Urvashi Seminar 7

8/2/2019 Urvashi Seminar 7

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DISSEMINATED INTRAVASCULAR COAGULATION

Disseminated intravascular coagulation (DIC) is a syndrome inwhich either the extrinsic or intrinsic or both pathways areactivated to produce multiple fibrin clots in small blood vessels.The resultant reduction of the coagulation factors and plateletsresults in bleeding. This process is accompained by activation offibrinolysis which further accentuates the bleeding tendency. Mostof the DIC syndromes are acute and generalised. It is a complexsystemic thrombohaemorrhagic disorder involving the generationof intravascular fibrin and the consumption of procoagulants andplatelets.


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MECHANISM OF DIC

The various etiological factors activate:

Extrinsic and intrinsic pathways of coagulation whichresult in widespread clotting in small blood vessels.

As the fibrin monomers polymerise in the microvascularsystem, there is platelet entrapment in the thrombusresulting in thrombocytopenia in addition to markedreduction of clotting factors (consumption coagulopathy).


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Fibrinolytic system is activated simultaneously,

resulting in lysis of fibrin, forming split products

fibrin degradation products (FDP) which furtherinhibits clot formation resulting in widespread

bleeding.

Tissue factor pathway inhibitor (TFPI an

anticoagulant mechanism is disabled in DIC.

Protein C+S are anticoagulant proteins. TNF-a and IL-

1 in sepsis incapacitate protein C and further reduced

via consumption.


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DIC develops acutely when sudden exposure of procoagulants occurs .

Compensatory haemostatic mechanisms are quickly overwhelmed. Chronic DICrefelects a compensated state that develops when blood is intermittently

exposed to tissue factor .Compensatory mechanism in liver and bonemarrow are not

overwhelmed andfrequently occurs in solid tumours.

Obstetric complicationsIn these cases, tissue thromboplastin released from placental tissue activate FX

therby initiating the coagulation sequence and widespread intravascular coagulation

.

2. Malignancies

In AML-M3, leukemic promyelocytes liberate tissue thromboplastin leading yo

DIC thus accentuating the bleeding due to thrombocytopenia.

3. Infections

In Gram Negative septicemia, bacteria liberates endotoxins which activate FXII,thus initiating the intrinisc coagulation cascade and finally widespread DIC.

ACUTE DIC VS. CHRONIC DIC


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SECONDARY FIBRINOLYSIS

Following DIC ,fibrinolysis gets activated and FDPs- D- Dimerformed have an inhibitory effect on clot formation. Plasmin formed

inactivates FV, VIII, IX and XI which further inhibit intrinsic

coagulation cascade.


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CLINICAL MANIFESTATIONS OF ACUTE DIC

Abrupt onset of bleeding in the form of ecchymoses, petechiae.

Bleeding from sites of venipuncture.

Gum bleeding, epistaxis and gastrointestinal haemorrhage.

State of shock may develop.

Acute renal failure is not uncommon.

Signs and symptoms of intracranial bleeding and pulmonaryhaemorrhage.

Clinical features of underlying diseases are present.


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LAB FINDINGS IN DIC

Thrombocytopenia because of utilization of platelets inmicrothrombi.

PT , APTT because of fibrinogenand plasmic digestion ofvarious factors.

FDP +, D-Dimer+.

Thrombin time (TT) because of fibrinogen .

Presence of fragmented RBCs in the peripheral smear.

Intravascular haemolysis may occur.


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Diagnosis/Lab Findings

TestPlatelet count

Fibrin degradation product

(FDP)

Factor assayProthrombin time (PT)

Activated PTT

Throbimn time

Fibrinogen

D-dimer

Antithrombin

AbnormalityDecreased

Increased

DecreasedProlonged

Prolonged

Prolonged

Decreased

Increased

Decreased (Otto,2001)


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Principle of test for presence of

FDPD-Dimer

Latex particles are coated with specific

antibodies to purified FDP.

Serum / Plasma of the plasma is taken and latex

particles (coated with Ab) are added.

Presence of agglutination suggests the presence

of FDP (D-Dimer).

D-Dimer test is specific in diagnosing DIC.


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Detection of fibrinogen/fibrin Degradation products using a

latex aggultination.

Principle:-

A suspension of latex particles is sentisized with specific antibodies to the purifiedFDP fragments D and E. The suspension is mixed on a glass slide with a dilution of

a serum to be tested. Agrregation indicated the presence of FDP in the sample. By

testing different dilutions of the unknown sample, a semiquantitative assay can be

performed.

Reagents :-

Venous blood.

Collected into a special tube (provided with the kit) containing the antifibrinolytic

agent and thrombin.

Test kit.(Oxoid Ltd.)

Positive and negative controls.

Provided by the manufacturer.

Glycine buffer.


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Make 1 in 5 and 1 in 20 dilution of aerum in glycine buffer.

Mix 1 drop of each serum with 1 drop of latex suspension on a glass slide.

Rock the slide gently for2 minute while looking for macroscopic agglutination.

If a posiyive reaction is observed in the higher dilution, make doubling dilutions

from the 1 in 20 dilution until macroscopic agglutinationcan no longer be seen.

Method :-

Allow the tube with blood to stand at 37C until clot retraction

commences. Then centrifuge the tube and withdraw the serum for

testing. It is important that the fibrinogen in the sample is completely

clotted or this will be detected by the test. This may be a problem in the

presence of heparin or a dysfibrinogenaemia or high levels of FDPs.

Addition of a few drops of 100 u(NIH)/ml thrombin will enhance

clotting in these cases.


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INTERPRETATION

Agglutination with a 1 in 5 dilution of serum indicates a concentration ofFDP in excess of 10 ug/ml;agglutination in a 1 in 20 dilution indicates FDP

in excess of 40 ug/ml.

NORMAL RANGE

Healthy subjects have an FDP concentration of less than 10 ug/ml.

Conditions between 10 and 40 ug/ml are found in a variety of conditionsincluding :

Acute venous thromboembolism

Acute myocardial infarction

Severe pneumonia

After major surgery

High levels are seen in systemic fibrinolysis associated with DIC and

thrombolytic therapy with streptokinase


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SCREENING TESTS FOR FIBRIN

MONOMERS

PRINCIPLE

When thrombin acts on fibrinogen, some of the monomers do not polymerise butgive rise to soluble complexes with plasma fibrinogen and FDP. These complexescan be associated in vitro by ethanol or protamine sulphate.

REAGENTS

PPP (PLATELET POOR PLASMA)From the patient and a control.

POSITIVE CONTROL

This is prepared by adding 0.1 ml of thrombin (0.2 NIH units/ml ) to 0.9 ml ofcontrol plasma and incubating at 37C for 30 minutes.

Fibrin threads formed during theincubation are removed by centrifugation.

PROTAMINE SULPHATE

1% (10g/l)

ETHANOL

50% (v/v) in water


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METHODProtamine sulpate test .

Add 0.05 ml of Protamiine sulphate to 0.5ml of patients plasma and to 0.5ml ofpositive control plasma.

Incubate undisturbed at 37C for 30 minutes.

A positive result is indicated by the formation of a fine fibrin network or fibrinstrands.

The presence of amorphous material only is a negative result.

Ethanol gelation testAdd 0.15 ml of ethanol to 0.5 ml of patients plasma and to 0.5 ml of the positivecontrol plasma at room temperature (c 20C).

After gentle agitation inspect the tubes at 1 minute intervals.

A positive result is the formation of a definite gel within 3 minute.

INTERPRETATION Positive gelatin tests are found in the following scenarios :

The early stages of the acute DIC

After major surgery

Severe inflammatory illness, in particular lobar pneumonia

Liver disease


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DETECTION OF CROSS LINKED

FIBRIN D-Dimers USING A LATEX

AGGLUTINATION METHODPRINCIPLE

The latex agglutination method used to detect crosslinked fibrin D-Dimersis identical to the test previously described to FDP, but in this case

thelatex beads are coated with a monoclonal antibody directed specificallyagainstfibrin D-Dimer in human plasma or serum.

Because there is no reaction with the fibrinogen,the need for serum iseliminated and measurements can be performed on plasma samples.

REAGENTS

Several manufacturers market kits for the measurement of D-Dimers. These usually contain the latex suspension, dilution buffer, and positiveand negative controls.


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METHODThe manufacturers protocol should be followed.

Undiluted plasma is mixed with one drop of latexsuspension on a glass slide and the slide is gently rocked forthe length of time recommended in the kit.

If macroscopic agglutination is observed, dilutions of the

plasma are made until agglutination can no longer be seen.

INTERPRETATION

Agglutination with the undiluted plasma indicates aconcentration of D-Dimers in excess of 200 mg/l.

The D-dimer level can be quantified by multiplying thereciprocal of the highest dilution showing a positive result by200 to give a value in mg/l.


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NORMAL RANGE

Plasma levels in normal subjects are < 200 mg/l.There has been much study of D-dimr assays as a useful way ofexcluding thrombosis, but there is naturally a compromisebetween senstivity and specificity especially when a rapidturnaround time is required.

Lack of an international standard and the poor correlationbetween kits mean that the use of kits for this purpose should bevalidated individually.

Number of kits using ELISA methods for the detection of D-dimer,s are now available that have greater senstivity but are

more cumbersome to perform.Latex test using automated analysers may provide an acceptablecompromise.

These tests have now been incorporated into clinical guidelinesaccording to their sensitivity .


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ASSESSMENT OF

FIBRINOLYTIC SYSTEM

Euglobulin lysis time

Euglobulin fraction contains

plasminogen,plasminogen activators and

fibrinogen.The test is a measurement of fibrinolytic activity.


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MANAGEMENT

Principles of treatment in DIC are:

Prompt treatment of the primary disease.

Heparin therapy.

Platelet transfusions.

Replacement therapy with coagulation factors.

Fresh frozen plasma.


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OTHER COAGULATION

DISORDERSHAEMORRHAGIC DISEASE OF THE NEWBORN

Commonly seen in the economically deprived populations.

Due to vitamin k deficiency in the neonate. Delayed colonization of

the gut affects the synthesis of Vitamin k.Bleeding occurs from umbilical cord stump or melena on 2nd/3rdday after birth.

Intracranial bleeding and large imtramuscular haemorrhages may

occur.PT ,APTT .

There is dramatic response to parenteral Vitamin k therapy.


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LIVER DISEASE Hepatic disease results in deficicncy of factors synthesized in liver likeII, VII,IX and X.

Severs liver disease leads to low levels of FV and fibrinogen also.

These factors are required for both extrinsic and intrinsic pathways andtherefore:

PT- prolonged APTT-prolonged

Gastrointestinal haemorrhageis the common manifestation and occursfrom peptic ulcer, or gastritis.

Spontaneous superficial haemorrhage is the clinical presentation insome cases.


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HOW TO INVESTIGATE A CASE

OF BLEEDING DIATHESIS

The focus in such cases is on :History

Physical examination

Blood counts and physical examination

Platelet count

Coagulation profilePT,APTT, TT, fibrinogen assay and FDPDDimer test

Platelet function tests to rule out functional disorder e.g. von Willebrandsdiaease.


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HISTORYAge of onset of bleeding

Whether bleeding is spontaneous induced or traumatic bleeding

Severity and duratiom of haemorrhage

History of drug intake like aspirin chlorthiazide

Past history of dental extraction ,minor surgery and traumatic bleeding

Menstrual history in females

Family history of bleeding disorder


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EXAMINATIONSite of bleedingskin /mucous membrane/intramuscular

/joint.

Platelet disorder : Petechiae,epistaxis,gingival bleeds+

Coagulation disorder : Haematomas, haemarthrosis,

ecchymosis+ ; bleeding in GIT and genitourinary systemnot uncommon.


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3. BLOOD EXAMINATION

Complete haemogram and peripheral smearexamination to rule out acute leukemia,macrocyticanaemia, aplastic anaemia etc. and other causes ofthrombocytopenia.

4. PLATELET COUNT

If low, all secondary causes of thrombocytopeniashould be ruled out before labelling the case as ITP

acute or chronic. Giant platelets observed in BernardSouliersyndrome.


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5. COAGULATION PROFILE

PT : DIC, Vit k deficiency, obstructive jaundice,oral

anticoagulant therapy to be considered.

APTT : DIC, haemophilia A and B, vWD to be

considered. TT : In DIC and fibrinogen deficiency.

Fibrinogen assay : Fibrinogen is reduced in DIC,

hypo/afibrinogenemia.FDP-D-Dimer : +ve in DIC.


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PLATELET FUNCTION TESTS

Platelet aggregation studies with ADP, collagen,epinephrine and ristocetin to rule out vWD,

storage pool defect and Bernard-Soulier syndrome

should be carried out.

The above tests help in arriving at a diagnosis in

bleeding disorders.

In inherited bleeding disorders, it is necessary to

carry out family studies and also tests for detection

of carriers of haemophilia A/B.


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Urvashi Seminar 7

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