Approach to diagnosis of bleeding disorders

APPROACH TO BLEEDING DISORDERS

PRESENTED BY –DR. BISWAJEETA SAHA

Normal hemostasisMechanism by which bleeding from an injured

vessel is arrested by formation of a thrombus.Functions-To maintain the blood in fluid stateTo prevent clots in intact vesselsTo arrest bleeding in injured vessels

Components-Blood vesselsPlateletsPlasma coagulation factorsFibrinolytic system

STAGES OF HEMOSTASIS

INJURY

VESSEL WALL+PLATELET

FORMATION OF PLT PLUG

ACTIVATION OF PLASMA COAGULATION FACTORS

FORMATION OF STABLE FIBRIN CLOT

PRIMARY

SECONDARY

DISSOLUTION OF FIBRIN CLOT BY FIBRINOLYSIS

STAGES OF HEMOSTASIS PRIMARY

Platelet & vessel wall mediated

Occurs within seconds of injury

Forms Platelet plug

Prevent blood loss from capillary,arterioles and venules

SECONDARY

Coagulation factors mediated

Takes several minutes for completion

Forms stable fibrin plug

Prevents blood loss from large vessels

COAGULATION PATHWAYS

FIBRINOLYSIS

PLASMINOGEN

PLASMIN

FIBRIN

FDP,D-DIMERS

INHIBITORY EFFECTS ON CLOT FORMATION

PLASMINOGEN ACTIVATORS

KALLIKREIN

XIIa

PLASMINOGEN ACTIVATORS

t-PA UROKINASE

STREPTOKINASE

CAUSES OF BLEEDING

1.Vessel wall disorders

2.Platelet disorders: quantitative or functional.

3.Coagulation factor: deficiency or inhibitors.

4.Combination of these.

VASCULAR DISORDERS

ACQUIRED

CONGENITAL

•SENILE PURPURA

•VASCULAR PURPURA

•HENOCH SCHONLEIN PURPURA

•HEREDITARY HEMORRHAGIC TELENGIECTASIA

•EHLERS DANLOS SYNDROME

PLATELET ABNORMALITIES

QUALITATIVE

QUANTITATIVE

•THROMBASTHENIA

•BERNARD-SOULIER SYNDROME

•DRUGS(ASPIRIN,TXA2,INDOMATHACIN

•THROMBOCYTOPENIA

•THROMBOCYTHEMIA

DISORDERS OF COAGULATION HEREDITARY HEMOPHILIA A(factor VIII deficiency) HEMOPHILIA B(factor IX deficiency) von WILLEBRAND DISEASE DISORDERS OF FIBRINOGEN-

HEREDITARY AFIBRINOGENAEMIA

HYPOFIBRINOGENAEMIA

DYSFIBRINOGENAEMIA

ACQUIRED DISSEMINATED INTRAVASCULAR COAGULATION(DIC) LIVER DISEASE VIT K DEFICIENCY MASSIVE TRANSFUSION OF STORED BLOOD ACQUIRED INHIBITORS OF COAGULATION HEPARIN OR ORAL ANTICOAGULANT THERAPY RENAL DISEASE

DIAGNOSIS OF BLEEDING DISORDERS

HISTORY

CLINICAL EXAMINATION

LABORATORY INVESTIGATIONS

APPROACH TO A PATIENT OF BLEEDING DIATHESIS

, 1.Clinical evaluation: History. Clinical features.

2.Laboratory approach:First line screening tests. Second line specific tests.

CLINICAL EVALUATIONHISTORY: Age of first manifestation,

Family history of bleeding, Spontaneous or after trauma, Time of manifestation after injury, Ease with which bleeding is controlled, Drug history.

INHERITED DISORDERS

Early age of presentationFamily history positiveMore severBleeding is the dominant

featureSingle factor defect

ACQUIRED DISORDERS

Later age of presentationFamily history usually negativeLess severClinical picture is dominated by

the underlying disorder e.g.DICMultiple hemostatic defect

Findings Disorders of Platelet

Disorders of Coagulation

i) Petechiaeii) Superficial

ecchymosis

iii) Deep dissecting hematomas

iv) Haemarthrosisv) 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

LABORATORY METHODS PREANALYTICAL VARIABLES

Collection of SamplePlastic or polypropylene syringes should be used.Venous blood is preferred over capillary blood.Don`t use pressure cuff or use for <1 min.Blood sample from a indwelling line or catheter should not be

used.Mix thoroughly with anticoagulant by inverting the container

several times.Keep the sample on crushed ice until delivered to the laboratory.

AnticoagulationMost commonly: Trisodium citrate (1:9).

Unacceptable: Oxalate,Heparin, EDTA.

Centrifugation: Preparation of Platelet Poor Plasma(PPP)

Centrifuged at 4000 rev/min at 4 C (for most of the tests) and at room temp. for Platelet function testing, LAC and APCR tests.

PPP should be kept at room temp. for PT,LAC & factor VII assay,and at 4 C for other assay.

Platelet count should be <10,000/mm, it is best achieved by double centrifugation or filtration through a 0.2micron filter.

End-point detection Detecting clot formation as the end-point depends on the rate of its

formation: the shorter the clotting time the more opaque is the clot and the easier to detect.

A slowly forming clot appear as mere fibrin wisp, which are difficult to detect by eye or machine.

Always try to adopt a uniform convention in detecting the end point. Automated analyser are not always correct, they may be spurious. The Coagulometer must detect long clotting times reliably and reproducibly.

Different techniques used: Electromechanical Photo optical Electrochemical etc.

LABORATORY INVESTIGATIONS

TESTS FOR COAGULATION FACTORS

I. Prothrombin Time(PT)

II. Activated Partial Thromboplastin Time(APTT)

III. Thrombin Time(TT)

IV. Fibrinogen assay

TESTS FOR PLATELET

I. Platelet count

II. Bleeding Time(BT)

PROTHROMBIN TIME(PT) SignificanceReflects overall activity of the Extrinsic Pathway.Most sensitive to changes in Factor V,VII,X.Lesser to Factor I & II.

Principle Platelet poor plasma+Tissue Thromboplastin+Calcium

In Presence of F VII Extrinsic pathway is activated & clot

formed

Normal Range

11 to 16 seconds(with rabbit thromboplastin)

10-12 seconds(with human thromboplastin)

Interpretation

Causes of prolonged PT

1. Deficiency of Factor VII,X,V,II,I

2. Vit K deficiency

3. Liver disease esp.Obstructive Jaundice

4. Oral anticoagulants

5. DIC

ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT)

SignificanceReflects efficiency of Intrinsic Pathway.Sensitive to changes in Factor VIII,IX,XI,XII.Also sensitive to heparin & circulating anticoagulants.

PrincipleThe test measures the clotting time of plasma after the activation of contact

Factors(Kaolin/Silica/Ellagic acid) and the addition of phospholipid and

CaCl2, but without added tissue thromboplastin.

So it indicates the overall efficiency of the Intrinsic pathway

Normal range

26 to 40 seconds.

Interpretation

Causes of prolonged APTT

1. Deficiency of Factor VIII(Hemophilia A).

2. Deficiency of Factor IX(Hemophilia B).

3. DIC.

4. Heparin therapy.

5. Circulating anticoagulants.

6. Liver disease.

7. Massive transfusion of plasma depleted stored blood.

THROMBIN TIME(TT)

Significance

Asses the final step of coagulation i.e. conversion of fibrinogen to fibrin in presence of thrombin.

Bypasses Extrinsic & Intrinsic pathway.

PrincipleThrombin is added to plasma and the clotting time is

measured.TT is affected by the concentration and reaction

of fibrinogen and by the presence of inhibitory substances

including fibrinogen/fibrin degradation products(FDPs) and heparin.

Normal rangeA patient’s TT should be within 2 s of the control

(i.e. 15–19 sec). Times of 20 s and longer are definitely abnormal.

Interpretation

Causes of prolonged TT

1. Disorders of fibrinogen-

Afibrinogenaemia. Hypofibrinogenaemia.

Dysfibrinogenaemia.

2. Presence of FDP- DIC or Liver disease.

3. Unfractioned heparin therapy.

4. Hypoalbuminaemia.

5. Paraproteinaemia.

.

Fibrinogen Assay

Usually done by 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.

Normal range 1.8 to 3.6 g/l

InterpretationSensitive to inherited Dysfibrinogenaemia. Insensitive to Heparin unless the level is very high(>0.8µ/µl).High level of FDP(>190µg/ml)may also interfare with the result.

Platelet count & Bleeding Time Platelet count must be done in a suspected bleeding disorder. PBS must be examined for size & staining properties of plt.

BLEEDING TIME(BT)

Significance

Assess Primary Hemostatic defect(vessel wall or platelet).

Dependent on adequate functioning of plt. & Bl.Vs.

Methods

I. Ivy’s

II. Duke’s-not recommended.

III. Template

Range

Ivy’s method: 2 to 7 mins.

Template method: 2.5 to 9.5 mins.

InterpretationCauses of prolonged BTI. THROMBOCYTOPENIA.

II. VWD.

III. PLATLET FUNCTION DISORDER.

IV. DISORDERS OF BLOOD VESSEL.

DIAGNOSIS OF VESSEL WALL DISORDER

LABORATORY TESTS: TPC,PT, APTT,TT are usually normal. The only test of any use is BT. BT may be normal or increased.

HESS` CAPILLARY FRAGILITY TEST: Cuff is wrapped in upper arm and pressure is

maintained midway b/w systolic and diastolic BP for 15 minutes. 4 cm below the elbow joint, a circle of 2.5 cm diameter is drawn on the anterior aspect of forearm.

Upto 10 new hemorrhagic spots are normal. But >20 new spots are always pathological.

This is positive in increased capillary fragility, ITP.

THROMBOCYTOPENIA

SECOND LINE INVESTIGATIONS

Relevant second line investigations are carried out with each of the patterns of abnormalities in first line tests.

1 PT-N

APTT-N

TT-N

FIBRINOGEN-N

PLATELET-N

Interpretation1. Normal hemostasis.

2. Disorders of platelet function(cong or acquired).

3. Vascular disorders of hemostasis.

4. Factor XIII deficiency.

5. Mild von Willebrand disease.

6. Disorder of fibrinolysis.

7. Administration of LMWH.

SECOND LINE INVESTIGATIONS

Clot solubility test. Specific factor assay for suspected factor

deficiency(factor XIII). PFA 100 system.

CLOT SOLUBILITY TEST

PRINCIPLE—

Fibrin clot in presence of factor XIII & thrombin is stable as A result of cross- linking.

But in absence of factor XIII clot dissolves rapidly.

Interpretation—

CLOT NOT DISSOLVED IN 24HRS-

F XIII PRESENT

CLOT DISSOLVES –

F XIII ABSENT

PLATELET FUNCTION ANALYZER(PFA)100

In vitro system for measuring plt- vwf fuction.Assesses both plt adhesion & aggregationMore sensitive than BT to asses Primary hemostasis.The membrane is coated with collagen & epinephrine or

collagen & ADP.It reproduces platelet vwf function under high shear rate.Time req. for closure of full aperture is closure timeNormal closure time: 1 to 3 mins.Interpretation:-

Thrombocytopenia.

von Willebrand Disease.

Plt. Function abnormality.

2 PT—LONG

APTT—N

TT—N

FIBRINOGEN—N

PLATELET COUNT-N

Interpretation

1. Factor VII deficiency.

2. Liver disease.

3. Vit K deficiency.

4. At the start of oral anticoagulant therapy.

5. Mild deficiency of Factor II, V, X.

SECOND LINE INVESTIGATIONS

1. Mixing test.

2. Factor VII assay.

3. Liver function test.

3 PT—N

APTT—LONG

TT—N

FIBRINOGEN—N

PLT COUNT--N

Interpretation1. Congenital deficiency of F VIII, FIX,prekallikrein or HMWK.

2. von Willebrand disease.

3. Heparin-either pt. Is on t/t or contaminated sample.

4. Circulating anticoagulants-

Specific (Anti factor VIII).

Non-specific(Antiphospholipid Ab).

Second line investigations

5. Mixing test.

6. Factor VIII & factor ix assay.

Mixing studyCORRECTION TEST USING PT or APTTPRINCIPLEUnexplained prolongation of PT or APTT can be

investigated with simple correction test by mixing the pt`s plasma with normal plasma.

Correction (should be within few seconds) indicates a possible factor deficiency, whereas failure to correct suggests the presence of an inhibitor.

METHODPerform a PT and/or APTT on control, patient`s,and a

50:50 mixture of the control and pt`s plasma.

PT IS PROLONGED

TREAT WITH HEPARINASE

PT NORMALIZES

HEPARIN IS THE CAUSE

PT REMAINS PROLONGED

PERFORM MIXING STUDY

1:1 MIX OF PATIENT’S AND NORMAL PLASMA

PT NORMALIZES

PT INITIALLY NORMALIZES,SUBSEQUENTLY PROLONGED

PT REMAINS PROLONGED

FACTOR I,II,V,VII,X DEFICIENCY

FACTOR V INHIBITOR

INHIBITORS

APTT IS PROLONGED

TREAT WITH HEPARINASE

APTT NORMALIZES

HEPARIN IS THE CAUSE

APTT REMAINS PROLONGED

PERFORM MIXING STUDY

1:1 MIX OF PATIENT’S AND NORMAL PLASMA

APTT NORMALIZES

APTT INITIALLY NORMALIZES,SUBSEQUENTLY PROLONGED

APTT REMAINS PROLONGED

FACTOR VII,IX,X,XI,XII DEFICIENCY

FACTOR VII INHIBITOR

INHIBITORS(LAC)

CORRECTION TEST USING TTPRINCIPLE The tests use certain physiochemical properties of reagents to bind to inhibitors

or abnormal molecules and normalise the prolonged TT.

INTERPRETATIONTT of

test plasma corrected with Interpretation Saline Normal

plasma ProtaminSO4 Toluidine blue

No Yes No No Deficiency

No Variable No Yes Dysfibrinogenemia of liver disease No Variable Yes No High level of FDP

DETECTION OF CIRCULATING ANTICOAGULANT

PRINCIPLE Circulating anticoagulants or inhibitors may act immediately or be time

dependent. To detect both type of inhibitors, normal plasma and test plasma samples are

tested immediately after mixing and also after incubation together at 37C for 120 min.

4PT—LONG

APTT—LONG

TT-N

FIBRINOGEN—N

PLT COUNT—N Interpretation

1. Vit k def.

2. On oral anticoagulants.

3. Liver dis.

4. Rare congenital or acq. Deficiency of factor v,x,ii .

5. Combined factor V+VIII deficiency.

Second line investigations

6. Mixing test.

7. Specific factor assay.

8. Liver function test.

5 PT—LONG

APTT—LONG

TT—LONG

FIBRINOGEN—N/ABNORMAL

PLT COUNT--N

Interpretation

1. Unfractionated heparin

2. Hypofibinogenaemia

3. Afibrinogenemia

4. Dysfibrinogenemia

5. Systemic hyperfibrinolysis with increased FDP e.g. DIC

6. Some cases of liver disease.

Second line investigations

7. Replitase or ancord time

8. D-dimer level.

REPTILASE OR ANCORD TIME

REPTILASE & ANCORD are purified enzymes from snake.

May be used to replace Thrombin in TT test. Snake venom is not inhibited by heparin- Normal time for clotting in presence of Heparin. Clotting time will be prolonged in presence of raised

FDPs/decreased Fibrinogen.

6 PT-LONG

APTT-LONG

TT-N

FIBRINOGEN-N/LOW

PLT COUNT-LOW

Interpretation1. Massive transfusion of stored/plasma depleted blood.

2. DIC.

3. Chronic liver disease esp.cirrohis.

Second line investigations

4. Specific factor assay.

5. Peripheral blood smear.

6. Bone marrow aspirate.

7 PT—N

APTT—N

TT—N

FIBRINOGEN—N

PLT COUNT—LOW

Interpretation

1. Thrombocytopenia.

2. Heparin use.

Second line investigations

3. Peripheral blood smear.

4. Bone marrow aspirate.

8PT—LONG

APTT—LONG

TT—LONG

FIBRINOGEN—LOW

PLT COUNT—LOW

Interpretation

1. DIC

2. Acute liver necrosis with DIC.

Second line investigations

FDP or D-Dimer assay.

FDP ASSAY Plasminogen

Plasmin

Fibrinogen/ FDP X,Y,D,E

Fibrin

Principle

A Suspension of latex particles sensitized with specific Ab to FDP fragments D & E.

Suspension mixed on a glass slide with a dilution of test serum. Agglutination indicates presence of FDPs.

Interpretation of FDP Assessment

Agglutination with 1in 5 dilution:-FDP >10µg/ml

Agglutination with 1 in 20 dilution:-FDP>40µg/ml

Normal range- <10 µg/ml

10-40 µg/ml- Acute myocardial Infarction.

Acute venous thromboembolism.

Acute pneumonia.

>40 µg/ml- DIC.

Thrombolytic therapy with Streptokinase .

D-DIMER ASSAY

Identical to FDP except latex particles are coated with a Monoclonal Antibody specifically directed against Fibrin D-dimer in human.

Normal range <200mg/l.

DIAGNOSIS OF vWD.

TESTS DONE1.Factor VIII:C concentration,2.vWF:Ag concentration,3.Ristocetin Cofactor Assay(vWF:RCo),4.Collagen binding activity(vWF:CB),5.Multimeric analysis of vWF:Ag.

vWF:RCo Assay: It measures the ability of the Ristocetin(developed as an

antibiotic) to promote the interaction b/w vWF & Platelet membrane gp Ib-IX.

Multivalent ristocetin-dependent binding of vWF creates interplatelet bridges leading to platelet clumps(agglutination) which is measured by an Aggregometer.

vWF:CB Assay: Is complementary(rather than alternative) to vWF:RCo assay. It is an ELISA based system, giving a greater precision.

DIAGNOSIS OF DIC• Finding a prolonged PT and APTT with a reduced TPC & Fibrinogen level is usually DIC,

until proven otherwise.• The diagnosis of DIC is made by the presence of a confirmatory test that shows the

simultaneous presence of thrombin and plasmin formation.

D-dimer Assay:• It is the best test for diagnosing DIC. Value >2000ng/ml is consistent with DIC.• It is the confirmatory test that shows that both thrombin and plasmin have been formed.• It measures plasmin-cleaved, insoluble,cross-linked fibrin that originally arose from

thrombin cleavage of fibrinogen.• D-dimer assay is characteristic but not pathognomonic for DIC.

FDP Assay:• FDPs only indicate plasmin-cleaved fibrinogen, soluble fibrin or insoluble fibrin.• It does not indicate plasmin-cleaved,insoluble,cross-linked fibrin.

Fibrin monomer assay:• Fibrin monomer is the large molecular mass of fibrinogen that remains after release of

fibrinopeptide A & B.• Elevated in early stage, but can be absent in sever DIC. Hence unreliable.