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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
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
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
Thrombin Fibrinogen
(Stable fibrin )
XIII
Principle
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
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.
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
Filter
bleeding After trauma or surgery. 3.The hemorrahagic disorders are either inherited or acquired.
3.Prothrombin time
4.Partial thromboplastin time 5.Fibrinogen determination
small blood vessels close off to stop bleeding (the condition of the blood vessels) and platelet function
18 18
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
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
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
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
PTpatient International Normalized Ratio (INR) INR PT meannormal ISI = international Sensitivity Index
ISI
populations prophylactic therapy for DVT: INR= 2.0 3.0 artificial heart valve: INR=3.0 - 4.0
optimal amount of phospholipids and surface activator.The addition of calcium ions triggers the coagulation process,and the clotting time is then measured
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
Clinical significance
Depressed Value are formed
Acquired hypofibrinogenemia.
Congenital hypofibrinogenemia. Elevated Values Hyperfibrinogenemia Cardiovascular disease.
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
LA Clot Time LlLA 2 Confirm ation LA 2 Clot Time Normal LA Ratio See table 2
Mixing study
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
ABN
ABN
ABN
ABN
ABN
ABN
N
ABN
LA + Factor deficient
Other inhibitor
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)
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.
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
severe deficiency of factor VIII or Factor IX. Plasma selected for this purpose should have normal PT or means.
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
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