A patient has a history of recurrent gum bleeding since childhood. While platelet counts and coagulation tests are normal, an inherited platelet disorder is suspected. The doctor's diagnostic approach would include a detailed bleeding history, physical exam looking for bruises/petechiae, and first-line screening tests like a bleeding time and platelet function analyzer. Further specific tests may include light transmission aggregometry to assess platelet aggregation in response to various agonists and help identify potential defects in adhesion, activation, secretion or aggregation. Interpreting the different waveforms is important to determine abnormalities consistent with disorders like Glanzmann's thrombasthenia or storage pool disease.

1.  by
Dr.saima mansoor bugvi
Haematologist
CH & ICH lahore

2. A patient with recurrent episodes of gum
bleed since childhood is referred to you
Platelet counts and coagulation profile is
normal. suspecting an inherited disorder.
What will be your diagnostic approach?
Explain the rationale.

3. Give under lying defects of two major
inherited platelet function disorders
What is the principle of platelet function
test
How will you interpret its different wave
forms Pitfalls of the technique

4.  Detailed history of the patient
bleeding from any other site besides gum
bleeding pts with platelet dysfunction may
have history of epistaxis and if female
menorrhagia
 Excessive bleeding in response to major
nicks is suggestive of platelet dysfunction and
VWD
 Ask about fatique, tiredness the bleeders
usually suffer from anaemia due to chronic
blood loss

5. Assess the extent of haemorrhages
against the background of trauma
Past history of bleeding during hemostatic
challenge tooth extraction
surgery, trauma, child birth

6. Obtaining objective confirmation of the
subjectivei nformation conveyed in the
bleeding history is valuable.
Objective data include
• Previous hospital or physician visits for bleeding
symptoms,
• Results of previous laboratory evaluations,
• Previous transfusions of blood products for bleeding
episodes, and
• A history of anemia and/or previous treatment with
iron.

7. Drug history
esp non- prescription drugs
Herbal medicines poses particular
problems
patients do not readily share
active ingredient difficult to determine
Ginkgo biloba and ginseng can cause
platelet dysfunction
Ask about dietary supplements

8. A detailed family history draw pedigree for
at least two generations, inherited platelet
disorders are autosomal recessive in
inheritance
History of consanguineous marriage,
consanguineous marriages are more
chances of expressing autosomal
recessive disorders

9. Look for the pallor
Look for bruises and petechiae

10. First line screening tests
Second line specific test

11.  Assessing platelet number and size [MPV]
 Assessing platelet morphology – blood film
 Screening tests of platelet function Bleeding Time [BT] and PFA-
100
 Light Transmission Aggregometry e.g. classical Born
aggregometry
 Flow cytometry e.g. to quantitate the presence or absence of
platelet membrane glycoproteins
 measurement of platelet nucleotides [ADP, ATP and the ADP:ATP
ratio]
 Electron microscopy to look for the presence or absence of
platelet granules
 Flow analysis to look for platelet granules [mepacrine binding]
 Lumiaggregometry to look at platelet granule release.

12. Screening test for the presence of platelet
function defect
Patients with functional defects of platelets
can show BTs in excess of 20 minutes
With platelet counts greater than
100,000/ul the BT should be less than 08
minutes

13. A complete blood count (CBC) with
examination of the blood film can also be
helpful
Platelet morphology can help in diagnosing
disorders as bernard soulier syndrome
gray platelet syndrome

14.  Direct measurements of platelet
activation/aggregation are possible using an
aggregometer or flow cytometer.
 The aggregometer provides a graphic display
of the wave of platelet aggregation in
response to agonists such as ADP,
epinephrine or collagen and the agglutination
response to ristocetin
 Specific functional defects respond differently
to these agonists.

17. Platelets have a complex ultrastructure
comprising a multitude of molecules and
the malfunctioning of any of these may
give rise to a specific disease .

18.  Platelets participate in haemostasis by adhering to
exposed elements of the subendothelial matrix.
They then spread onto the subendothelial surface,
become activated, release the contents of their
storage organelles, and aggregate to each other.
Abnormalities in any of these stages – adhesion,
activation, secretion and aggregation
 – may give rise to congenital disorders of platelets.
Patients
 suffering from any of these diseases usually show
a bleeding diathesis with a prolonged bleeding
time and a normal platelet count.

20. 1. GLANZMANS THROMASTHENIA
2. STORAGE POOL DEFECT

21.  The platelets contain defective or low levels of glycoprotein
IIb/IIIa (GpIIb/IIIa), which is a receptor for fibrinogen.
 As a result, no fibrinogen bridging of platelets to other
platelets can occur,
 (GpIIb/IIIa), also known as αIIbβ3, which is an integrin
aggregation receptor on platelets.
 This receptor is activated when the platelet is stimulated by
ADP, epinephrine, collagen, or thrombin.
 GpIIb/IIIa is essential to blood coagulation since the activated
receptor has the ability to bind fibrinogen (as well as von
Willebrand factor, fibronectin, and vitronectin), which is
required for fibrinogen-dependent platelet-platelet interaction
(aggregation)

23. Platelet aggregation may be defined as the
interaction of activated platelets with one
another and occurs after adhesion of
platelets to the wall of the injured blood
vessel.
 A series of factors are capable of inducing
platelet aggregation and may be classified
as primary and secondary platelet -
aggregating agents.

24. Primary aggregating agents are those
factors, such as ADP, adrenaline and
thrombin, able to directly induce platelet
aggregation independently of their ability to
release intraplatelet ADP or to induce the
production of prostaglandins.

25.  Mutations within the genes that code for α
IIb β3 subunits have been described in GT
patients.

26.  Type I GT is characterized by the lack of surface -
 detectable α I Ib β3 complex and a profound defect
in platelet aggregation and clot retraction
 Platelets of patients suffering from type II GT have
detectable, but mark-edly reduced, amounts of the
αI Ib β 3 receptor on their surface, usually 10 –
20% of normal values
 a series of patients with a variant form have been
described who present near - normal levels of the
αI Ib β 3 complex, which is dysfunctional in that
platelets, when activated, can neither aggregate
nor bind fibrinogen

27.  With a Storage Pool Disease (SPD), there may not be enough
of a certain type of granule, the granule may be abnormal, or
there may not be enough of the chemicals it is supposed to
hold.
 In Delta Storage Pool Disease, the delta granules (also called
dense granules) are affected.
 In Alpha Storage Pool Disease, it is the alpha granules. Alpha
SPD is also called Gray Platelet Syndrome. This is because
the platelets of someone with Alpha SPD look gray when
viewed under a microscope.
 It is possible to have Alpha/Delta SPD in which both types of
granules are affected.
 When platelets are not able to store chemicals or secrete
them when needed, they can’t let other platelets know to come
and help form a plug. It takes longer for a clot to form. SPD
usually results in mild to moderate bleeding symptoms.

29.  Defects of secondary a ggregation
 Secondary aggregation disorders are more
frequent than
 primary aggregation disorders and the most
common in this
 category are the storage pool defi ciency
(SPD) syndromes. SPD
 syndromes may be classifi ed in a system
that takes into account
 the content of both dense and α granules

30.  The disorder is heterogeneous and the term
 SPD includes a group of disorders having as their common
 feature a diminution in secretable substances stored in
platelet
 granules.
 A storage pool disease
 Patients with δ – SPD or α δ - SPD usually have absent
ADP - and adrenaline- induced
 secondary aggregation waves, although the primary waves
are
 Present Collagen - induced aggregation is absent or
 markedly reduced, whereas ristocetin - induced
agglutination is
 normal.

31.  The light absorbance of PRP decreases as
platelets aggregate.
 The amount and the rate of fall are dependent
on platelet reactivity to the added agonist
provided that other variables, such as
temperature, platelet count and mixing speed,
are controlled.
 The absorbance changes are monitored on a
chart recorder.

32.  Light transmission aggregometry (LTA) is
regarded as the gold standard of platelet
function testing and is still the most used test
for the identification and diagnosis of platelet
function defects.
 Platelet rich plasma (PRP) is stirred within a
cuvette located between a light source and a
detector. After addition of a various panel of
agonists, such as collagen, ADP, thrombin,
ristocetin, epinephrine, and arachidonic acid,
the platelets aggregate and light transmission
increases.

33.  The platelet aggregation pattern is thought as
a primary response to an exogenous agonist,
followed by a secondary response to the
release of dense granule contents. This
biphasic response can be masked if high
concentrations of agonists are added.
Parameters measured include the rate or
slope of aggregation (%/min) and the
maximal amplitude (%) or percentage of
aggregation after a fixed period of time,
usually 6–10 min

34.  Platelet aggregation is studied by means of a
platelet aggregometer, Used Principle:
1. Photo-optical Method
2. luminescence technology (Platelet
Lumiaggregometry)
3. Electrical Impedance Method

39.  Arachidonic acid: used to assess the viability of the thromboxane
pathway.
 Thrombin: reacts with several membrane sites to induce full
aggregation and secretion of organelle contents independent of the
prostaglandin or ADP pathways.
 ADP: binds to a specific platelet membrane receptor and causes
platelet activation and release of dense granule stored ADP. Shows
biphasic aggregation.
 Epinephrine: binds to specific receptor and causes ADP secretion, but
does not cause aggregation in storage pool disorder or release defects.
 Collagen: Shows no primary wave of aggregation and depends on
intact membrane receptors, membrane phospholipase pathway
integrity and normal cyclooxygenase and thromboxane pathway
function.
 Ristocetin: requires vWF and intact surface membrane including a
functional vWF receptor site (GPIb).

41. Primary Response
Primary response is the reversible aggregation of platelets
by the aggregating agent. The appearance of a biphasic
reaction, showing both primary and secondary response,
can occur for some agonists at low concentrations
Secondary response is the result of enhancement of the
initial aggregation process caused by the release of
endogenous ADP and the formation of thromboxane A2.
the secondary response
Parameters measured include the rate or slope of
aggregation (%/min) and the maximal amplitude (%) or
percentage of aggregation after a fixed period of time,
usually 6–10 min
is irreversible.

44.  The most obvious abnormalities in this series of aggregation
traces is a lack of aggregation to all agonists except ristocetin
 There are two possible explanations:
1. Glanzmann's Thrombasthenia [GTT] in which there is a
defect in the GpIIb/IIIa receptorby lack of or reduction in platelet
aggregation to all agonists because fibrinogen cannot bind to
produce a platelet aggregate.
Platelet responses to ristocetin and vWF are normal, and platelet
secretion to collagen and thrombin remains existent, although at
a reduced level because there is no additional burst in secretion
resulting from platelet aggregation.
 2. Afibrinogenaemia.

46.  These aggregation traces are essentially the
inverse of those seen in Q1. That is aggregation to
all agonists except ristocetin. This suggests that
the problem lies with the GpIb receptor.
Remember the GpIb receptor is involved in the
binding of Von Willebrand factor and therefore this
pattern of traces would suggest either Bernard
Soulier Syndrome [BSS] or VWD [probably severe
Type 1 or Type 3.]
 The binding of platelets to VWF via the GpIb
receptor is critical for the binding of binding of
platelets to the damaged vascular endothelium.

48. The obvious abnormalities in these series
of aggregation traces are the lack of
second wave aggregation with ADP and
adrenaline [remember these are weak
agonists] but in addition the aggregation
with collagen and ristocetin is abnormal
[reduced.]
This suggests either a platelet storage pool
disorder or an abnormality of platelet
granule release.

49. Platelet
Aggregation

50.  ADP
• reversible 1o wave
• if ADP is released, then 2o wave
• abnormal with aggregation and release problems
 Epinephrin
• similar to ADP
 Collagen
• direct release so only one wave of aggregation
 Ristocetin
• antibiotic
• aggregation only with vWF and GP-Ib

51. Platelet function tests are still labor
intensive and time-consuming and require
special equipment and experts of
specialized laboratories.
A review of recent and regular medications
is also required

52.  The main disadvantage of platelet function studies
is the use of PRP instead of the whole blood under
relatively low shear conditions, and, in the absence
of red and white cells, it does not accurately
simulate primary hemostasis.
 It also requires large sample volume and is time-
consuming and there are many preanalytical and
analytical variables that affect the LTA results.
 The LTA technique is not standardized, despite the
fact that guidelines have been published.

53. There are many preanalytical and analytical
variables that affect the LTA results
Sample quality is critical
Fibrinogen levels are important
Agonists must be prepared fresh daily
Thrombocytopenia makes result
interpretation difficult
Complete patient history is essential