Approach to a Bleeding Disorder, Laboratory Diagnosis of Hemophilia, Coagulation Disorder

1. Dr. Sourabh Mandwariya
Pathologist & Nodal Officer Central Lab,
Indira Gandhi District Hospital, Mandsaur

2.  Bleeding disorders are very common
 Haemophilia A & vWD most common
 Bleeding patient can be a nightmare
 Proper evaluation & anticipation is needed

3.  Normal physiological mechanism for keeping
the blood in fluid state in vascular system
and for prevention of hemorrhage by
complex interaction of blood vessel walls,
platelets and plasma proteins.
 Primary Hemostasis (Platelet plug formation)
 Secondary Hemostasis (Stable fibrin clot)

4.  Three main components
 Blood Vessel Wall
 Constriction of blood vessel
 Endothelial cell –
 von Willebrand factor (vWF)
 Tissue Factor
 Platelet activating factor

5.  Life span – 7 to 10 days
 Function
 Adhesion
 Release Reaction (ADP, TxA2, Serotonin)
 Aggregation

7.  Coagulation Factors
 Coagulation Inhibitors
 Fibrinolytic Systems

8.  Coagulation Factors
I. Fibrinogen
II. Prothrombin
III. Tissue Factor, Thromboplastin
IV. Calcium
V. Labile Factor
VI. No Factor
VII. Stable Factor
VIII. Antihemophilic globulin; Antihemophilic factor
IX. Christmas Factor
X. Stuart-Prower Factor
XI. Plasma Thromboplastin Antecedent
XII. Hageman Factor
XIII. Fibrin Stabilizing Factor; Laki-Lorand Factor
Prekallikrein –Fletcher Factor
High Molecular Weight Kininogen – Fitzgerald Factor

9.  Coagulation Factors
 Three Groups
1. Fibrinogen Group: I, V, VIII, XIII
2. Vitamin-K Dependent: II, VII, IX, X
3. Contact Group: XI, XII, HMWK, Prekallikrein

10.  Extrinsic Pathway
 Intrinsic Pathway
 Common Pathway

12.  Coagulation Inhibitors

13.  Fibrinolytic System

16.  Inherited Disorder of Coagulation
 Hemophilia A (Classical Hemophilia, F VIII
Deficiency)
 Point Mutation or deletions of F VIII gene (Long arm of
the X chromosome)
 X-linked recessive disorder
 Primarily affecting males; females are carriers but do
not manifest the disease.
 Incidence – 1:10000

17.  Inherited Disorder of Coagulation
 Hemophilia A (Classical Hemophilia, F VIII Deficiency)
 Three Types
 Mild - > 5%(Excess Bleeding only after major trauma or
surgery)
 Moderate – 1-5%(Excess Bleeding after mild to
moderate trauma; occasional hemarthrosis;
spontaneous bleeding infrequent)
 Severe - < 1% (Frequent and spontaneous deep tissue
hematomas, Hemarthroses, Intracranial hematomas, )

18.  , Hemarthroses

21.  Acquired Inhibitors of Coagulation
1. Specific
 Against F VIII (Multi transfused patient, Old Age, RA,
Postpartum females)
2. Non-specific
 SLE, Neoplasia, HIV

22.  Is it a congenital or acquired bleeding
If congenital - Factor defi / Platelet / vWD
If Acquired - Surgical / Coagulopathy / Drugs
 Is bleed present at presentation
 Special situations - On drugs / Pregnancy /
Neonate
 Other Organ Involvement

23.  Clinical Evaluation
 Easy Bruising
 Spontaneous Bleeding
 Multiple Sites Bleeding
 Repeated Episodes of Excessive Bleeding
 Restart of bleeding hours or days following injury
 Similar past or family history
 Poor wound healing
 Bleeding is out of proportion to the degree of trauma.

24.  Clinical Evaluation
 Petechia - ≤2 mm in diameter
 Purpura - ≥3 mm but <1 cm in diameter
Both in Primary Hemostatic disorder
 Ecchymosis (Bruise) - >1 cm in diameter
(Defective hemostasis or trauma)
 Hematoma – Large area of hemorrhage
(Coagulation Disorder)
 Hemarthrosis – Coagulation Disorder Specially –
Hemophilia.

25.  Clinical Evaluation
 Muco-cutaneous bleeding (Purpiura, Petechia, Gums Bleeding,
Epistaxis, GI Bleeding, Menorrhagia)- Platelet Disorders, vWD
 Cephalhematomas, hemarthrosis, Intramuscular Hematoma, Intra
Cranial Bleeding, Retroperitoneal Haemorrhages – Hemophilia,
Afibrogenemia, vWD
 Umblical Stump Bleeding – Afibrogenemia, F XIII Defecicency
 Defective wound healing – F XIII Defeciency
 Recurrent Sever Epistaxis – Hereditary hemorrhagic Telangietasia

26.  Clinical and Laboratory
evaluation both are
necessary.
 Detailed Family History
 Family Tree

27.  Clinical Evaluation
 History of bleeding only in males and positive
family history on maternal side – X linked
Disorders (F VIII & F IX deficiency)
 Autosomal Recessive Disorders – Both males and
females are affected, History of consanguinity)
 Autosomal Dominant Disorders – Both sex, Older
generation, in one parent.

29.  Laboratory Evaluation
 CBC
 Peripheral Smear
 Bleeding time
 Clotting Time
 Prothrombin Time
 Activated Partial Thromboplastin Time

30.  Prothrombin Time (PT)
 Extrinsic (F VII) and Common (F X, F V,
Prothrombin & Fibrinogen)

31.  Prothrombin Time (PT)
 Sample Collection
 Should not use Glass Syrynge
 Trisodium Citrate (3.2%) Anticoagulant (1:9)
 Mix by gentle inversion 5 times
 Centrifuged 3000 RPM for 20 minutes
 Within 02 hrs of Blood collection test should be carried
out

32.  Prothrombin Time (PT)
 Method
 100 µl plasma + 100 µl Thromboplastin Reagent (TF
and Phospholipids)
 Incubate 37ºC for 1 minute
 Add 100 µl Calcium Chloride
 Record time for clot formation
Normal Range – 11 to 16 Seconds

33.  Prothrombin Time (PT)
 Prolongation of PT
1. Oral Anticoagulants
2. Liver Disease
3. Vit K Deficiency
4. DIC
5. Inherited Deficiency of factors of extrinsic and
common pathway

34.  Activated Partial Thromboplastin Time (aPTT)
 Intrinsic (F XII, F XI, HMWK, Prekallikrein, F IX &
F VIII) and Common Pathway

36.  Activated Partial Thromboplastin Time (aPTT)
 Method
 Phospholipid Reagent and Calcium chloride solution in
glass tube at 37ºC
 100 µl plasma and 100 µl Kaloin Solution at 37ºC for 10
minutes
 Add 200 µl Phospholipid and Calcium Chloride Mix.
Note the clotting time.
Normal Range – 30-40 seconds

37.  Activated Partial Thromboplastin Time (aPTT)
 Prolongation of aPTT
1. Hemophilia A or B – Always perform PT along
with aPTT
2. Monitor Heparin Therapy (Activate
Antithrombin III)
3. Inhibitors of Coagulations

38.  Mixing Studies (For distinguish between factor
deficiency and factor Inhibitors)

39.  Interpretation of screening test
1. Only Thrombocytopenia
 Hematological Disease
2. Selective Prolongation of Bleeding Time
 Platelet Function Disorder
 vWD
 Vascular Disorder
3. Selective Prolongation of aPTT
 Intrinsic Pathway
 Heparin Therapy
 Inhibitors
4. Prolongation of aPTT and Bleeding Time
 vWD

40.  Interpretation of Screening Test
5. Selective prolongation of PT
 F VII Deficiency
 Early Vit K Deficiency
 Oral Anticoagulant
6. Prolongation of both PT and aPTT
 Common Pathway Factors Deficiency
 Dysfibrinogenemia
 Liver Disease
7. All Screening test abnormal
 DIC
8. All screening test normal
 Mild form of vWD
 Platelet Function Defect
 Vascular Disorder
 Mild Coagulation Factor Deficency

41.  Specific Test for Hemostasis
1. Factor VIII assay (Normal 50-150%)
 log/log Graph
2. Platelet Aggregation Studies
3. Detection of Fibrinogen/fibrin degradation product
4. Detection of D-dimers
5. Estimation of fibrinogen
6. Platelet Glycoprotein analysis

42.  Specific Test for Hemostasis
 Normal Graph Paper

43.  Specific Test for Hemostasis
 log/log Graph

44.  Mild Congenital disorders can present late in life
 Every bleed in Pediatric age group is not Cong
 Combinations are possible
 History / General examination / Routine Inv
 Specialized investigations needed
 Repeat testing may be needed
 Obstetric bleeding to be tackled very aggressively
 Neonatal evaluation can be tricky

45.  Thank You