1. Lecture:8 [Path] Platelet disorders
Objectives
The objective of the lecture is to discuss the evaluation of a patient with bleeding disorder;
classification of hemorrhagic disorders; idiopathic thrombocytopenic purpura and disorders of platelet function
Learning Outcomes
 At the end of the lecture, students should be able to
Discuss the work up of a patient with hemorrhagic disorders
Classify hemorrhagic disorders due to vascular defects (non-thrombocytopenic purpura)
Classify hemorrhagic disorders due to platelet deficiency (thrombocytopenia)
Discuss the definition, pathogenesis, morphology and clinical course of idiopathic thrombocytopenic purpura
Classify disorders of platelet function

2. Haemostasis
DEF:-Process of forming a barrier to blood loss at the site of injury is haemostasis
Stages of Haemostasis
Primary Haemostasis – Blood vessels and Platelets
Secondary Haemostasis – The Coagulation system
Tertiary Haemostasis-Fibrinolysis
Defect in Any Stage-----Haemorrhage

3. Stable Hemostatic Plug
Hemostasis
BV Injury
Tissue Factor
Neural
Coagulation
Cascade
Blood Vessel
Constriction
Platelet
Aggregation
Primary hemostatic plug
Reduced
Blood flow
Fibrin formation
Stable Hemostatic Plug

4. Coagulaton cascade

5. Work Up of a case of HAEMORRHAGIC Disorder

6. Test for Primary Hemostasis
Platelet Count
Bleeding time
Duke’s method-ear lobule or Ant. Forearm two pricks 4 mm deep with special Lancet.
Normal=3-5 min
Ivy method--two 10 mm long and 1mm deep incisions on fore arm with a blade.BP Apparatus cuff pressure 40 mm Hg.
Normal -3-10min
Platelet aggregation tests

7. Bleeding Time Determination

8. Bleeding Time Increased in
Thombocytopenia
Thrombocytopathies=Bernard Soulier Disease and Glanzman’s Thrombosthenia
DIC
Aspirin and other cyclo-oxynase blockers

9. Test for secondary haemostasis
Whole blood clotting time-( Lee and white method)=
5ml of blood is placed in a test tube at 37 degree centigrade in water bath.
Clotting time min
if prolonged-severe def. of clotting factors
Capillary method=

10. Other Tests For Secondary HAEMOSTSIS
ProthombinTime
Russel viper venom Test
Activated Partial Thromboplatin Time(aPTT)
Thrombin Time
Factor assays
Fibrin split products(FDP)
Circulating anticoagulant

11. Surface Injury Thrombin or XIIa TF + VII XI XIa TFPI X Xa IX IXa +PT X Xa IX
IXa +
VIIIa
IXa:VIIIa X Xa + Va
Prothrombin
Xa:Va
Thrombin
APTT
Fibrinogen
Fibrin

12. Prothrombin Time (PT) IT Is A Test For Extrinsic Pathway
Normal secs
Reagent =thromboplastin,ca,citrated plasma

13. Prothrombin Time Prolonged PT is seen in Deficient factor activity
Congential or acquired def. of factors mainly Fator ii v vii x
Presence of coagulation factor inhibitors
Liver disease and Obstructive Jaundice
Oral anticoagulant therapy
Vit k def
Too much citrate

14. Prothombin TIME Expression Of PT 1. In Seconds
2.INR-Intrnational Normalisation Ratio.
Why INR?=same sample measured by different Laboratories can give very different PT value in seconds.-due to different reagent source and different instrument type.

15. Prothrombin Time
INR–was established by the WHO and International Committee on Thrombosis and Hemostasis for reporting the results of PT test.
All PT results are standardized by this calculation.
INR=(patient PT/control PT) ISI

16. ISI=International sensitivity index
Given by manufacturer of each reagent and instrument combination
It reflects the reagent sensitivity to different Vit K dependent factor.

17. Proposed Therapeutic range of PT for monitoring Oral Anticoagulants(ISTH)
Pimary and secondary prevention of venous thrombosis
Active venous thrombosis,pulmonary embolism,prevention of recurrent venous thrombosis

18. Russel Viper Venom Test(RVVT)
Russel viper venom acts as thromboplastin and directly activates Factor X.
RVVT is used to differentiate def of factor VII and X
Dilute RVVT is used for testing Lupus anticoagulant

19. Activated Partial thromboplastin Time Test(APTT)
IT IS A Test for Intrinsic pathway
Normal=30-35 sec
Reagents
Surface activator:
Silica
Kaolin
Phospholipids
Calcium

20. Activated Partial Thromboplastin Time(APTT)
Test for screening abnormalities in the intrinsic pathway -xii,xi,ix,viii,
Prolonged APTT may be seen in
Deficient factor activity-Factor xii,xi, viii and ix inherited or aquired
Consumption (DIC)
Presence of coagulation factor inhibitors
Presence of lupus anticoagulant
Non fractionated Heparin therapy
Too much citrate

21. If both PT and APTT are abnormal
Heparin contamination
Deficiency of the factors in the common pathway
Presence of circulating inhibitors

22. Thrombin Time Reagent=Thrombin (Ca)+citrated plasma Normal= 8-10 sec
Thrombin T ime Abnormal in
Inherited and aquired Hypofibrogenemia/ dysfibrogenemiaand afibrogenemia
Non fraactionated heparin therapy
Fibrin degradation products( FDP) due to fibrinolysis

23. Fibrinolysis Primary- Pregnangy complication
Secondary –Disseminated Intravascular Coagulation.
Thrombolytic therapy
PT& APTT Abnormal

24. Clinical Features of Bleeding Disorders
Platelet Coagulation disorders factor disorders
Site of bleeding Skin & Mucus Membrane Deep in soft tissue
Epistaxis,GIT,Vaginal Muscle & joints
2.Petechiae Yes No
3.Ecchymoses (“bruises”) Small, superficial Large, deep
4.Hemarthrosis / muscle bleeding Extremely rare Common
5.Bleeding after cuts & scratches Yes No
6.Bleeding after surgery or trauma Immediate, Delayed (1-2 days),
usually mild often severe

25. Clinical Terms used for skin bleeds
Petechae--- < 2mm .Generally Platelet Disorder
Purpura mm .Platelets & Vascular
Echymosis > 10 mm. Blue purple coloraton of skin and mucosa due to rupture of vessel in subcuaneous tissue. Generally coagulation disorder
Bruise—( layman’s term) also called contusion caused by blunt trauma.It is haematoma. Can occur in skin ,muscle ,bone
Haematoma---Collection of blood anywhere.
Purpura due to vasculitis is palpable
All these lesions donot blanch on pressor

26. Petichae
<2mm

27. Petechae

28. Purpura
2-10mm

30. echymosis
>10mm

31. Bruise

32. Intracranial bleed

33. Classification of Haemorrhagic/Bleeding disorders Disorder
Haemorrhagic disorder due to vascular abnormality
Haemorrhagic disorder due to platelet abnormality
Haemorrhagic disorder due to coagulation factor abnormality

34. Bleeding/Haemorrhagic Disorders – Vessel wall abnormalities
Also called non-thrombocytopenic purpura
Common but not usually serious.
Produce petechiae/purpura in skin & mucous membrane.
Sometimes nose bleeds, menorrhagia, GI bleeds.
Routine tests are usually normal

35. Bleeding Disorders – vessel wall abnormalities-Causes
Infections=Meningococcemia, infective endocarditis, rickettsioses (either vasculitis or DIC)
Drug reactions=immune leucocytoclastic vasculitis
Scurvy & Ehlers-Danlos syndrome Lack of collagen support
Senile purpura & Cushing’s Syndrome
Henoch – Schonlein purpura (immune complex disease)
Colicky abdominal pain, polyarthralgia & acute glomerulonephritis
Hereditary hemorrhagic telangiectasia
AD disorder with tortuous thin blood vessels. Nose bleeds, bleeding in oral cavity, eyes etc
Systemic Amyloidosis

36. Henoch – Schonlein – Purpura ( Allergic)
Etiology-NOT KNOWN.Most cases follow pharyngitis or vaccination
Pathogenesis :
Immunological damage to vessel wall(vasculitis) due to immune complex deposition in vascular wall containing IgA +c3.
Clinical :Triad of symptoms—purpura,
abdominal pain and arthritis.Renal involvement ---haematuria
Pathology :Fibrinoid necrosis of vessel wall Neutrophilic vasculitis. IgA and complement deposits

37. Role of Platelets in Haemostasis
Formation of primary haemostatic plug
Formation of secondary haemostatic plug
Healing of injured tissue

38. Bleeding Disorder Due to Quatitative & Qualitative Defect In Platelets

39. Quantitative Defect Thrombocytopenia
Platelet count of less than /mm3
Clinical bleeding symptoms generally not seen until platelet count < 50000/mm3
Possibility of severe and spontaneous bleeding is high if count <

40. Thrombocytopenia - Causes
Decreased production
Megakaryocyte hypoplasia – Amegakaryocytic thrombocytopenia
Replacement of normal marrow by leukaemia, cancer metastasis
Aplastic anaemia
Increased peripheral destruction/utilization
Immune mediated
Immune (Idiopathic) thrombocytopenic purpura
Transplacental thrombocytopenia
Drugs
Non-immune mediated
DIC, HUS, TTP
Increased splenic sequestration - Hypersplenism
Dilution-multiple blood transfusion
Multifactorial causes-Vit B12 def, FA Def, Cirrhosis,

41. Immune /Autoimmune /(Idiopathic) Thrombocytopenic Purpura
Most common cause for thrombocytopenia
Acute ITP
Chronic ITP

42. Acute ITP Most often in children 2 to 6 years of age
No sex predilection
Generally follows a viral infection by 1 to 3 weeks.
Autoantibody fomation against platelet antigen
Presents with abrupt onset of easy bruising, petechiae on the extremities and bleeding from mucus membranes
No fever, no splenomegaly

43. Acute ITP – Lab Findings
Platelet count – usually < 20000/mm3
Peripheral smear – Normal except for thrombocytopenia
Bleeding time – Prolonged
Bone marrow – Increase in megakaryocyte young and old with ronded margins.

45. Acute ITP – Course
Most patients (90%) – spontaneous remission within 2 to 6 weeks
Recurrences very uncommon
Treatment – symptomatic, corticosteroids

46. Chronic ITP Seen in young women Male to Female ratio is 1:3
Onset is insidious.
Platelet count is generally between to 80000/mm3
Autoantibody titer is higher than in acute ITP – directed toward GPIIb/IIIa
Lab investigation findings similar to acute ITP

47. Chronic ITP – Course and Treatment
Chronic disease, spontaneous remissions uncommon
Few cases have alternating periods of remissions and exacerbations – Intermittent ITP
Treatment – Corticosteroids, Splenectomy

48. Platelet function disorder Qualitative Defect Inherited Acquired

49. Inherited Defect Defects of Adhesion Defects of Platelet aggregation
Bernard-Soulier Syndrome: inherited (AR) defect in adhesion of platelets to subendothelial collagen due to lack of Ib-IX (vWF receptor). PBS—Giant platelets
Defects of Platelet aggregation
Glanzman’s throbasthenia: AR disease, characterized by failure of platelets to aggregate with ADP due to lack of IIb-IIIa (fibrinogen receptor)
Disorders of Platelet Secretion
Storage pool disease: Failure of release of granule bound ADP & impaired secretion of prostaglandins
Alpha granule def—gray platelet syndrome
Dense granule def-Delta storage Pool disease

50. Giant Platelets

51. Acquired Defect
Drugs
Aspirin,non steroid antiinflammatory drugs ingestion
Inhibits prostaglandin synthesis by its inhibitory action on COX
Uremia, paraproteinaemia

53. Lecture:9 [Path] Bleeding disorders related to abnormalities in clotting factors
Objectives
The objective of the lecture is to discuss coagulation disorders- hemophilia and Von Willebrand disease
Learning
Outcomes
At the end of the lecture, students should be able to
Classify coagulation disorders
Describe the inheritance, clinical manifestations, types and diagnosis of hemophilia
Describe inheritance, clinical manifestations, types and diagnosis of Von Willebrand disease

54. Secondary Haemostasis The Coagulation System
Coagulation factors interact in a series of enzymatic reactions to convert the soluble protein fibrinogen to insoluble fibrin
All the reactions except conversion of fibrinogen to fibrin require a phospholipid membrane
The phospholipid membrane is provided by injured vessels and activated platelets

55. Classification of Coagulation Disorder
Inherited coagulation disorder
Hemophilia A and B( Christmas diesease)
Von-Willebrand disease
other factor deficiency V ,X,XI.,II,I
Acquired
Liver Disease
Vit k def.
Over dose of warfarin anticoagulant
DIC

56. Hemophilia
X – linked recessive disorder characterized by deficiency in Factor VIII (Hemophilia A) or Factor IX (Hemophilia B)
Overall incidence is 1 in 5000 males
80% have Factor VIII deficiency
In approximately 30%, there is no positive family history

57. Inheritance of Hemophilia AX X X Y
1st generation
Carrier
Female
Normal
Male
Hemophiliac
Male
Normal
Female

58. INHERITANCE

59. Genetics of Hemophilia A
In Hemophiliacs, the activity of Factor VIII ranges from complete deficiency to as high as 30 % of normal.
This variation is because of the fact that the Factor VIII gene mutation is of different types
Female carriers of hemophilia gene have a slightly reduced Factor VIII concentration

60. Clinical Features :
Easy bruising, massive bleeding after minor injury,
particularly to joints (hemarthrosis), hematomas, ecchymoses;
Recurrent bleeding into joints leads to deformities
Petechiae are ABSENT

61. Hemophilia – Clinical Features
Factor VIII (U/dL)
Bleeding tendency
< 2
Severe, frequent spontaneous bleeding into deep tissues and internal organs
2 - 10
Moderately severe, some spontaneous bleeds after minor trauma
>
Mild, bleeding only after significant trauma

62. Intra articular bleed

63. Haemophilia

64. Joint Bleed In Haemophilia

65. Lab Investigations(Haemophilia-A)
Screening tests
Bleeding Time --Normal
Coagulation time – Prolonged
Prothrombin time – Normal
Activated Partial Thromboplastin time – Confirmatory tests
Substitution of aPTT with Factor VIII – APTT- N
Factor VIII assays

66. Hemophilia A - Treatment
Mainstay of treatment is replacement of Factor VIII
Fresh Frozen plasma
Cryoprecipitate
Factor VIII concentrate

67. Hemophilia B (Christmas Disease)
Indistinguishable from hemophilia A
Deficiency of factor IX
In 14% of patients factor IX is present but non-functional
X-Linked recessive;
Incidence 1:100,000 males;
Clinical Features :
Easy bruising, massive bleeding after minor injury, particularly to joints (hemarthrosis), hematomas, ecchymoses;
Recurrent bleeding into joints leads to deformities
Petechiae are ABSENT

68. Hemophilia B (Christmas Disease)
Lab. Findings
BT – normal
Clotting Time--Abnormal
PT – normal
APTT- Prolonged
Confirmatory Tests
APTT – prolonged & corrected by normal serum
Factor IX is decreased

69. Von Willebrand Disease
Autosomal dominant inheritance pattern
Males and females are affected equally
Incidence - 1/10,000
Clinical features - mucocutaneous bleeding

70. VWF VWF gene : short arm of chromosome 12
VWF gene is expressed in endothelial cells and megakaryocytes
Most vWF is secreted by these cells.
Some vWF is stored
Weibel-Palade bodies in endothelial cells
Alpha granules of platelets

71. Functions Of vW Factor in Hemostasis
Carrier protein for Factor VIII (FVIII)
Protects FVIII from proteolytic degradation
Localizes FVIII to the site of vascular injury
Anchors platelets to subendothelium
Bridge between platelets

73. vWD Classification 3 major subclasses
Type I: Partial quantitative deficiency of vWF
Mild-moderate disease
70%
Type II: Qualitative deficiency of vWF
Mild to moderate disease
25%
Type III: Total or near total deficiency of vWF
Severe disease 5%
Additional subclass
Acquired vWD

74. vWD Screening Tests aPTT PT
BLEEDING TIME:- Historically, bleeding time is a test used to help diagnose vWD
Not currently recommended for making the diagnosis of vWD because it Lacks sensitivity and specificity
aPTT
Mildly prolonged
Normal PTT does not rule out vWD
Prolongation is secondary to low levels of FVIII PT
Usually within normal ranges

75. Additional Assays Multimer analysis PFA-100 closure time
Screens platelet function in whole blood
Prolonged in vWD, except Type 2N
FVIII activity assay
Ristocetin induced platelet agglutination(RIPA)

76. Clinical Manifestations
Most with the disease have few or no symptoms
Generally,Mild mucocutaneous bleeding
Severe hemorrhage only with trauma or surgery
Types II and III: Bleeding episodes may be severe and potentially life threatening

77. Disease may be more pronounced in females because of menorrhagia
Bleeding often exacerbated by the ingestion of aspirin
Severity of symptoms tends to decrease with age due to increasing amounts of vWF

78. Treatment of von Willebrand Disease
Cryoprecipitate
Source of fibrinogen, factor VIII and VWF
Only plasma fraction that consistently contains VWF multimers
DDAVP (deamino-8-arginine vasopressin)
 plasma VWF levels by stimulating secretion from endothelium
Duration of response is variable
Not generally used in type 2 disease
Dosage 0.3 µg/kg / 12 hr IV
Factor VIII concentrate (Intermediate purity)
Virally inactivated product

79. Disseminated Intravascular Coagulation
Def:-Thrombo-hemorrhagic disorder occurring as a secondary complication in a variety of conditions, characterized by formation of thrombi throughout the microcirculation;consumption of coagulation factors and clinically by a bleeding diathesis and hemolytic anaemia
Synonym – Consumptive coagulopathy

80. Disorders Associated with DIC
Massive tissue injury
Accident,multiple injuries , crush injury
Burns
Obstetric complications
Abruptio placentae
Amniotic fluid embolism
Retained dead fetus
Infections
Gram Negative septicaemia
Meningococcemia
Neoplasms
Disseminated carcinomas
Acute promyelocytic leukemia
Miscellaneous
Snake bite, giant hemangiomas, vasculitis, heat stroke etc.

81. DIC - Pathogenesis 5 Step Process
Tissue injury OR endothelial cell injury
Release of procoagulants into the blood
Excessive activation of the coagulation cascade
Widespread thrombosis and secondary fibrinolysis
Consumption of clotting factors and platelets-leading to decreased clotting factors and platelets

82. DIC - Pathology Schistocytes
Hyaline microthrombi in arterioles and venules
Platelets and fibrin
Thrombocytopenia
Prolonged PT and PTT
Decreased fibrinogen
Increased D-Dimer (fibrin deradation product)
Anemia
Schistocytes

83. DIC - Clinical Features
Stroke
Renal Failure
Extremity infarction
Deep venous thrombosis
Bleeding

84. Fibrin thrombus in the alveolar capillary

85. Glomerular Fibrin thrombi

86. Peripheral smear findings in DIC

87. Lab Diagnosis of DIC Peripheral Smear examination
Fragmented RBCs (Schistocytes)
Nucleated RBCs
Thrombocytopenia

88. Lab Diagnosis of DIC Bleeding time - Prolonged
Clotting time - Prolonged
Prothrombin time - Prolonged
Activated partial thromboplastin time - Prolonged
Fibrin degradation products - elevated

89. DIC - Treatment Block coagulation or Control bleeding?
Treatment should be individualised
Fresh frozen plasma
Anticoagulants - heparin
Platelet transfusion