Coagulation Study

8/3/2019 Coagulation Study

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What is Coagulation

Coagulation is the process of blood clotsforming to stop the bleeding by converting

soluble fibrinogen in to insoluble fibrin andrepair the damaged blood vessels .This processis called hemostasis.

This process involves 20 plasma proteins and 12blood cloting factors.



2. Coagulation process

3

• Twelve factors are involved in the coagulationprocess. 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 diminishedthe 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.

6



12 FACTORS

7

Factor I Fibrinogen Factor VIII Antihemophilic

globulin

Factor II Prothrombin Factor IX Partial

thromboplastin

component

Factor III Thromboplastin Factor X Stuart-Prower

factor

Factor IV Calcium Factor XI Plasma

thromboplastin

antecedent

Factor V Labile or proaccelerin Factor XII Hageman factor

Factor VII Stable factor or proconvertin Factor XIII Fibrin-stabilizing

factor



COAGULATION COMPLEX

.

Platelet Adhesion•shape change

• release

ADP release.

ThrombxineA2Serotinin

Platelet Aggregation

Coagulation

•Fibrin formation

3 sec

10 sec

5 min



Coagulation Pathway Intrinsic Pathway Extrinsic Pathway

XII XIIa VIIa VII

XIa XI

IX IXa

Prothrombin Thrombin

Fibrin Fibrinogen

(Stable fibrin ) XIII XIIIa

TTTISSUE FACTOR

V VIII Ca++

X Xa

Ca++ Va



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 sampledue to increase turbidity.

Photoid observe the scattered light and convertsdetected intensity into electrical signals and thenmicroprocessor compute the clotting time of sample.

Sample

Photoids

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 tochanges is sample color by para-nitraniline pigments.

Light pass the pass without interupted and photoidreceived the transmitted light and coverts the intensity in

to electrical signals.

Sample

PhotoidFilterLED(LightEmiting Diode)



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

stablizing reagents and antibody and exposed to light(575nm).

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 lightand converts into electrical signals.

LED(LightEmiting Diode

FilterSample

Photoi



Clinical approach for bleeding

disorder

1.The patients who have to go under major surjery.

2.Patients who have history of spontaneousbleeding

After trauma or surgery.

3.The hemorrahagic disorders are either inheritedor 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 15minutes 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.( Ivy’s Method)

This is a test that measures the speed at whichsmall blood vessels close off to stop bleeding (thecondition of the blood vessels) and plateletfunction

18 1



Perform the Test

• A blood pressure cuff is placed onthe upper arm and inflated. Twoincisions are made on the lowerarm. These are about 10 mm (less

than 1/2 inch) long and 1 mm deep(just deep enough to causeminimal bleeding).

• The blood pressure cuff isimmediately deflated. Blotting

paper is touched to the cuts every 30 seconds until the bleeding stops.The length of time it takes for thecuts to stop bleeding is recorded.

http://www.nlm.nih.gov/medlineplus/ency/article/003398.htm




Normal Values

The bleeding stops within 1 to 9 minutes (what isconsidered 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 (includingaspirin).

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 tubeafter every 30 seconds of intervals and the time isnoted 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 thecoumarin type of anticoagulation drugs, such as warfarin.

Principal of the Method

The coagulation process is triggered by incubationof 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 3seconds make sense.

Percentages : 60~140% . ( It means theprothrombin 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

International Normalized Ratio (INR)ISI = international Sensitivity Index

INR target ranges are specified by patientpopulations

prophylactic therapy for DVT: INR= 2.0 -3.0

artificial heart valve: INR=3.0 - 4.0

ISI

meannormal

patient

PT

PT INR



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 topinpoint exactly which factors is defective or

deficient



Principle of the method Factors of the intrinsic coagulation system are

activated by incubating the plasma with theoptimal amount of phospholipids and surfaceactivator.The addition of calcium ions triggers the

coagulation process,and the clotting time is thenmeasured

Reference value: 22~34 seconds



Clinical Significance in PTTIncrease PTT

Lack of factor VIII------Hemophilia A

Lack of factor IX------ Hemophilia B DIC

Presence of non-specific inhibitor lupus likeanticoagulant.

In patient receiving oral anticoagulant.

Decrease PTT

No diagnostical significance



Determination of Fibrinogen

Fibrinogen (Factor I ) determination gives ideaabout coagulation stage 3 defects.

Fibrinogen is a plasma protein which is convertedfrom a soluble protein to an insoluble polymer by

the action of thrombin resulting in the formationfibrin 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 orclotting factor such as prothrombin.

LA is used to help to determine the cause of anunexplained 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 clottingtime 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 IScreening

LlLA 2Confirm

ation

Mixingstudy

lLA notdetected

Seetable 2

LA Present

LLA Present

Normal

LA I Clot Time

Normal

LA 2 Clot Time

Abnormal

LA Ratio

Normal

LA Ratio

LA Clot TimeProlonged

LA 2 Clot TimeProlonged



Table 2(Mixing Study) This test should carried out 50:50 mixture of test plasma and normal

plasma

LA I LA 2 Diagnosis

PatientPlasma

Mix Patient+Normal

PatientPlasma

MixPatient+

Normal

N N N N LA notdetected

ABN ABN N N LA Present

ABN N ABN N Factordeficient

ABN ABN ABN N LA + Factordeficient

ABN ABN ABN ABN Other

inhibitor



Disease of Coagulation disorder

Von- Willebrand disease This is most common heriditary coagulation

disease.

There is defect in Von-Willebrand factor ,whichbinds glycoprotein to collagen,this binding helpsin the activation of platelets and formation of hemostasis.

Factor VIII is also done to know thisdeficiency.(because it is bond with vWF.F VIII PT APTT BT PLATELETS

Decreased Unaffected Prolonged Prolonged Unaffected



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 APTT BT CT PLT F VIII F IX

Hemophilia A Unaffected Prolonged Unaff ected Prolonged Unaff ected Decreased

Hemophilia B Decreased



CORRECTION STUDIES

Principle

Unexplained prolongation of the PT and aPTT can

be investigated with simple correction tests by mixing the patient plasma with normalplasma,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.

R t



Reagents

The reagents which can be used for mixing test areas 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 onequal volume of mixture (50:50) of additive andtest Plasma.





Barium sulphate(100mg to 1 ml) is mixed withcontrol plasma and keep it at 37 c̊ 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 clottingfactor.

Preapartion of adsorbed plasma



3. Aged serum

Normal control blood is allowed to clot and theserum seperated(after atleast 4 hours at 37 c̊ or 48hour’s old aged serum).During clottingFI,FII,FV,FVII are consumed.

Adsorbed plasma will contain factorsFI,FV,FVII,FXI,FXII and FXIII.

Aged Serum will contain factorsFVII,FIX,FX,FXI,FXII and FXIII.



Factor VIII and Factor IX deficient plasma

This plasmas is taken from patients with isolatedsevere deficiency of factor VIII or Factor IX.

Plasma selected for this purpose should have normalPT or means.

Such plasma can be lyopholised(freeze-dried) for longterm storage and stored at -35 c̊ for at lest 3 months



Interpretation of resultDefectin testPlasma

PT APTT Aged Serum AdsorbedPlasma

NormalPlasma

VIII Abnormal Abnormal No Correction Correction Correction

IX Abnormal Abnormal Correction No correction Correction

XI,XII Abnormal Abnormal Correction Correction Correction

Inhibitor Abnormal Abnormal No correction No correction Nocorrection

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



Interpretation of result

Mixing with Factor VIII and FIX Deficient Plasma

Defect intest plasma

PT APTT F VIII def plasma

FIX def plasma

Normalplasma

VIII Abnormal Abnormal Nocorrection

Correction Correction

IX Abnormal Abnormal Correction No

correction

Correction



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Coagulation Study