Approach to Bleeding disorders

Tasneem V. Bisht
24/07/2014
What is a bleeding disorder?
Prolonged / increased bleeding following
apparent minor injury
Spontaneous/excessive/recurrent bleeds from
same/multiple sites
Bleeding into joints, muscle
Family history of any of these
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
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

SCURVY

HENOCH SCHONLEIN
PURPURA
HEREDITARY
HEMORRHAGIC
TELENGIECTASIA

EHLERS DANLOS
SYNDROME
PLATELET
ABNORMALITIES
QUALITATIVE
QUANTITATIVE
THROMBASTHENIA

BERNARD-SOULIER
SYNDROME

DRUGS(ASPIRIN,TXA2,IND
OMATHACIN
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.



HISTORY OF BLEEDING
Age of onset of symptoms. In childhood possible
inherited disorder. Recent onset acquired disorder
Type of bleeding. Mucosal and skin versus deep
tissues and haemarthrosis.
Delayed wound healing / umbilical bleeding.
Severity of bleeding tendency.
Family history. Sex linked recessive haemophilias.
Autosomal other disorders
Drug history
INHERITED DISORDERS
Early age of presentation
Family history positive
More sever
Bleeding is the dominant
feature
Single factor defect
ACQUIRED DISORDERS
Later age of presentation
Family history usually negative
Less sever
Clinical picture is dominated by
the underlying disorder
e.g.DIC
Multiple hemostatic defect
Finding Disorders of Coagulation Disorders of Platelets or
Vessels
Petechiae Rare Characteristic
Deep dissecting
hematomas
Characteristic Rare

Superficial ecchymoses Common; usually large and
solitary
Characteristic; usually small
and
multiple
Hemarthrosis Characteristic Rare
Delayed bleeding Common Rare
Bleeding from
superficial cuts and
scratches
Minimal Persistent often profuse

Sex of patient 8090% of inherited forms
occur only in male patients
Relatively more common in
females
Positive family history Common Rare (exc. vWF , hereditary
hemorr.
telangiectasia)
LABORATORY FIRST LINE INVESTIGATIONS
TESTS FOR COAGULATI ON 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. Platelet morphology
III. Bleeding Time(BT)











PROTHROMBIN TIME(PT)
Significance
Reflects 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)
I nterpretation
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)
Significance
Reflects efficiency of Intrinsic Pathway.
Sensitive to changes in Factor VIII,IX,XI,XII.
Also sensitive to heparin & circulating anticoagulants.
Principle
The 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.
I nterpretation
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.
Principle
Thrombin 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 range
A patients TT should be within 2 s of the control
(i.e. 1519 sec). Times of 20 s and longer are definitely abnormal.

I nterpretation
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
I nterpretation
Sensitive to inherited Dysfibrinogenaemia.
Insensitive to Heparin unless the level is very high(>0.8/l).
High level of FDP(>190g/ml)may also interfare with the result.

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

BLEEDING TIME(BT)
Significance
Assess Primary Hemostatic defect(vessel wall or platelet).
Dependent on adequate functioning of plt. & Bl.Vs.
Methods
I. Ivys
II. Dukes-not recommended.
III. Template
Range
Ivys method: 2 to 7 mins.
Template method: 2.5 to 9.5 mins.

I nterpretation
Causes of prolonged BT
I. THROMBOCYTOPENIA.
II. VWD.
III. PLATLET FUNCTION DISORDER.
IV. DISORDERS OF BLOOD VESSEL.


MEAN PLATELET VOLUME and PDW
Inc. in
myeloproliferative dis
Peripheral plt destruction
Reticulated platelets
Index of plt production
DIAGNOSIS OF VESSEL WALL DISORDER

LABORATORY TESTS: Plt count,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 5-7 minutes.
1 cm below the elbow joint, a circle of 3 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.

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
Interpretation
1. 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 & aggregation
More sensitive than BT to assess 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 time
Normal closure time: 1 to 3 mins.
Interpretation:-
Thrombocytopenia.
von Willebrand Disease.
Plt. Function abnormality.


2
PTLONG
APTTN
TTN
FIBRINOGENN
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
PTN
APTTLONG
TTN
FIBRINOGENN
PLT COUNT--N
Interpretation
1. 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

1. Mixing test.
2. Factor VIII & factor IX assay.


Mixing study
CORRECTION TEST USING PT or APTT
PRINCIPLE
Unexplained 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.

METHOD
Perform 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 PATIENTS 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 PATIENTS 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)
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.
4
PTLONG
APTTLONG
TT-N
FIBRINOGENN
PLT COUNTN
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
1. Mixing test.
2. Specific factor assay.
3. Liver function test.







5
PTLONG
APTTLONG
TTLONG
FIBRINOGENN/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
1. Replitase or ancord time
2. 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.


CORRECTION TEST USING TT
PRINCIPLE
The tests use certain physiochemical properties of reagents to bind to inhibitors
or abnormal molecules and normalise the prolonged TT.

INTERPRETATION

TT 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

6
PT-LONG
APTT-LONG
TT-N
FIBRINOGEN-N/LOW
PLT COUNT-LOW
Interpretation
1. Massive transfusion of stored/plasma depleted blood.
2. DIC.
3. Chronic liver disease esp. cirrohis.

Second line investigations
1. Specific factor assay.
2. Peripheral blood smear.
3. Bone marrow aspirate.



7
PTN
APTTN
TTN
FIBRINOGENN
PLT COUNTLOW
Interpretation
1. Thrombocytopenia.
2. Heparin use.
Second line investigations
1. Peripheral blood smear.
2. Bone marrow aspirate.


THROMBOCYTOPENIA

8
PTLONG
APTTLONG
TTLONG
FIBRINOGENLOW
PLT COUNTLOW
Interpretation
1. DIC
2. Acute liver necrosis with DIC.

Second line investigations
FDP or D-Dimer assay.






FDP ASSAY
Plasminogen

Plasmin

Fibrinogen/
Fibrin FDP X,Y,D,E

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 >10g/ml
Agglutination with 1 in 20 dilution:-FDP>40g/ml

Normal range- <10 g/ml
10-40 g/ml- Acute myocardial Infarction.
Acute venous thromboembolism.
Acute pneumonia.
>40 g/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 DONE
1.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.

CORRECTION STUDIES
To rule out the presence of an inhibitor.
To pinpoint the deficient factor using
correction by adsorbed plasma and aged
serum.
To do correction studies using specific factor
deficient plasma


Adsorbed plasma contains FI, FV, FVIII, FXI
and FXII.
Aged serum contains F VII, FIX, FX, FXI, and
FXII.
CORRECTION STUDIES
AP PT FVII AS
FV,FVIII,XI,XII FVII, IX, X, XI, XII

Normal
aPTT prolonged PT and no corr. FII

Corr. by ads. pl Corr .by aged serum
PT PT PT PT
Normal Abnormal Normal Abnormal
FVIII FV FIX FX


Corrected by both
FXI or FXII