What is Coagulation

Coagulation is the process of blood clots forming to stop the bleeding by converting soluble fibrinogen in to insoluble fibrin and repair the damaged blood vessels .This process is called hemostasis.
This process involves 20 plasma proteins and 12 blood cloting factors.

2. Coagulation process

Twelve factors are involved in the coagulation process. Pic 1 Most of factors are manufactured by liver. Vitamin K correlated factor: II, VII, IX, X .

PHASES OF HEMOSTASIS
.There is four phases of hemostasis 1.Constriction of blood vessel(Which diminished the blood flow) 2.Formation of Platelets plug. 3.Formation of clot due to thrombin converted in to fibrin. 4.Formation of stable hemostatics plug or thrombus by plasmin.
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12 FACTORS
Factor I Factor II Fibrinogen Prothrombin Factor VIII Factor IX Antihemophilic globulin Partial thromboplastin component

Factor III

Thromboplastin

Factor X

Stuart-Prower factor
Plasma thromboplastin antecedent Hageman factor

Factor IV

Calcium

Factor XI

Factor V

Labile or proaccelerin

Factor XII

Factor VII

Stable factor or proconvertin

Factor XIII

Fibrin-stabilizing factor
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COAGULATION COMPLEX
Platelet Adhesion shape change . release 3 sec ADP release. ThrombxineA2 Serotinin

Platelet Aggregation

10 sec

Coagulation Fibrin formation

5 min

Coagulation Pathway
Intrinsic Pathway

Extrinsic Pathway
TTTISSUE FACTOR

XII XIa IX

XIIa XI IXa
V VIII
X

VIIa

VII

Ca++ Xa

Ca++

Va

Prothrombin Fibrin XIIIa

Thrombin Fibrinogen

(Stable fibrin )

XIII

Coagulation analyzer( CA -1500)

Principle

Analyzer worked on three Principle. 1.Clotting Method.

2.Chromogenic Method.
3. Immunology Method.

1.Clotting Method
Its is dteremine the clotting time by measuring

changes in the intensity of light scattered by a sample due to increase turbidity. Photoid observe the scattered light and converts detected intensity into electrical signals and then microprocessor compute the clotting time of sample.
Photoids Sample

LED(Light Emiting Diode)

2.Chromogenic Method
When sample is incubate with reagents and exposed to

light (405 nm).There is changes in light obserbance due to changes is sample color by para-nitraniline pigments. Light pass the pass without interupted and photoid received the transmitted light and coverts the intensity in to electrical signals.

LED(Light Emiting Diode)

Sample Filter Photoid

3.Immunology Method
Sample has been warm for a certain period of time with

stablizing reagents and antibody and exposed to light(575 nm). There is change in light absorbance which is caused by antigen,antibody reaction.Light is transmitted through sample and reaches to photoid,photoid receives the light and converts into electrical signals.
Sample Photoi

LED(Light Emiting Diode

Filter

Clinical approach for bleeding disorder

1.The patients who have to go under major surjery. 2.Patients who have history of spontaneous

bleeding After trauma or surgery. 3.The hemorrahagic disorders are either inherited or acquired.

Sreening for bleeding disorders

1.Bleeding time 2.Clotting time

3.Prothrombin time
4.Partial thromboplastin time 5.Fibrinogen determination

Procedure for sample collection

1.Venous sample should be collected in (light blue tube)3.2% sodium citrate of ratio is 1:9. Centrifuge the blood specimen 1500 x g for 15 minutes at room temperature. 2.Reject sample if collect in 3.8 % sodium citrarate. 3.Needle size should be 20-25 gauze to prevent cloting or hemolysis. 4.Visisble hemolysis sample should be rejected. 5.Test must be performed with in 4 hour,s of collection.

1.Bleeding Time.( Ivys Method)

This is a test that measures the speed at which

small blood vessels close off to stop bleeding (the condition of the blood vessels) and platelet function

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Perform the Test

A blood pressure cuff is placed on the upper arm and inflated. Two incisions are made on the lower arm. These are about 10 mm (less than 1/2 inch) long and 1 mm deep (just deep enough to cause minimal bleeding). The blood pressure cuff is immediately deflated. Blotting paper is touched to the cuts every 30 seconds until the bleeding stops. The length of time it takes for the cuts to stop bleeding is recorded.

Normal Values

The bleeding stops within 1 to 9 minutes (what is considered normal varies from lab to lab, depending on how the test is measured).

Elevated Values
A vascular (blood vessel) defect
Thrombocytopenia (low platelets) Severe liver disease

Von Willebrand's disease

Drugs that may increase bleeding times include dextran, indomethacin, and salicylates (including

aspirin). DIC Aplastic anaemia.

CT (Clotting Time)Capillary or Wright

Principle

Blood is collected in a capillary tube after finger prick and the stop watch is started.The formation of fibrin string is noted by breaking the capillary tube after every 30 seconds of intervals and the time is noted at the first appearance of the fibrin string.
Normal Range 4 10 minutes

Perform test

Wipe fingertip with 70% alcohol swab.

Make a deep (1 mm) incision with sterile lancet and start

the stopwatch. Wipe of the first blood drop and collect blood in capillary tube. After every 30 seconds break off about 1 cm of capillary to find out the fibrin formation. When fibrin string appears,stop the stopwatch and note the time.

Elevated Values
This is generally used in severe clotting disorder. 1.Any deficiency of Factor.

2.Or hyperheparinemia.

3. Prothrombin Time
PT measure the factors in Extrinsic way such as Factors II,V, VII and X

II, VII , IX, X, are manufactured by liver and required Vitamin K.

PT alse used to measure the effectiveness of the

coumarin type of anticoagulation drugs, such as warfarin. Principal of the Method The coagulation process is triggered by incubation of plasma with the optimal amount of thromboplastin with calcium.The time to formation of the fibrin clot is then measured.

Measurement
Reference values : 12~15 seconds, over the control 3 seconds make sense. Percentages : 60~140% . ( It means the prothrombin activity) PT ratio : the ratio of the PT to control. International Normalized Ratio (INR) INR : PT ratio convert to INR according ISI (International Sensitivity Index) . Recommendations value: INR 2.0~3.0

Clinical Significance of PT
Increased PT : liver diease or damage such as cirrhosis of liver. Unable to absorb Vitamin K from gastrointestinal tract. True deficiency of Vitamin K. Treatment of Oral Anticoagulant. DIC Billary Obstruction. Decreased PT No diagnostical significance

Prothrombin Time
Monitor warfarin therapy

Monitor heparin/warfarin crossover

Target times are set by

PTpatient International Normalized Ratio (INR) INR PT meannormal ISI = international Sensitivity Index

ISI

INR target ranges are specified by patient

populations prophylactic therapy for DVT: INR= 2.0 3.0 artificial heart valve: INR=3.0 - 4.0

4. Partial Thromboplastin Time

PTT demonstrate the lack of factors, except factor VII. PTT test the function of Instrinsic clotting system. PTT is useful to screen of Factor deficiency of stage I.
The purpose of PTT is to monitor heparin therapy. If the PTT is abnormal, further test are needed to pinpoint exactly which factors is defective or deficient

Principle of the method

Factors of the intrinsic coagulation system are activated by incubating the plasma with the

optimal amount of phospholipids and surface activator.The addition of calcium ions triggers the coagulation process,and the clotting time is then measured

Reference value: 22~34 seconds

Clinical Significance in PTT

Increase PTT Lack of factor VIII------Hemophilia A Lack of factor IX------ Hemophilia B DIC Presence of non-specific inhibitor lupus like anticoagulant. In patient receiving oral anticoagulant.
Decrease PTT No diagnostical significance

Determination of Fibrinogen
Fibrinogen (Factor I ) determination gives idea about coagulation stage 3 defects. Fibrinogen is a plasma protein which is converted from a soluble protein to an insoluble polymer by the action of thrombin resulting in the formation fibrin clot.
Principle

Reference Range 200 to 400 mg/dl

Clinical significance
Depressed Value are formed

Acquired hypofibrinogenemia.
Congenital hypofibrinogenemia. Elevated Values Hyperfibrinogenemia Cardiovascular disease.

LUPUS Anticoagulant(LA I/LA2)

Lupus anticoagulant is non-specific inhibitors

autoantibodies against phospholipids complexes or clotting factor such as prothrombin. LA is used to help to determine the cause of an unexplained thrombosis,recurrent fetal loss or prolongation of APTT. Principle The test uses russel viper venom to directly activate factor X to induce clotting. Presence of LA prolongs LAI screening. Then the addition of LA 2 reagents corrects the clotting time Factor II,V,&X deficiency. Prolongation of both LA I and LA2,start mixing studies.

EXPECTED VALUES
Normal ranges of LAI screening reagent is 31-34 seconds. LA2 confirmation reagent is 30-38 seconds. The ratio of LAI/LA2 was in the range of 0.8-1.2
If ratio

LA I screening is greater than 2.0 LA strongly present LA 2 screening If ratio LA I screening is between 1.5- 2.0 LA moderately present LA 2 screening If ratio LA I screening is greater than 1.2-1.5 LA weekly present LA 2 screening

INTERPRITATION OF RESULT
Figure 1

Suspected LA

LA I Screening

Normal
LA I Clot Time

lLA not detected

Prolonged LLA Present Normal LA 2 Clot Time

LA Clot Time LlLA 2 Confirm ation LA 2 Clot Time Normal LA Ratio See table 2

Prolonged LA Present Abnormal LA Ratio

Mixing study

Table 2(Mixing Study)

This test should carried out 50:50 mixture of test plasma and normal

plasma
LA I
Patient Plasma Mix Patient+ Normal Patient Plasma

LA 2
Mix Patient+ Normal N N N

Diagnosis

N ABN ABN

N ABN N

N N ABN

LA not detected LA Present Factor deficient

ABN
ABN

ABN
ABN

ABN
ABN

N
ABN

LA + Factor deficient
Other inhibitor

Disease of Coagulation disorder

Von- Willebrand disease This is most common heriditary coagulation disease. There is defect in Von-Willebrand factor ,which binds glycoprotein to collagen,this binding helps in the activation of platelets and formation of hemostasis.
F VIII PT APTT BT Factor VIII is also done to know this PLATELETS Unaffected

deficiency.(because it is bond with vWF. Decreased Unaffected Prolonged Prolonged

HEMOPHILIA
Hemophilia is usually inherited,it is rarely acquired. In hemophilia there is defect of Factor VIII and IX. Two types of Hemophilia 1.Hemophilia A (There is defect in Factor VIII)

2.Hemophilia B (There is defect in Factor IX)

PT Hemophilia A Hemophilia B Unaffe cted APTT Prolo nged BT Unaff ected CT Prolo nged PLT Unaff ected F VIII Decreased Decreased F IX

CORRECTION STUDIES
Principle Unexplained prolongation of the PT and aPTT can be investigated with simple correction tests by mixing the patient plasma with normal plasma,adsorbed plasma or with aged serum. Correction indicates a possible factor deficiency where as failure to correct,suggests the presence of inhibitors,lupus anticoagulant etc.

Reagents
The reagents which can be used for mixing test are

as follows. 1.Normal Plasma 2.Adsorbed Plasma 3.Aged serum Factor VIII deficient plasma Factor IX deficient Plasma The abnormal PT and aPTT test are repeated on equal volume of mixture (50:50) of additive and test Plasma.

1.Normal Plasma
The normal plasma contains all the coagulation factors and therefore mixing test for normal plasma

will only identify the presence of an inhibitor or a factor deficiency.The factor which is deficient in patients sample can be identify by correction test using the following plasmas.

2.Adsorbed plasma
Barium sulphate and ammonium hydroxide have the

property of adsorbing certain plasma proteins wihich include the four vitaminK realted factors,namely FII,FVII,FIX,FX.

Preapartion of adsorbed plasma

Barium sulphate(100mg to 1 ml) is mixed with control plasma and keep it at 37 c for 3 minutes.

The mixture is then centrifuged immediately (1700g for 3 min at room temperature).
Take the supernatent plasma and check for PT. Note: This adsorbed plasma should have a PT values > 60 seconds with sensitive reagents.

Care must be taken in the adsorption time,as over adsorption will result in loss of other clotting factor.

3. Aged serum
Normal control blood is allowed to clot and the serum seperated(after atleast 4 hours at 37 c or 48

hours old aged serum).During clotting FI,FII,FV,FVII are consumed.

Adsorbed plasma will contain factors FI,FV,FVII,FXI,FXII and FXIII. Aged Serum will contain factors FVII,FIX,FX,FXI,FXII and FXIII.

Factor VIII and Factor IX deficient plasma

This plasmas is taken from patients with isolated

severe deficiency of factor VIII or Factor IX. Plasma selected for this purpose should have normal PT or means.

Such plasma can be lyopholised(freeze-dried) for long

term storage and stored at -35 c for at lest 3 months

Interpretation of result
Defect in test Plasma VIII PT APTT Aged Serum Adsorbed Plasma Correction Normal Plasma Correction Abnormal Abnormal No Correction

IX

Abnormal

Abnormal Correction

No correction

Correction

XI,XII

Abnormal

Abnormal Correction

Correction

Correction

Inhibitor

Abnormal

Abnormal No correction

No correction

No correction

Note: Correction shows Possibility of factor deficiency whereas failure to correct shows inhibitors.

Interpretation of result
Mixing with Factor VIII and FIX Deficient Plasma
Defect in PT test plasma VIII Abnormal APTT F VIII def plasma No correction Correction FIX def plasma Correction Normal plasma Correction

Abnormal

IX

Abnormal

Abnormal

No correction

Correction

Thank You