated – so circumvents problem of preactivation)

2.      Stage 2 – addition of aliquot from stage 1 to normal plasma – Generation of thrombin  and fibrin.  In chromogenic addition of chromogenic Xa substrate is used to quantify Xa in the stage 1 sample.


§        Advantages

§        Totally deficient plasma not needed

§        Claimed to have better sensitivity and precision than one stage - able to measure very high and low levels of FVIII (i.e. in concentrates)

§        Circumvents the problem of FVIII preactivation by preactivating all the VIII (by incubating with IXa, phospholipid and Ca) and then assaying this in a separate system by its ability to generate Xa.

§        High dilutions minimise interferences (e.g. from lupus anticoagulants)

§        Disadvantages

§        Complex and technically difficult


Assays for the detection of inhibitors

§   Bethesda unit defined as the amount of inhibitor that will neutralise 50% of the factor VIII:C in normal plasma (ie with 100% FVIII) after 2 hours at 37oC

§   The strength of the inhibitor is the reciprocal of the dilution that resulted in a reduction of FVIII activity to 50%.  (ie if the dilution to 1:16 caused a 50% reduction the inhibitor has a strength of 16 BU).


§   The test plasma is made up in a variety of dilutions (ranging from undiluted to 1:50) and mixed with an equal volume of normal plasma, which is then incubated at 37oC and tested for FVIII activity.

§   The % of residual FVIII activity is calculated as a percentage of the activity in the normal plasma


§   The inhibitor activity in the diluted sample is then calculated by the formula:


§   Which is more easily visualised on the graph below (NB the graph is on semilog paper and 100% = 0; 50% =1 and 25% =2)



§        The strength of the inhibitor is then calculated by dividing the FVIII Inhibitor activity in the diluted sample by the amount it has been diluted.

§        For example

§        1:5 dilution gives a residual FVIII activity of 33%

§        From the above formula/graph there is a FVIII inhibitor activity of 1.6 BU in this sample. 

§        The dilution is then compensated for by dividing the inhibitor activity by the dilution ie 1.6 by (1/5 ie 0.2) = 8.0

§   If the strength of the inhibitor changes with the dilutions it has complex kinetics (as opposed to simple kinetics where the strength is constant at different dilutions).

§   The control is either mixed with buffer (original Bethesda) or FVIII deficient plasma at pH 7.4 (Nijmegen modification – prevents false positives at low inhibitor levels)


Clot solubility testing

§   Clots formed in the presence of FXIII and thrombin are stable for at least 1 hour in the presence of urea (5mmol/l).

§   This assay is only sensitive for FXIII deficiency up to 5iU/dL. (i.e. it has a poor sensitivity)

§   Patient plasma is mixed with thrombin and forms a clot which is then put into urea solution and left for 24h, with inspection at regular intervals.  (normal clot will be intact at the end of 24h).

§   ELISA based assays are more sensitive but have poor reproducibility and their role is not clear.


Euglobulin clot lysis time

§        Investigates fibrinolytic potential

§        Plasma is diluted and acidified and a precipitate (euglobulin) form which consists of tPA, plasminogen and fibrinogen

§        PAI-1 and a2-AP are left in solution

§        Precipitate is redissolved, fibrinogen clots with thrombin, and the time for clot lysis is measured

§        Pre and post venous occlusion times

§        Venous occlusion increases tPA release and shortens the lysis time

§        Reduced PAI-1, a2-AP shortens the lysis time (increased sensitivity with venous occlusion)

§        Can also assay tPA, a2-AP and PAI-1



§   Detected by using latex agglutination

§   Latex particles attached to antibodies to purified FDP fragments are mixed with the patients serum.  Aggregation indicates the presence of FDPs.  This can be made semiquantitative using different dilutions of the  patient’s sample.



§   Latex agglutination method - identical to above except that the beads are coated with a monoclonal antibody to the fibrin D-dimer

§   Since there is no reaction with fibrinogen the need for serum is eliminated and measurements can be performed on plasma samples.

§   D-dimer estimation can also be performed by ELISA, which have a greater sensitivity but are more complex to perform.


Genetic testing

§   The genes involved can be tracked in most families leading to certainty about carrier status



Investigation of vascular disorders of haemostasis (e.g Ehlers-Danlos, pseudoxanthoma elasticum)

§   In general the tests of coagulation are unhelpful apart from the bleeding time.

§   Careful history and examination are most likely to give the diagnosis.  Particular attention should be paid to previous scars and associated signs of the inherited syndromes.

§   Confirmation of the diagnosis requires analysis of collagen from cultured fibroblasts or DNA analysis


Bleeding time

§        Blood pressure cuff inflated around the arm at 40mmHg.  Standard incision (6mm long and 1mm deep) is made on the volar surface of the forearm. Blot off the blood exuding from the cut every 15s. 

§        Ivy’s method uses a microlance but is otherwise fairly similar.

§        Normal range is 2-7 min.

§        Interpretation

§        Thrombocytopenia (Plts < 50 may result in very prolonged bleeding time)

§        Platelet function defects

§        vWD

§        Vascular abnormalities

§        Occasionally severe deficiency of factor V or XI or afibrinogenaemia



§        Instrument aspirates blood sample through a capillary and a microscopic aperture cut into a membrane which is coated in collagen and either epinephrine or ADP.

§        The time taken to close the aperture is reported as the closure time.

§        Sensitive to

§        Platelet adherence and aggregation abnormalities (but may miss minor disorders such as secretion defects)

§        vWD

§        Glycoprotein Ib

§        Glycoprotein IIb/IIIa


VWD investigations – see vWD

Platelet aggregometry – same




§   Whole blood

§   Rotating pin to imitate sluggish venous flow and activate coagulation

§   Speed and strength of clot formation is measured

§   Paraemeters measured

§        R time – reaction time/ rate of initial fibrin formation

§        Increased – deficiencies of clotting factors or inhibitors

§        Decreased – hypercoagulable

§        K – clot formation time / fibrin build up and cross linking

§        Prolonged – deficiencies of plateletets / fibrinogen / clotting factors

§        Αlpha angle – speed at which clot forms

§        Reduced with deficiencies of platelets/ fibrinogen

§        Maximum amplitude – fibrin strength

§        Any abnormality leading to reduced fibrin clot (quantitative or qualititative)

§        A60 – measures clot lysis

§        Reduced in hyperfibrinolysis




Interpretation of results in bleeding patient


Factor levels up to 50% will usually give normal results


1. PT/APTT/TT/Fibrinogen/Platelets count all normal


§        Normal haemostasis

§        Platelet function disorder (congenital or acquired)

§        Mild vWD

§        Factor XIII deficiency

§        Disorder of vascular haemostasis

§        Mild / masked coagulation deficiency

§        Disorder of fibrinolysis (antiplasmin or PAI-I deficiency)

§        LMW heparin


1.      PFA 100

2.      RiCo / VIII levels

3.      Clot solubility test

4.      Xa level

5.      Euglobulin clot lysis time


2. Prolonged PT with normal APTT / TT / Fibrinogen / Platelets


§   Factor VII deficiency

§   Start of oral anticoagulation

§   Possibly lupus anticoagulant

§   Mild deficiencies of II, V, X in some cases may just prolong the PT


1.      Mixing test

2.      VII level (II, V, and X possibly)

3.      Lupus anticoagulant

4.      LFTs


3. Prolonged APTT with normal PT/TT/ Fibrinogen / Platelets


§   Congenital factor deficiencies of the intrinsic pathway (VIII, IX, XI and XII)

§   vWD

§   Prekallikrein and high molecular weight kininogen (HMWK) deficiency

§   Mild deficiencies of II, V, and X with some reagents

§   Inhibitors

§   Heparin but TT usually prolonged – confirm with reptilase time

§   Lupus anticoagulant


1.      Mixing studies

2.      VIII, IX, XI, XII levels

3.      vWF Ag and Ristocetin cofactor

4.      2 minute APTT to exclude contact factors (usually 5 minute incubation time, but APTT more sensitive to contact factor deficiency with a short incubation time)


4. Prolonged PT and APTT (normal TT / Fib / Platelets)


§   Vitamin K deficiency

§   Warfarin

§   Liver disease

§   II, V, and X deficiencies

§   V and VIII deficiency


1.      Mixing studies

2.      If correct – II, V and X levels

3.      Vitamin K levels


5. Prolonged PT / APTT / TT (normal Fib / Platelets)


§   Heparin

§   Hypofibrinogenaemia

§   Liver disease

§   Hyperfibrinolysis


1.      Reptilase time

2.      FDPs or D-dimers


6. Prolonged PT / APTT /with low platelets (normal TT / fib norm or decreased)


§   Massive transfusion

§   Chronic liver disease

§   DIC


7. Prolonged PT, APTT, TT and reduced platelets and fibrinogen


§   DIC

§   Liver failure

Investigations not normally necessary as diagnosis usually obvious (FDP / D-Dimers, consider dual pathology ? blood film)