This document summarizes key components of hemostasis including primary and secondary hemostasis. It describes platelet adhesion, activation, aggregation and secretion. Tests for evaluating hemostasis are outlined including bleeding time, platelet function analyzer, and assays for factors, fibrinogen, D-dimer and FDP. Causes and interpretation of abnormal results are provided for tests such as PT, APTT, TT and specific assays of platelet function and coagulation factors.

1. APPROACH TO BLEEDING
DISORDERS
DR. NOOPUR PATIL
JR1 PATHOLOGY.

2. HEMOSTASIS:
• Arrests bleeding from injured site.
• Maintain blood in fluid state in normal vessels.
Components of Haemostasis : -
• Vessel wall
• Platelets
• Coagulation cascade
(F-III)
(Fibrin stabilising factor)

3. PRIMARY HEMOSTATSIS
Inactive platelets

Active platelets
Sequence : (i) Adhesion
(ii) Activation
(iii) Aggregation
(iv) Secretion

4. ADHESION : Attachment of platelets to something
besides other platelets.
Injury to endothelium

Exposes the subendothelial collagen fibers

Platelet attach to the collagen fibers

They require gpIb/IX receptor & vwf for this
attachment

Thus vwf acts as a bridge between platelets and
collagen fibers

5. Adhesion defect occurs in
- Bernard Soulier Disease
- Von Willibrand Disease
PLATELET ACTIVATION :
Once platelet attaches to collagen

A number of MORPHOLOGIC & FUNCTIONAL changes
occur

Platelet activation
It includes changes in :
1. Metabolic biochemistry
2. Shape
3. Surface receptor
4. Membrane phospholipid orientation

6. Platelet aggregation
It is attachment of platelet to one another.
2 phases:
(i) PRIMARY
(ii) SECONDARY
PRIMARY AGGREGATION
Injury

Damaged tissues and endothelial cells

Release agonists like ADP

Stimulate primary aggregation

If stimulus is weak,

Primary aggregation is reversible

7. SECONDARY AGGREGATION
Activated platelets

Release their own ADP & TXA2

Continue the stimulation process

Platelet aggregation is strong
Fibrinogen and Ca2+ are needed for aggregation to occur.
Both are plasma constituents
Fibrinogen acts as a bridge between platelets

8. SECRETION / RELEASE :
Secretion and secondary aggregation go side
by side.
Platelet cytoplasm has 3 types of granules
1. Dense bodies
2. α-granules
3. Lysosomal granules
Dense bodies :
 ADP
 ATP Mediators of
 Ca2+ platelet function
 Serotonin

9. α-granules : 2 major groups of protein
Group I  Hemostatic proteins
eg: - Fibrinogen
- VWF
Group II  Non-hemostatic proteins
eg: - PF4
- PDGF
Lysosomal granules : hydrolytic enzymes
All these leads to formation of primary hemostatic
plug.

10. Primary
haemostasis
Secondary
haemostasis
i) Platelet mediated
ii) Occurs within
seconds of injury
iii) Important in
preventing blood
loss from
capillaries, small
arterioles and
venules
iv) Forms platelet
plug
i) Coagulation factors
mediated.
ii) Takes several minutes
for completion
iii) Important role in
larger vessels.
iv) Forms a stable fibrin
plug.

11. SPECIFIC TEST FOR HEMOSTASIS
• PLATELET AGGREGATION TEST.
• COAGULATION FACTOR ASSAY TEST.
• ESTIMATION FOR FIBRINOGEN.
• TEST FOR FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS.
• TEST FOR D-DIMER.
• PLATELET GLYCOPROTEIN ANALYSIS.

14. Petechiae Ecchymoses
Haematoma Haemarthrosis

16. BLEEDING TIME(BT):
Time needed for the formation of primary haemostatic plug.
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(as it can cause a large local
hematoma)
III. Template
Range
Ivy’s method: 2 to 7 mins.
Template method: 2.5 to 9.5 mins.

17. DUKE’S METHOD:
Site  Ear lobe
Procedure  Clean the site

Dry

Puncture with lancet

Start the stop watch

After every 30 seconds collect the drop of blood at one corner of
filter paper

DO NOT TOUCH the skin with paper

When bleeding ceases stop the watch

Record the time
Normal time  2- 7 mins

19. IVY’S METHOD :
Site  Volar aspect of forearm
Procedure  Blood pressure cuff (40 mmHg)

Clean the area, dry

With a lancet 2 skin punctures are made about 5 cms apart

Start stop watch

Blot the blood from each site on separate piece of filter paper

Record the time(average of both the
time is taken)
Avoid a site with superficial veins
Disadvantages : depth of incision is
still NOT standardized
(approx 2-2.5mm)
Advantage : 2-3 pricks, thus more
standardized than Duke’s method

20. TEMPLATE METHOD :
Same as Ivy’s method except that instead of a
lancet a disposable device is used that
contains either a spring loaded blade which
descends vertically into the epidermis or a
blade which cuts the epidermis as it makes a
rotary arc

Thus the cut is standardized(6-9mm long and
1mm deep)
Causes of prolonged BT
I. THROMBOCYTOPENIA.
II. VWD.
III. PLATLET FUNCTION DISORDER(GLANZMANN
THROMBASTHENIA AND BERNARD SOULIER SYNDROME.
IV. DISORDERS

21. PLATELET FUNCTION ANALYZER(PFA)100:
In vitro system for measuring plt- vwf fuction.
 Assesses both plt adhesion & aggregation
 More 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 time
 Normal closure time: 1 to 3 mins.

24. Platelet count :
I. Manual Method
 Rees Ecker Method
 Ammonium Oxalate Method
 Microscopic Method
II. Automated Platelet count
 Semiautomated
 Fully Automated
III. Indirect Method

25. Rees Ecker Method:
SAMPLE  VENOUS/ CAPILLARY BLOOD
VENOUS BLOOD IS PREFERRED
ANTICOAGULANT  EDTA
DILUTING FLUID 
 Sodium citrate 3.8 g
 Formaldehyde (40%)0.2 ml
 Brilliant cresyl blue 0.1 ml
 Distilled water 100 ml

26. Mix the sample properly

Using a red cell pipet, draw blood to 0.5 mark and the diluting
fluid to 101 mark

Gently mix the pipet for 3-5 minutes

Discard the 1st 3 or 4 drops from the pipet

Charge the Neubauer’s chamber

Allow it to stand for 15 minutes

Count the platelets in the corner 4 large squares of Neubauer’s
chamber

Platelets are visible as small highly refractive particles
Total platelet count 
n x dilution factor x depth factor
area counted
n x 200 x 10=n x 500 plts/mm3
4

27. AMMONIUM OXALATE METHOD/ BRECKER
CRONKITE METHOD:
Here ammonium oxalate is used as a diluting fluid
Ammonium oxalate 1 gm
D/W 100 ml
stored at 40C
Advantage : It causes lysis of RBCs which can be sometimes
confused with platelets.
Normal Range : 1.5 – 4.5 lakhs/mm3
280± 130 x 109/L
Platelet count per ul=
No. of platelet counted x dilution factor(100)
Area in mm2 (1)x depth of chamber (0.1)
=no. of platelet counted x 1000

28. Microscopic Method :
Wright stained peripheral smear

Count the number of platelets seen per oil immersion field
8-25 platelets/oil immersion field is normal
The previous 2 methods should always be accompanied with
microscopic method

Thus we have a double check on platelet count
On PS we also get an idea about
- Platelet morphology  Normal size is 1-3 µm
Giant platelets > 5 µ Platelet satellism

29. 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.
 PT is used for controlling anti coagulant therapy.
Principle
Platelet poor plasml(0.1ML)+Tissue Thromboplastin reagent(0.1ml)+
after 1 min add Calcium chloride(0.1ml)
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)

30. 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

31. 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(0.1ml) after the
activation of contact Factors(0.1m) (Kaolin/Silica/Ellagic acid) and
after 10mins the addition of phospholipid(0.2ml) and CaCl2, but
without added tissue thromboplastin.
So it indicates the overall efficiency of the Intrinsic pathway.
Normal range
26 to 40 seconds.

33. 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.

36. 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 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

37. 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

40. Divided into 6 main groups:-
1) Adhesion test
2) Aggregation test
3) Assessment of granular content
4) Assessment of release reaction
5) Investigation of Prostaglandin pathways
6) Test of platelet coagulant activity
Specific Platelet function tests :

41. Adhesion test : The adhesiveness of blood
platelets is measured in vitro by their
ability to adhere to the glass surfaces.
SALZMAN METHOD/ RETENTION IN A GLASS
BEAD COLUMN:-

42. Sample: Whole blood in 1 vaccutainer
according to routine procedure
Whole blood into 2nd vaccutainer via the glass
bead collecting system.
Platelet count is performed on both the
samples.
% of platelet
adhesiveness
=
Plt count without
Glass beads
Plt count with
Glass beads
- X 100
Plt count without
Glass beads
Normal value – 26-60% of platelet adhesiveness
Decreased adhesiveness – vW disease
Bernard Soulier Disease
Increased adhesiveness - venous thrombosis
Diabetes mellitus
Following splenectomy

43. Aggregation tests :
Invitro tests done to check the ability of
platelets to aggregate with certain agonists.
Indications : When a patient has increased
BT in presence of normal platelet count.
Sample : platelet rich plasma (PRF)

44. ADP AND EPINEPHRINE INDUCE A BIPHASIC CURVE,WHEREAS COLLAGEN,ARACHIDONIC ACID
AND RISTOCETIN INDUCE A SINGLE WAVE OF AGGREGATION.

46. CLOT SOLUBILITY TEST(F XIII
QUALITATIVE ASSAY):-
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

47. 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).

48. Normal range
1.8 to 3.6 g/l
Interpretation
 Sensitive 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.

49. 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.

51. Agglutination with 1 in 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.