PARVEEN SINGH
Guided by
Mr. S.K.BOSE SIR
Mr. ISHWAR BIHANA SIR
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Blood vessels Platelets
Plasma
coagulation
factors
InhibitorsFibrinolytic
system
COMPONENTS OF
HEMOSTASIS

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Bachelor of Chinese Medicine, The
University of Hong Kong
Hemostatic response

ABNORMAL BLEEDING
This may result from:
 Vascular disorders
Thrombocytopenia
 Platelet Disorder
Defective platelet function
 Defective coagulation
 Defective Fibrinolysis
• The pattern of bleeding is relatively predictable
depending on the aetiology.
• Vascular and platelet disorders tend to be associated
with bleeding from mucous membranes and into the skin
whereas in coagulation disorders the bleeding is often
into joints or soft tissue
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• GOOD CLINICAL HISTORY/DATA
 hereditary or acquired?

Liver disease
Malignancies
Renal disease
Poor nutrition
Medical history

• HISTORY OF MEDICATIONS
intake of analgesics ,
 anticoagulant therapy???
History of blood transfusion

BLEEDING DISORDERS
Inherited bleeding
disorders
Acquired bleeding
disorders
• Hemophilia A and B
• von Willebrand’s disease
• Platelet disorders
• Fibrinolytic disorders
• Other factor deficiencies
• Liver disease
• Vitamin K deficiency/warfarin
overdose
• DIC
• Thrombocytopenia
• Renal Failure
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VASCULAR BLEEDING DISORDERS
• The vascular disorders are a heterogeneous group of
conditions characterized by easy bruising and
spontaneous bleeding from the small vessels.
• The underlying abnormality is either in the vessels
themselves or in the perivascular connective tissues.
• Most cases of bleeding caused by vascular defects alone
are not severe.
• IN THESE CONDITIONS THE STANDARD SCREENING TESTS
ARE NORMAL.
• THE BLEEDING TIME IS USUALLY NORMAL AND THE
OTHER TESTS OF HAEMOSTASIS ARE ALSO NORMAL.
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INVESTIGATIONS OF THROMBOCYTOPENIA
• Platelets have proved more difficult to count than
either red or white cells. Historically techniques for
platelet enumeration fall into four groups:
• Direct methods by hemocytometry
• Indirect methods in which red cells were counted
directly and their proportion to platelets determined
in a stained blood film;
• Semi-automated methods in which counts were
performed electronically on platelet -rich plasma;
• Fully automated methods
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Assessment of the platelet count is essential as
numerical deficiency or defect in their function may
lead to bleeding. The normal platelet count at all
ages is 150–400 × 10 /L of whole blood.
The four main recent analytical procedures for
platelet counting are
Manual counting using phase contrast microscopy.
 Impedance analysis,
 Optical light scatter/fluorescence analysis using
various commercially available analyzers,
 And immunoplatelet counting by flowcytometry
(anti CD41 and anti CD61)
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MANUAL COUNTING USING PHASE
CONTRAST MICROSCOPY
In 1953 the manual phase contrast microscopy
method was developed, which enabled platelets to
be easily discriminated from lysed red cells within a
counting chamber or hemocytometer .
It may still be necessary to use manual counting
methods in the routine laboratory if the platelet
count is low or if there are atypical platelets
present in the sample.
This method became the International Council for
Standardization in Haematology (ICSH) reference
method in 1988
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THE FUNCTIONS OF PLATELETS
In homeostasis include;
Maintenance of vascular integrity
Initial arrest of bleeding by platelet plug
formation
Stabilization of haemostatic plug by contributing
to fibrin formation
Platelet must be adequate in number and function
to participate optimally in homeostasis.
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SCREENING TESTS
WORK UP FOR
PLATELET FUNCTION TEST
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Bleeding Time
Finger Prick Smear
PBF (shape and size examination)
Clot Retraction Test
CONFIRMATORY TESTS
Platelet Adhesion Test
Platelet Aggregation Test
PF-3 Availability Test

CONFIRMATORY PFTs
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PLATELET ADHESION TEST
• Platelet adhesiveness is determined in vitro by
measuring the retention of platelets by a column of
glass beads.
• It is a complex phenomenon depending not only on
the adhesion property of platelets but also the ADP
released from RBCs, the conc. of plasma fibrinogen ,
the viscosity of the blood , the rate of flow through the
column , the number & size of the glass beads and the
type of plastic used to construct to the column.

Platelet Aggregation Tests
Principle:-
• Platelet functions are determined either in whole blood or
platelet rich plasma.
• It is proportional to changes either in turbidity or impedance.
• The lesser the turbidity or the greater the impedance, better
the platelets in responding to various aggregating agents.
• The aggregation rxns can be measured and plotted using one
of several instruments that measure and record the optical
density.
Method:-
Platelet Rich Plasma
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1.Base line
2.Addition of agonist
3. Shape change
4.Primary wave
5.Secondary wave

THE ‘CLOTTING SCREEN’
Basic tests of coagulation are often performed with no
specific diagnosis in mind and in the absence of any
clinical indication of a haemostatic disorder.
• OUR CURRENT PRACTICE IS TO PERFORM
• PT,
• APTT,
• FIBRINOGEN ASSAY
• Platelet Count
• D-Dimer
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PROTHROMBIN TIME
Principle;
• The PT test measures the clotting time of
recalcified plasma in the presence of an optimal
concentration of tissue extract (thromboplastin)
and indicates the overall efficiency of the
extrinsic clotting system.
• It is sensitive to changes in factors V, VII, and X,
and less so to factor II (prothrombin).
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Activated partial thromboplastin time
Principle
• The test measures the clotting time of plasma after the activation of contact
factors and the addition of phospholipid and CaCl2, but without added tissue
thromboplastin, and so indicates the overall efficiency of the intrinsic pathway.
• To standardize the activation of contact factors, the plasma is first preincubated
for a set period with a contact activator such as kaolin, silica or ellagic acid.
• During this phase of the test, factor XIIa is produced, which cleaves factor XI to
factor XIa, but coagulation does not proceed beyond this in the absence of
calcium.
• After recalcification, factor XIa activates factor IX and coagulation follows. A
standardized phospholipid is provided to allow the test to be performed on PPP.
• The test depends not only on the contact factors and on factors VIII and IX but
also on the reactions with factors X, V, prothrombin.
• It is also sensitive to the presence of circulating anticoagulants (inhibitors) and
heparin
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THROMBIN CLOTTING TIME
PRINCIPLE
• The thrombin time reflects the reaction between thrombin
and fibrinogen.
Thrombin
Fibrinogen Fibrin
• It is prolonged when the fibrinogen level is very low (less
than 1.0 g/l); in the presence of heparin and heparin-like
substances; in the presence of other inhibitors, such as
fibrin degradation products (FDPs); and when fibrinogen is
qualitatively abnormal (dysfibrinogenemia), including both
congenital and acquired.
NORMAL RANGE
• A patient’s TT should be within the control (i.e. 15–19 s).
Times of 20 s and longer are definitely abnormal. 25

MEASUREMENT OF FIBRINOGEN
CONCENTRATION
• Numerous methods of determining
fibrinogen concentration have been devised,
including clotting, immunological, physical
and nephelometric techniques, and all tend
to give slightly different results, because of
the heterogeneous nature of plasma
fibrinogen.
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CLAUSS ASSAY
PRINCIPLE
• Dilutions of standard normal plasma with known
fibrinogen content are prepared in glyoxaline buffer.
• The clotting time is measured after the addition of
thrombin, and a graph is constructed.
• The clotting time is proportional to the concentration of
fibrinogen, and the 1/10 dilution is taken to represent the
value in the standard preparation.
• The test plasma is diluted 1/10, and the result read from
the standard line.
• Normal Range: 2.0 – 4.0 g/l
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SECOND-LINE INVESTIGATIONS
If all the first-line investigations are normal in a patient who continues
to bleed from the site of an injury or after surgery (or has a history of
such bleeding), there are several possible diagnoses:
 von Willebrand disease (VWD) in which the factor VIII is not
sufficiently low to cause prolongation of the APTT. This is quite
common in mild cases
 A mild coagulation disorder that is below the sensitivity of the
routine tests to detect or that has been masked by the administration
of blood products or by high levels of other factors: for example, mild
factor VIII deficiency of 30 iu/dl
 Factor XIII deficiency
 Bleeding from a severely damaged vessel or vessels with normal
haemostasis
 A disorder of fibrinolysis such as antiplasmin or PAI-1 deficiency
 Administration of LMWH. 28

INVESTIGATION OF A BLEEDING
DISORDER RESULTING FROM A
COAGULATION FACTOR DEFICIENCY
• When the screening tests indicate that an individual
has a coagulation defect, the plasma concentration of
the coagulation factors should be assayed.
• Such assays establish the diagnosis of the deficiency
or defect and they assess its severity; they also can be
used to monitor replacement therapy and to detect
the carrier state in families in which one or more
members are affected by a congenital bleeding
disorder.
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Correction Tests Using the PT
or APTT
• Unexplained prolongation of the PT or APTT can be
investigated with simple correction tests by mixing the
patient’s plasma with normal plasma.
• Correction indicates a possible factor deficiency,
whereas failure to correct suggests the presence of an
inhibitor, but interpretation should initially be cautious
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Assays Based on PT & APTT
• Essentially, the assay consists of comparing the ability of dilutions
of a standard or reference plasma and test plasma to correct the
prothrombin time & APTT of a plasma known to be totally
deficient in the clotting factor being measured.
• In a factor VIII assay, the plasma is deficient in factor VIII.
• Clotting factors INTRINSIC, EXTRINSIC & COMMON PATHWAY may
be assayed in a similar way.
• Plot results on semi log graph paper, with percent factor activity
on the x axis and seconds on the y axis. Draw a best-fit line.
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Urea clot solubility test
PRINCIPLE
 Fibrin clots formed in the presence of factor XIII and thrombin
are stable (as a result of crosslinking) for at least 1 h in 5 mol/l
urea, whereas clots formed in the absence of factor XIII dissolve
rapidly.
 0.2 ml patient plasma + 0.2 ml thrombin.
 Incubate at 37°C for 20 min and add 3 ml urea solution (5 molar)
for 24 hour.
 In absence of factor XIII, the clot is dissolved.
Test for factor XIII

VON WILLEBRAND FACTOR
 VWF is produced in endothelial cells and
megakaryocytes. It has two roles It promotes
platelet adhesion to subendothelium. it is the carrier
molecule for factor VIII, protecting it from premature
destruction. Th e latter property explains the
reduced factor VIII levels found in VWD.
 To diagnosis of von willebrand
disesae(VWD),probably the most common
congenital bleeding disorders, requires a number of
special test at the lab level. among them vwf-ag
determination is essential and must be performed
on every pateint to reach proper diagnosis.
depending upon the lab findings. 34

VW -types
• Type I
– most frequent, quantitave defect (decreased VWf)
• Type II
– qualitative defect (abnormal VWf )
• Type III
– severe, rare, (absence of VWf )

Laboratory findings
 The PFA - 100 test .This has largely replaced the bleeding
time test
Factor VIII levels are often low. If low, a factor VIII/VWF
binding assay is performed.
 The APTT may be prolonged.
 VWF levels are usually low.
 There is defective platelet aggregation by patient plasma
in the presence of ristocetin (VWF: Rco). Aggregation to
other agents (adenosine diphosphate (ADP), thrombin or
adrenaline) is usually normal.
Collagen - binding function (VWF: CB) is usually reduced.
 The platelet count is normal except for type 2B disease
(where it is low).
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In the PFA - 100 test
• Citrated blood is aspirated through a
capillary tube onto a membrane coated
with collagen/ADP or collagen/adrenaline.
• Blood flow is maintained. Platelets begin to
adhere and aggregate, primarily via VWF
interactions with GPIb and GPIIb/IIIa,
resulting in occlusion of the aperture.
• Full platelet function tests and VWF
screening may be required to exclude
abnormal platelet function, even if the PFA
- 100 test is normal.
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Treatment Approaches to
the Bleeding Patient
• Red blood cells
• Platelet transfusions
• Fresh frozen plasma
• Cryoprecipitate
• Amicar
• DDAVP
• Recombinant Human factor VIIa

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