COAGULATION PROFILE

Dr.Mohamid Afroz Khan

HEMOSTASIS

Hemostasis

The termination of bleeding by mechanical or chemical means or by the complex coagulation process of the body, which consists of vasoconstriction, platelet aggregation, and thrombin and fibrin synthesis.

HEMOSTASIS

Primary

Platelets

Secondary

Coagulation Cascade

PRIMARY HEMOSTASIS

Endothelial damage

Release of Von

Willebrand Factor (vWF)

Platelets attach to vWF via GP Ib/IX

Degranulation attracts more

plateletsPlatelet plug

SECONDARY HEMOSTASIS

• Platelet aggregation initiates secondary hemostasis through the coagulation cascade.

• Coagulation cascade is initiated by the intrinsic or extrinsic pathway.

• The final cascade results in fibrin deposition cross-linking platelets and clot formation

Coagulation Cascade

HISTORICAL PERSPECTIVE

• ‘Coagulation cascade’ based upon the waterfall hypothesis of Ratnoff & Davies and MacFarland.

• In 1977, Osterud and Rappaport recognized that factor VIIa is able to activate factor IX to factor IXa.

• Broze and colleagues factor VIIa–tissue factor complex cannot directly activate factor X but has to go through factor IX activation.

• Gailiani and Broze found that formed thrombin can activate factor XI, resulting in amplification of the coagulation system under stress.

APPROACH TO COAGULATION DISORDERS

Clinical approach

1. Is the bleeding significant ?

2. Local Vs Systemic ?

3. Platelet Vs Coagulation disorder?

4. Inherited Vs Acquired ?

Findings Disorders of Platelet

Disorders of Coagulation

i) Petechiaeii) Superficial

ecchymoses

iii) Deep dissecting hematomas

iv) Hemarthosisv) Bleeding from the

superficial cuts & scratches.

vi) Positive family history

vii) Bleeding from mucous membrane

CharacteristicCharacteristic, usually small & multipleRare

RarePersistent often profuse

Rare

Prominent

RareCommon, usually large & solitary

Characteristic

CharacteristicMinimal

Common

May occur

History & Clinical Examination

Primary HemostasisSecondary Hemostasis

Disorder of secondary hemostasis

aPTT, PT,

Prolonged aPTT

Normal PT

Factor XI Factor XII,

PK & HMWK

Prolonged aPTT & PT

Disorders of fibrinogenFactor IIFactor VFactor X

Combined def of Vit K Dependent Factors

Normal aPTT Prolonged PT

Factor VII

Normal aPTT and

PT

Factor XIII

Laboratory approach

• Large number of tests are essential to diagnose spectrum of bleeding disorders.

• An investigative approach becomes cost effective and patient friendly

Bleeding disorder

Defect / Deficiency in plasma

coagulation proteins

True protein

deficiency

An abnormal

protein

Missense, Deletion &

translocation of DNA

Inhibitor to active site of protein

Immunoglobulins

Enhanced clearance of protein

Result of antigen antibody

complex formation

Defect in platelet number or function

Defect in adhesive interaction between

platelet & vessel wall

A short screening profile

2 To 7 minutes

Abnormalities in Short Screening

Tests

Extended screening

tests

Specialized Diagnostic

Tests.

ASSESSMENT OF COAGULATION PROTEINS

(Assessment of secondary hemostasis)

General considerations

Sample collection• Venous blood is used• 21-gauge for adults• 22- or 23-gauge needle for infants• The blood should be mixed with sodium

citrate anticoagulant in the proportion 9 parts blood: 1 part anticoagulant.

• This should be 0.109M (3.2% trisodium citrate dihydrate)

• Anticoagulant solution can be stored at 4°C for up to three months.

• Use plastic or siliconized glass

• Test within 4 hours.

. • Storage at -70°C or lower is preferable for

further testing

• 40 year old cascade hypothesis still has merit in explaining mechanisms of clot formation in screening tests.

• The coagulation proteins are classified as members of intrinsic or extrinsic pathway.

Activated partial thromboplastin time (aPTT)

• Principle –plasma is incubated with an activator (which initiates intrinsic pathway

of coagulation by contact activation).Phospholipids and calcium are then added and clotting time is measured

• Reagents-kaolin 5 gm/l

-phospholipid

-calcium chloride 0.025 mol/l

• Method –

1.Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 370 C

2.Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution.Incubate at 370 C in the waterbath for 10 minutes.

3.After exactly 10 minutes,add 0.2 ml of phospholipid-calcium chloride mixture,start the stopwatch,and note the clotting time.

Normal range- 30- 40 seconds

Causes of prolongation of APTT

1.Haemophilia A and B.

2.Deficiencies of other coagulation factors in intrinsic and common pathways.

3.Presence of coagulation inhibitors

4.Heparin therapy

5.DIC

6.Liver disease

Test Methodology

Normal Values and Critical Limits• 24 - 37 sec • Statistically, the aPTT is slightly lengthened in

young individuals and slightly shortened in older populations.

• Premature infants have prolonged aPTT values which return to normal by 6 months of age.

Interferences• Lipemia and hyperbilirubinemia interfere with

the detection of clot formation by photo-optical methods.

Prothrombin Time and INR

Principle- tissue thromboplastin and calcium are added to plasma and clotting time is determined.The test determines the overall efficiency of extrinsic and common pathways.

Reagents-

1.Thromboplastin reagent

2.Calcium chloride 0.025 mol/litre

• Method-

1.Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 370 C.

2.Add 0.1 ml of thromboplastin reagent and mix.

3.After 1 minute, add 0.1 ml of calcium chloride solution.Immediately start the stopwatch and record the time required for clot formation.

Test Methodology

• The result is reported in seconds (prothrombin time), or as a ratio compared to the laboratory mean normal control (prothrombin ratio, PTR).

• Normal Value: 11-16 seconds

Understanding the INR

• Need for standardization of PT results.

• The International Normalized Ratio (INR) introduced by WHO.

• Calibration system was developed to relate any PT ratio to a WHO standard.

• International Reference Preparation (IRP).

• ISI correlates the sensitivity of commercial thromboplastin preparations to the IRP.

• By definition, the ISI of the first IRP was 1.0

• An additional term, the INR, was introduced to compare a given prothrombin ratio measurement to the IRP.

• Thus, the INR represents the prothrombin time which would have been obtained if the IRP had been used as a reagent in the test.

Calculating the INR

• ISI is supplied by manufacturer.

• If the ISI is known, the INR is easily calculated by the following formula:

• INR should be maintained in the therapeutic range for the particular indication (INR of 2-3 for prophylaxis and treatment of deep venous thrombosis;INR of 2.5-3.5 for mechanical heart valves).

• Therapeutic range provides adequate anticoagulation for prevention of thrombosis and also checks excess dosage,which will cause bleeding.

Thrombin Time (TT)

• To perform this assay, purified exogenous thrombin is added to plasma to determine the time for clot formation.

• Direct measure of fibrinogen function.• Used to asses if there is defect in fibrinogen

function.

• Prolonged in hypofibrinogenic states and in dysfibrinogenemia.

• The normal range is 15 – 17 seconds.

Fibrinogen Assay (Clauss Technique)

• Principle-diluted plasma is clotted with a strong thrombin solution;the plasma must be diluted to give a low level of any inhibitors (e.g.FDPs and heparin).A strong thrombin solution must be used so that the clotting time over a wide range is independent of the thrombin concentration.

• Make dilutions of the calibration plasma in veronal buffer to give a range of fibrinogen concentrations (i.e. 1 in 5; 1 in 10; 1 in 20 and 1 in 40)

• 0.2 ml of each dilution is warmed to 370C & 0.1 ml of thrombin solution added & clotting time measured

• A calibration curve prepared by plotting clotting time in seconds against the fibrinogen conc in g/l

• The normal range is approximately 1.8–3.6 g/l

Mixing studies

• Used to distinguish between factor deficiencies factor inhibitors.

• If APTT is prolonged, patient’s plasma is mixed with an equal volume of normal plasma

• Plasma samples found to have abnormal screening tests (i.e. PT/APTT) may be further investigated.

• Abnormal screening tests are repeated on equal volume mixtures (termed 50:50 below) of additive and test plasma.

• The following agents can be used for mixing tests:

1. Normal plasma

2. Aged plasma (deficient in factors V and VIII)

3. Adsorbed plasma(Adsorbed plasma is deficient in factors II, VII, IX, and X - vitamin K-dependent factors).

4. FVIII-deficient plasma

5. FIX-deficient plasma

Individual Factor Assays

• One staged assays based on PT and APTT

• Comparing the ability of dilutions of a standard or reference plasma and test plasma to correct the PT or APTT of a plasma known to be totally deficient in the clotting factor being measured.

• Functional FVII activity is measured by a PT-based, FVII deficient plasma clotting assay

• Use of r-human thromboplastin will yield results that are more likely to reflect in vivo FVII levels

• Therapeutic options include FFP, prothrombin complex concentrates, and recombinant FVIIa

• For major surgery, plasma FVII levels of at least 20% are sufficient

One-Stage Assay of Factor VII (based on PT)

• Principle-The assay of factor VII is based on the PT. Assay compares the ability of dilutions of the patient’s plasma and of a standard plasma to correct the PT of a substrate plasma

• It is easily adapted to assay of prothrombin, factor V or factor X

Reagents• PPP from the patient• Standard/reference plasma

• Factor VII-deficient (substrate)plasma-Commercial or from a patient with known severe deficiency

• Barbitone buffered saline• Thromboplastin

Method• Prepare 1 in 5, 1 in 10; 1 in 20 and 1 in 40

dilutions of the standard and test plasma in buffered saline

• 0.1 ml of each dilution +0.1 ml of deficient (substrate) plasma

• Mix and allow to warm to 370 C• Add 0.1 ml of dilute thromboplastin and start

the stopwatch• Record the clotting time

Calculation of Results• Plot the clotting times of the test and standard

against concentration of factor VII on graph paper

The normal range is 50–150 iu/d

One stage assay of Factor VIII (based on APTT)• Principle- based on APTT according to the

bioassay principle.• Reagents-PPP. From the patient

Standard/reference plasma, factor VIII deficient plasma (substrate),barbitone buffered saline,reagent for APTT,plastic tubes,ice bath.

Method-place the APTT reagent and CaCl2

37 degree celsius and patient’s,standard and substrate plasma in the icebath until used

• Make 1 in 10 dilutions of the test and standard plasma in buffered saline in plastic tubes in the ice bath

• Using 0.2 ml volumes, make doubling dilutions in buffered saline to obtain 1 in 20 and 1 in 40 dilutions.

• Place 0.1 ml of three dilutions in glass tubes.• Add to each dilutions 0.1 ml of freshly

reconstituted or thawed substrate plasma and warm up at 37 degree celsius.

• Perform APTTs according to the laboratory protocol.

• The dilutions should be tested at 2-min intervals on the master watch.

• Normal range- 45- 158 iu/dl

Reduced factor VIII concentration is found in-

1.Haemophilia A

2.VWD,types I and III and some cases of type II

3.Congenital combined deficiency of factors VIII and V.

4.DIC

• In this example, the FVIII concentration in the test sample is 7% of that in the standard.

• If the standard has a concentration of 85 IU/dl, the test sample has a concentration of 85 IU/dl × 7% = 6 IU/dl.

Additional Special tests• FDP Assays-FDPs are fragments produced by

proteolytic digestion of fibrinogen or fibrin by plasmin.

• For determination of FDPs,blood is collected in a tube containing thrombin (to remove all fibrinogen by converting it into a clot) and soybean trypsin inhibitor (to inhibit plasmin and thus prevent in vitro breakdown of fibrin).

• A suspension of latex particles linked to anti-fibrinogen antibodies is mixed with dilutions of patients’s serum on a glass slide.If FDPs are present ,agglutination of latex particles occurs.

• Increased levels occurs in DIC,DVT,severe pneumonia and recent MI.

• D-dimer assay- D-dimer is derived from the breakdown of fibrin by plasmin and D-dimer test is used to evaluate fibrin by plasmin and D-dimer test is used to evaluate fibrin degradation.

• Blood sample can be either plasma or serum.• Latex or polystyrene microparticles coated

with monoclonal antibody to D-dimer are mixed with patient’s sample and observed for microparticle agglutination.

• D-dimers are raised in –

DIC,intravascularthrombosis

(MI,stroke,venous thrombosis,pulmonary embolism) and during postoperative period or following trauma.

• Reptilase time (assess the rate of fibrinogen → fibrin conversion in the presence of heparin.)

AUTOMATED COAGULOMETER

Caveron Alpha analyzer

• It is a fully automatic ,coagulation measuring instrument used to perform all plasma coagulation tests for routine and for research for in vitro diagnostic use.

• It can analyze samples using clotting,chromogenic and turbidimetric methods.

• The Caveron Alpha consists of an analyzer, a personal computer and an optional printer.

• It operates on the photometric measurement principle.

• This measurement method finds the coagulation time by an optical detection of the change of turbidity caused by the formation of fibrin fibres.

• For chromogenic methods the change of absorbance is detected after adding a chromogenic substrate after plasma and reagent incubation and for immunological methods also the change of absorbance is detected during the reaction of antigen and antibody complex formation.A calibration converts the results into concentration units.

THANK YOU