PLATELET PRODUCTION, STRUCTURE AND

FUNTION

1.

History
1841
Addison described platelets as extremely
minute
granules in the blood and as dust in
the PBS
Bizzozero coined the term platelets
1890 Howell came up with the term megakaryocyte
1906 James Homer Wright suggested that blood
plates are derived from the cytoplasm of
megakaryocytes
1970s Clonal assays of megakaryocytic progenitor
cells
1980s & 1990s Characterization of several
hematopoietic growth factors that
support the cell process
Present Statistics:
Approx. 1.5 million platelet
transfusion/ annum
9 M donors
4-5 bags per transfusion

2.

3.

4.

Maintains pool of megakaryocyte progenitors

BFU- Meg

Least mature

Larger, more complex colonies that includes

stenate collections of megakaryocytes and
contains up to several hundred cells

Hundreds of daughter cells

CFU- Meg

Cell that develops into a simple colony

containing 3-50 mature megakaryocytes

Scores of daughter cells

LD- CFU- Meg

First stage of endomitosis

Little/ non proliferative capacity

Transitional/ promegakaryoblast

Polyploidy is first established

Morphology is still indistinguishable

CFU- GEMM (Granulocyte, Erythrocyte, Monocyte,
Megakaryocyte)

Most primitive in vitro colony forming cells

Identification of Progenitor Megakaryocytes:

Immunologic probes/ cytochemical stains

MEGAKARYOPOIESIS

Immunologic Probes & Flow Cytometry

1.
2.
3.

Megakaryocytopoiesis Thrombopoiesis
Thrombocytopoiesis
The process of formation of thrombocytes
Site of production: Bone Marrow
30% of platelets are in the spleen, 70% at
circulation

Endomitosis/Endoruplication
-

Mitosis that lacks telophase and cytokines

Loss of spindle fiber orientation at telophase,
cytokines are arrested
Doubling of DNA without the cell dividing
8N, 16N, 32N, 64N ploidy
Single megakaryocyte 2,000- 4,000 platelets
108 megakaryocyte 1011 platelets per day

Stages of Platelet Development

Pluripotential stem
cell

Cytochemical Staining
-

Uses monoclonal antibody that will bamd to a

single receptor

1.
2.

PF4
GWF

Produced by megakaryocytes
Platelet GP (Glycoprotein)

GPIb (CD42b)
o Receptor for vWF & thrombin

GPIIb/IIIa (CD41)
o Receptor for vWF & fibrinogen ana III a

3.

CFU- GEMM

Megakaryoblast

Terminal Megakaryocyte Differentiation

Megakaryocyte

Platelets

Progenitor Megakaryocytes
Defined by culture colony characteristics
All three resembles small lymphocytes in the bone
marrow using Wright stained smear
BFU- Meg, CFU- Meg

Diploid

Participates in normal mitosis

Platelet peroxidise- ER progenitors or

megakaryoblast
Platelet peroxidise- DTS mature platelets

Platelet Specific Probes/ Immunologic Markers

Myeloid Stem Cell

CFU- Meg

CD32
HLA-DR
Platelet glycoprotein IIIa (GP IIb, IIIa, CD41)

Morphologically identifiable stages

Maturation time take about 5 days in the bone
marrow
8-11 days in circulation
Platelets are produced directly from
megakaryocyte cytoplasm
Chromatin pattern- basis of identifying maturation
stage
1.
2.
3.

MK-I: Megakaryoblast
MK-II: Promegakaryocyte
MK-III: Megakaryocyte

%
Diameter
Nucleus
Chromatin
Pattern
N:C ratio
Endomitosi
s
Cytoplasm

Identificatio
n

Note

Megakayobla
st
20
14-18um
Round
Fine
Homogenous
3:1
Ends

Promegakaryoc
yte
25
15-40um
Indented
Condensed

Basophilic
Non granular
w/ blunt
projections
(contains
gran & DMS)

Basophilic
Granular
(granules
adjacent to
nucleus)

GP Ib
vWF
adhesion
receptor(CD4
2)
Mpl
Cant be
distinguished
from myelo/
pronormobla
st

1:2
Absent

GP IV (CD36)

Eosinophilic
Granular
(granules
will
aggregate
and bud out
plts)
Fibrinogen

Nuclear
lobularity
becomes
apparent at 4N
(light
microscopy)

2.

3.

Largest cell
in the BM
No visible
nucleoli

Endomitosis
is complete
Irregular
shape

Regulated
by TPO

Full ploidy

4.

Localized to
the
albuminal
structure of
sinusoid
lining
endothelial
cells

Least
differentiate
d

Megakaryoc
yte
55
30-50um
Multilobed
Deeply
condensed
1:4
Absent

Fibrinogen- detected by immunostaining in fully

developed megakaryocyte
Polyploid- possess multiple chromosome copies within
a cell
Emperopolesis- myelocytic & erythrocytic precursors
cells cross the megakaryocyte cytoplasm to reach
sinusoid lumen
faux phagocytosis
Mepacrine nucleic acid dye in megakaryocyte flow
cytometry used to measure ploidy levels, stains alpha
granule phosphates

5.
6.
7.
8.

Hormones & Cytokines of Megakaryopoiesis

1.

TPO/ Trombopoietin

23% homology with EPO

mRNA for TPO is found in the kidney (primary

source), liver and smooth muscle

Binds megakaryocytes and mpl

Inversely proportional to platelet &

megakaryocyte mass

IL 3

Acts with TPO

Induces early differentiation of stem cell

IL 6

Acts with TPO

Enhances endomitosis, meg maturation & plt

release
IL 11

Enhances endomitosis, meg maturation & plt

release

Synthesized as Nuemega

Stimulates plt production in patients w/ chemo

induced thrombocytopenia
Kit ligand/ Mast cell growth factor/ stem cell factor
EPO
G-CSF
Granulocyte- Macrophage

Inhibitors of Megakaryocyte Growth

1.
2.
3.
4.
5.
6.
7.

PF4 (Platelet Factor 4)

B-T6
Neutrophil activating peptide 2
IL8
FOG
GATA I
NFE2

**5-7 diminish megakaryopoiesis at progenitor

endomytosis & terminal maturation phase

THROMBOPOIESIS
-

Platelet Shedding

Steps in the Release of Platelets

1.
2.
3.

Mpl receptor for TPO

V-mpl- viral oncogene associated with murine
myeloproliferative leukemia

Induce stem cell differentiation into

megakaryocyte progenitors
Induces proliferation and maturation
Induces platelet release
circulates in the plasma
Recombinant TPO:
commercially prepared
and used to
elevate platelet count both to
donors
and patients with neoplasms

Demarcation (invagination)
Fragmentation
Formation of microtubular action proplatelets

Proplatelets
-

Psuedopodial extensions of megakaryocytes that

progressively branch and thin out
Platelets are formed at the ends of proplatelets
through microtubular action
Newly released platelets are bigger, more active
metabolically and more effective hemostatically

Dwarf Micromegakaryocytes

Circulating, resting platelets

-

Biconvex
Round up on EDTA

Sequestered platelets
-

Abnormal and rarely found in any condition except

in Myeloproliferative/ myelodysplastic syndrome
Cytoplasm is pale blue, may contain pink granules
reminiscent of mature platelet

30% (2/3)
Sequestered from the white pulp of the spleen
Immediately available in times of demand

2.

Stroke: clot in the brain (blood flow is

blockedIn the supra- aortic vessel
MI: clot in the coronaries of the heart

Excessive bleeding (hemorrhage)

Terminologies:

Thrombocytopathy: Abnormal/disease of the

platelets
Thrombasthenia: platelet function
Thrombocytopenia: platelets number
Thrombocytosis: platelet number

Reticulated/ Stress platelets

-

Appear in compensation for thrombocytopenia

Appears in compensation for thrombocytopenia
Larger than ordinary mature circulating platelets
6 um diameter
MPV= 12- 14 fL
Round up on EDTA
Cylindrical and beaded on citrated whole blood
Carry free ribosomes & RER fragments
Thiozole orange: nucleic acid dye that binds RNA of
ER
Plt dense granules: false increase of reticulated plt
count

PLATELETS/ THROMBOCYTE
-

2-4um in diameter in average

Derived from fragmentation of the megakaryocyte
cytoplasm
Light blue to purple cytoplasm
Very granular (azurophilic granules)
Vary in size and shape
Chromomere:
granular, centrally located
Hyalomere: surrounds chromomere, non granular,
clear to light blue
Anuclear
Irregularly shape
formation of blood clots
Works hand in hand with RBC (RBC gives bulk to
the clot to easily stop the bleeding)

Reference Values
1.
2.

Platelet Count (150-400 x 109/L)

MPV (8-10fL)

Function
-

Hemostasis by forming hemostatic plugs to stop

loss from injured vessels to maintain integrity of
blood vessels

Platelet Dysfunction
1.

Formation of unwanted thrombus (clot)

Obstructs the vessel lumen

Arterial thrombosis: blood flow is blocked

especially in aortic vessel myocardial
infarction or stroke

PLATELET STRUCTURE
Peripheral
Glycocalyx
Glycoprotein
Plasma membrane
Cholesterol
Fatty acids
Carbohydrates
Submembranous
area

Sol-gel
Microtubules
Microfilaments
Actin
Myosin

Phosphatidylcholine
Sphingomyelin
Phosphatidylserine
Phosphatidylinositol
Phosphatidylethanola
mine

Organelle
Alpha granules
Fibrinogen
vWF
P-selectin
FV & VIII
binding protein
thromboglobu
lin
PF4
PDGF
Delta granules
ADP
ATP
Serotonin
Ca and Mg
Lysosomes

A. PERIPHERAL ZONE
1. Glycocalyx

20- 30 nm

Platelet membrane outer surface

Adhesive

Negative surface charge

Repels other plts, ec, other blood cells

Surrounds are rich in protein

Rich in glycoprotein and proteoglycans

Support surface glycosaminoglycans,

oligosaccharides & glycolipids

Absorbs albumin, fibrinogen & transports

them to storage organelles (endocytosis)
2. Plasma membrane

Selectively permeable

Made up of:
Cholesterol
o Maintains fluidity
o Controls passage
o For stability
Fatty acids
Carbohydrates
3. Submembranous area

Provides phospholipids (where clot

formation happens)

Neutral
Phosphatidyl choline
Sphingomyelin
Anionic/ Polar
Phosphatidylserine
Phosphatidylinositol
o Supply arachidonic acid

Unsaturated FA

Converted to prostaglandin &

thromboxane during plt
activation
o Release of ionic Ca++
Phosphatidylethanolamine
o Flips to outer space on activation
o Phospolipid surface on which
coagulation enzymes assembles

B. SOL-GEL ZONE
1. Microtubules

25 um in length

Mitotic spindle fibers

Functions:

Maintains platelet shape

Composed of the protein tubulin

(structural support for the normally
discoid cells)

When microtubules disassemble (ref temp/

treated with colchicines), platelets round
up but recovers original disc shape upon
warming

Contract on activation to encourage

expression of alpha granule contents

Provides rigidity to pseudopods

2. Contractile Microfilaments

5 um in length

Assists in cell division

Principal composition:
Platelet Actin
o Contraction
o Anchors plasma membrane

Proteoglycans

glycoproteins
o Present throughout cytoplasm (2030%)
o In resting plt: actin is globular and
amorphous
o cytoplasmic Ca++ conc. actin
filamentous & contractile
Desmin and Vimetin
o Intermediate microfilament that
connects actin and tubules
maintaining platelet shape
o Controls platelet shape change,
extension of psuedopods, secretion
of granular contents

Platelet myosin
C. ORGANELLE ZONE
1. Alpha Granules

50-80 per platelet

As the plasma activates, alpha granules

fuse with SCCS (Surface Connceted
Canaliculi System)
Participate in adhesion and aggregation
Support plama coagulation
Stains medium gray in osmium-dye TEM
Gray Platelet Syndrome

Inherited absence of granule contents

Large & light gray in wright-stained

films

Alpha granules protein content:

a.
b.
c.

d.

e.
f.

g.

Fibrinogen (CD1)

Promotes coagulation (intrinsic)

Von Willebrand Factor (VWF)

Promotes platelet adhesion &

aggregation
P selectin

Can bind P-Selectin Glycoprotein

(PSGL-1)
o PSGL-1 prevents exposure of tissue
factor

Also expressed by monocytes,

neurophil, injured endothelial cells that
leads to expression of circulating
factors
Factor V (Multimerin) and VII binding
protein

Labile factors

Factor V- cofactor in fibrin clot

formation

Factor VII binding protein- promotes plt

adhesion
-thromboglobulin

Used to monitor platelet activation in

some disease
Platelet Factor 4 (PF4)

Neutralizes heparin

Regulates vascular permeability

Regulates calcium mobilization from

bone

Chemotaxis of monocytes & neutrophil

Platelet- derived growth factor (PDGF)

Involved in repair of damaged blood

vessels

Stimulates mitosis in vascular smooth

muscle cells

2. Dense core granules/ Delta granules

2-7 per platelet

bulls eye

Stains black (opaque) with osmium-dye

TEM

Released directly in the plasma

Locus of stored, non metabolic pools

Storage Pool Disorder

Diminished delta granule contents

Arises with irregular plt production in

myeloproliferative neoplasm or
myelodysplastic syndromw

Occurs in inherited disease

characterized by albinism
o Chediak-Higashi syndrome
o Wiscott-Aldrich syndrome

X-linked recessive delta granule

deficiency

Characterized by severe
eczema
Does not affect wright stain plt
morphology
Plt fail to secrete when treated with
thrombin or TRAP

Twists spongelike throughout the platelet to

store additional quantities of hemostatic
proteins
Glycocalyx is less developed
Route for endocytosis & secretion of granular
contents on activation
In direct communication with the extracellular
environment

Dense core granules protein content:

a.

b.

c.
d.

ADP (Adenosine Diphosphate)

Supports neighboring plt. Aggregation

Receptors: P2Y, P2Y12

Aggregation: plt to plt

Adhesion: plt to collagen

ATP (Adenosine Triphosphate)

Source of energy

Detected using lumiaggregometry

luciferase luminescence
Serotonin

Vasoconstrictor
Calcium & Magnesium

Supports activation & coagulation

3. Lysosomes

Stains positive with:

Arylsulfatase

-glucuronidase

Acid phosphatase

Catalase

Digest vessel wall matrix components

during in vivo organization

Digest autophagic debris

HEMOSTASIS
-

Components of Hemostasis
A.

Cellular
1. Vascular intima
2. TF-bearing cells
3. Platelets
B. Biochemical
1. Coagulation cascade
2. Fibrinolytic proteins & inhibitors
Function of Hemostasis
-

MEMBRANOUS SYSTEM
1. Dense Tubular System

Derived from RER (Rough endoplasmic

reticulum)

Positive staining for platelet peroxidise activity

for arachinodic acid metabolism within platelet

Control center for platelet activation

Ca++ sequestering pump, providing low levels

of cytoplasmic Ca++ in the resting platelets

Bears series of enzymes

Phospholipase A2

Mobilizes arachidonic acid

Cyclooxygenase

Converts arachidonic acid to PGI2

Inactivated by Aspirin permanently

Thromboxane synthetase

Supports prostaglandin synthesis

Phospholipase C

Supports production of inositol

triphosphate (IP3) & Diacylglycerol
(DAG)
2. Surface Connected Canalicular System

Alpha granules

Heme: blood; stasis: stoppage

Process by which blood is maintained fluid within
the vessel walls
The ability of the system to prevent excessive
blood loss upon injury
Balance between activation and inhibition
Cessation of bleeding
Hemostasis involves interaction of
vasoconstriction, platelet aggregation, coagulation
enzyme activation

Limit blood loss resulting from injury

Maintain intravascular blood fluidity
Promote revascularization of thrombosed vessels
after injury

Factors Affecting hemostasis:

1.
2.
3.

Thrombocytopenia
Von Willebrand disease
Other platelet disorder
a. Bernard- Soulier Syndrome

Defect in GPIb receptors

b. Glanzmanns Disease

Lacks GPIIb/IIIa receptor

c. Afibrinogenemia

Complete absence of plasma fibrinogen

Fibrinolysis
-

Final event of hemostasis

Slow digestion of fibrin clot
Plasminogen

Bound to fibrin

Activated by TPA to plasmin

Plasmin degrades fibrin clots to fibrin

degradation products (X, Y, D, E, D-dimer)

Types of Hemostasis

Trigger

Compone
nt
Response
Process

Primary
Small injuries to blood
vessels
Dequamation of dying
or damaged EC
(procoagulant subs are
exposed)
Vascular intima
Platelets
(rbc, neutro,
monocytes)
Rapid, short lived
Blood vessel contracts
to seal the wound

Platelets fill the open

space to form a plug

Secondary
Primary hemostasis
Large wounds
Trauma, surgery
(exposed tissue factor)

Platelets
Coagulation cascade

Delayed, long term

TF exposed, FVII
activated

Extrinsic pathway

Fibroblast (scar tissue)

takes part in the
healing

PRIMARY HEMOSTASIS

B. Endothelial Cell Synthesizes:

1.

Prostacyclin (PGI2)

Synthesized through eicosanoid pathway

Inhibits/ prevents activation of platelets

Prevents platelet aggregation in healthy blood

vessels

Vasodilator

2.

Nitric Oxide

Counteracts vasoconstriction & maintains

healthy arterioles

Also secreted by Smooth Muscles, Neutrophil

& Macrophage

3.

Heparan sulfate

Intimal glycosaminoglycan

Retards coagulation by activating antithrombin3

Heparin prevents propagation of thrombin that

causes coronary thrombosis, once platelet is
activated, it will recruit other platelets

Thrombin: responsible for hydrolyzing

fibrinogen

Blood Vessel Layers

1.
2.
3.

Tunica adventitia

collagen fibers
Tunica media

smooth muscles & elastic fibers

Tunica intima

endothelium that lines the lumen of all vessels

a. Endothelial cell

Innermost vascular lining

Rhomboid and contagious

Should be smooth, unbroken surface

Damaged endothelial cells secrete vWF

& P-selectin (WBC & platelet binding)
b. Lamia propia

Smooth muscle
o Vasoconstriction

Connective tissue
o Composed of collagen & fibroblast
o Collagen plays a role in binding
vWF & platelets
o Fibroblasts produce collagen

Thrombin (inactivated prothrombin)

(hydrolyzes)
stupidity
Fibrinogen

Fibrin
Monomers

Fibrin Clot
4.

TFPI (Tissue Factor Pathway Inhibitor)

Inactivated Factor VIIa

Controls TF or extrinsic coagulation pathway

5.

Thrombomodulin

Protein that activates the protein-C pathway

(digests Factor V & Factor VIIIa) hindering the
intrinsic pathway of coagulation

Inhibitor of thrombin formation

6.

TPA (Tissue Plasminogen Activator)

Activates fibrinolytic system

VASCULAR INTIMA
-

Structure of blood vessel wall is important in the

regulation of blood flow

Vascular Intima Anticoagulant Property

-

The intact intima prevents intravascular thrombosis

Inhibits platelet and coagulation activation

Promotes fibrinolysis

Negatively charged surface

A. Non hemolytic function

Tissue barrier from

Collagen- promotes plt activation &

adhesion

TF- activates coagulation system

Basement membrane collagen
Collagen of matrix
Elastin
Fibronectin
Laminin
Vitronectin
Thrombospondin

Procoagulants Properties of Vascular Intima

1.

2.

Vasoconstriction

Constricts by mechanical or chemical stimulus

Mimimizes blood flow on injured site

Collagen

Flexible, elastic structural protein

Binds and activates platelets

Upon stimulation by collagen:

3.

4.

5.

6.

Platelets change in shape from discoid to

spherical
Extend psuedopods
Undergo internal contraction resulting in
centralization of their alpha granules &
dense core granules

vWF

Secreted by EC

600,000-20,000,000 D-glycoprotein

Necessary for platelet to adher to exposed

collagen
P-selectin

Secreted by EC

Adhesion molecule that promotes platelets and

leukocyte binding
Immunoglobulin-like adhesion molecule

Promotes leukocyte binding

ICAM (Intracellular Adhesion Molecules)

PECAM (Platelet Endothelial Cell Adhesion

Molecules)
Tissue Factors

Activates the coagulation system through

Factor VII

TF also appears on monocyte surface during

inflammation

nonplatelet
surfaces
Reversible
Seals endothelial
gaps
Some secretion of
growth factors
Requires intact
platelet
membranes,
Functional plasma
vWF
(vWF isnt
necessary in
arterioles for
adhesion)

1.
-

each other

granules contents

Irreversible
Platelet plug forms

Irreversible
Occurs during
aggregation
Platelet contents
are secreted
Essential to
coagulation

Platelet contents
are secreted
Requires intact
platelet
membranes &
platelet activation
pathways,
Normal fibrinogen
concentration,
Normal secretion
of granules

ADHESION
Binding of non platelet to platelet
Platelets roll and cling to non- platelet surfaces
Internal Ca++ reaches a threshold
Reversible
Secrete growth factors (PDGF)
Involvement of vWF GPIb (receptor site)

Requirements for Adhesion

1.
2.

GPIb
vWF

Stored in the alpha granules & weible

palade bodies of EC

Result
-

Fibrinolytic Properties of Vascular Intima

1.

Secretion of TPA (Tissue Plasminogen Activator)

Binds fibrin, activates nearby fibrin

Secreted by EC

Converts of plasminogen to form plasmin

Plasmin digests thrombus & restores blood flow

2.

Secretion of PAI-1 (Plasminogen Activator

Inhibition)

TPA control protein

Inhibits the activation of fibrinolytic system

3.

Thrombin bound thrombomodulin

Activates thrombin-activatable fibrinolysis

inhibitor which increases the tendency for
thrombus formation

FORMATION: PLATELET ACTIVATION

2.
-

Adhesion
Plts roll & cling to

Aggregation
Platelets adhere to

Secretion
Platelets discharge

Larger membrane surface area for biochemical

reaction
Greater chances of contact with other platelets
Pseudopods for jigsaw puzzle effect
Appearance of active GPIIb/IIIa receptors

Receptor site for fibrinogen

Seals endothelial gaps
Endpoint

White clot (platelet + vWF)

Red clot (fibrin + RBC + platelets)

AGGREGATION
Requires extensive damage to the blood vessel
Platelets to platelet adherence
Requires active conformation of GPIIb/IIIa,
fibrinogen and psuedopod formation
Requires redistribution of P-selectin to the surface
membrane

Activation by contact w/ agonist (promoter)

ADP

TXA2 (produced by alpha granules)

Phases of Aggregation:
a.

b.

3.
-

Primary

Reversible

Platelet adhere loosely to each other

Secondary

Irreversible

Release of substance that acts as agonist

SECRETION
Platelets discharge the contents of their granules
Irreversible
Occurs during aggregation
Essential to coagulation
Alpha granules and lysosomes content flow
through SCCS
Dense core granule contents are secreted through
the plasma membrane

Role in
Hemostasis
Promote
coagulation

Substance

Source

Comments on principal function

HMWK/fitzgerald factor
Fibrinogen
Factor V
Factor VII, vWF

granules
granules
granules
granules

Promote
aggregation

ADP
Calcium
PF4
Thrombospondin

Promote
vasoconstriction

Serotonin
Thromboxane A2
precursors

Promote vascular
repair
Other systems
affected

PDGF
2 thromboglobulin
Plasminogen
2 antiplasmin
C1 esterase inhibitor

Dense
bodies
Dense
bodies
granules
granules
Dense
bodies
Membraneo
us
phospholipi
ds
granules
granules
granules
granules
granules

Contact activation of intrinsic coagulation pathway

Converted to fibrin for clot formation
Cofactor in fibrin clot formation
Assists platelet adhesion to subendothelial to provide
coagulation surface
Promotes platelet aggregation
Promotes platelet aggregation
Promotes platelet aggregation
Promotes platelet aggregation

Promotes vasoconstriction at injury site

Promotes vasoconstriction at injury site

Promotes smooth muscle growth for vessel repair

Chemotactic for fibroblasts to help in vessel repair
Precursor to plasmin which induces clot lysis
Plasmin inhibitor, inhibits clot lysis
Complement system inhibitor

DIAGNOSTIC PROCEDURES FOR PRIMARY

HEMOSTASIS

1. CAPILLARY FRAGILITY TEST(CFT)

-

Torniquet test
To evaluate fragility of capillary walls
o Weak = venous pressure = rupture
o Scurvy (vitamin C deficiency)
To identify platelet deficiency, CFT correlates with
the degree of thrombocytopenia

Procedure

Procedure

Check blood pressure

Ex. 120/90 =

120+ 90
2

= 105 mmHg

Apply sphygmomanometer (5 mins)

Count petechiae fter 15-30 mins

Interpretation of Result
1
+
2
+
3
+
4
+

0-10
1120
2150
>50

2. BLEEDING TIME
A. Conventional Method/ Modified Ivy-Duke

Original test

1912: Duke
1941: modified by Ivy
Used among 0-3 years old patients
(finger) and adult patients with no site for
simplate method

Cleanse site (70% alcohol)

Skin puncture (2mm depth)
Start timer
Without touching the wound, blot drop of
blood with filter paper every 30 seconds
**Blotting the wound directly would yield
false increase result
Record at the nearest 30 seconds
Report as more than 20 minutes if bleeding
does not stop after 20 minutes

Reference Range 2-4 mins

B. Simplate Method
Procedure

Select site at the volar area of the arm

(muscular)
Place sphygmomanometer
Simplate/ surgicutt (5 mm wide, 1 mm
depth)
Inflate to 40 mmHg
2 punctures for quality control
Depress trigger and simultaneously start
the timer
Blot with filter paper every 30 seconds

Reference Range 2-9 minutes

**Above platelet count of 100x109/L should fall

within the reference range

C. In vitro Bleeding Time Device

Dode Behring PFA 100 (Platelet function

Analyzer)

High Sheer Flow System

Platelets occlude an aperture within

membranes coated with:
o Collagen/ epinephrine

For primary screening

o Collagen/ ADP

For differentiation of
dysfunction due to aspirin

Ultrega

Platelet Aggregometry
3. PLATELET ESTIMATION IN PERIPHERAL BLOOD
SMEAR
-

Ideal specimen requires a fresh drop of capillary

blood without anticoagulant.
Platelets from skin puncture: irregular in shape,
activated
Sample collected in EDTA may also be used
Films made after 5hrs from blood collection may
exhibit plenty of artifacts
Platelets from EDTA: round
Stain with Romanowsky stain
10-30 RBCs= 1-3 platelets

0-49,000 uL
50, 000- 99,000 uL
100,000- 149,000 uL
150,000- 199,000 uL
200,000- 400,000 uL
401,000- 599,000 uL
600, 000- 800,000 uL
>800,000 uL

Marked decrease
Moderate decrease
Slight decrease
Low normal
Normal
Slight increase
Moderate increase
Marked increase

Quality Assurance
1.

2.

Even cellular distribution

o No overlapping
o Space should be approximately diameter of
RBC
Platelet clumps or other abnormal cells in the
feathery edge (platelet satellitism)
o Examine entire smear for validation of
abnormally low platelet count

Verification of Low Platelet Count (<50,000/uL)

-

Vortex for 2 mins, run specimen on machine for

10,000 rpm
Recollect specimen on blue top (citrated blood)
PBS

Decreased Platelet Count

a.
b.

c.

Platelet clumps
o Also yields to false increase WBC count
Platelet satellitism
o Occurs in EDTA blood sample
o Platelet adheres on cell membrane of
neutrophils
o Use sodium citrate to avoid this phenomenon
Giant platelets

False Elevation of Platelet Count

a.

Cytoplasmic tags/ fragments

Small particles which lack platelet organelles

Result from the tendency of cytoplasmic

fragments to separate from leukemic blast cells

Same size and shape as platelets

Resembles platelets in Wrights stain

b.

Cryoglobulins

Proteins that tend to precipitate on low

temperature

Appears as small round globules

Exhibits in patients with cryoglobulinemia

(mycoplasma pneumonia infection)

Microscopic Examination
-

To evaluate quality of smear

To ascertain approximate number of platelets in
OIO (100X)
For the detection of rouleaux formation
For the detection of fibrin clots

Platelet Estimate:

platelets10 fields x 100 x 14,000

100

**answer must be compared with the result obtained

from the instrument. Any discrepancy must be
investigated
-

Examine under 100x OIO

Count 10 OIO fields
7-25 platelets per OIO field is considered adequate

Interpretation of Platelet Estimate

4. PLATELET COUNT
Reference Range 150,000- 400,000 uL (150400x109/L)
Thrombocytosis

Thrombocytopenia

Polycythemia vera

Thrombocytopenia

Idiopathic
thrombocythemia
CML
Splenectomy

purpura
Aplastic anemia
Acute leukemia
Gauchers disease
Chemotherapy and
radiation

Significant Platelet Levels

<150,000 uL
30,000- 50,000 uL
<30,000 uL

Methods for Platelet Count

<5,000 uL

A. Direct Method (Hemocytometry)

o Use RBC (thoma) pipet
o Count 25 small squares in center square
(1mm2)

Correlation between platelet count and bleeding

time

Total
Platelet Count= cells counted

1
area (1 ) x depth
10

( )

1. Light Microscopy
o Reese-Ecker

Sodium Citrate, BCB, d.H2O

Prevents coagulation and hemolysis

Preserves RBC

Provides necessary low specific gravity

to facilitate settling of platelet

Provides fixation to reduce the

adhesiveness of the platelets

1:200
o Guy & Leake

Sodium oxalate, formalin, crystal violet,

sodium citrate
2. Phase Contrast Microscopy
(recommended)
o Becker-Cronkite

Reference method

1% Ammonium oxalate (hemolyzes

RBC)

Utilizes phase contrast microscopy

1:100 dilution

15 minutes settling time

40x objective lens (2-4um, round/oval,

light purple sheen)

Dilute blood 1:20 if fewer than 50 are

counted

Dilute blood 1:200 if greater than 500

are counted

Avoid using finger prick specimen

o Unopette

Developed in 1950

Commercially prepared diluents

1:100 dilution
B. Indirect Method
o Platelets are counted in relation to 1,000 RBCs
in the blood smear

platelet count=
o
o
o

RBC count
1, 000 RBC x platelet counted

Dameckek: BCB, Na Citrate, formalin, sucrose,

d.H2O
Fonios: 14% Magnesium sulphate
Olefs

Abnormally low
Possible bleeding with
trauma
Possible spontaneous
bleeding
Severe spontaneous
bleeding

1. Normal PC, Prolonged BT

o Quality platelet abnormality

Aspirin

GpIIb/IIIa deficiency
x dilution factor

von Willebrand syndrome

Primary vascular abnormality

2. Decreace PC, Normal BT
o Autoimmune thrombocytopenia
3. Decrease PC, Very Prolonged BT
o Quantitative + qualitative platelet dysfunction

AGGREGOMETRY
-

Assess platelet adhesion, aggregation and

secretion
Instrument designed for measuring platelet
aggregation

Importance of Platelet Function Monitoring

1. Cardiovascular Disease
o Control of compliance
o Control of individual response to anti-platelet
o Treatment evaluation of drug interactions
o Evaluation of drug interactions
2. Hematology
o Assessment of platelet disorders
3. Research Applications
o Anti-platelet drugs
o Effect of aspirin/ clopidogrel
o Monitoring transfusions
Types of Aggregometry
A. Light Transmittance Aggregometry
As a plt aggregates form, more light passes
through the PRP and the tracing begins to move
towards 100% light transmission

Light transmission in proportion to the degree of

slope change
aggregation light
500 mcL specimen (PRP)
800-1200 rpm
Platelet deficiencies reflected in diminished/absent
aggregation
40% aggregation: lower limit of normal function

B. Lumiaggregometry
Simultaneous measurement of platelet aggregation
and secretion of ATP
As ATP is released it oxidizes a firefly derived
luciferin luciferase reagent to generate
chemiluminiscence proportional to ATP
concentration
May be performed using whole blood or PRP
Thrombin
o First agonist used
o Induces full secretion
Normal secretion induced by agonists other than
thrombin produces luminescence at 50% of that
resulting from thrombin
C.
-

Impedance Aggregometry
300-500 mcL specimen (whole blood)
impedance platelet aggregation
Non optical
Diluted in 1:1 saline to avoid spontaneous in vitro
platelet activation
o An electrode probe assembly is inserted into
cuvette containing WB sample
o Electrode probe assembly consists of 2 metal
wires (reusable probe with palladium
electrodes and disposable probe with goldplated electrode) that are immersed in the
sample
o AC voltage is applied to probe circuit
o An aggregating agent is added to the cuvette
and stimulated platelets aggregate to the
platelet monolayer of the immersed electrode
o Accumulation of platelets results in an increase
in electrical resistance within the circuit
o During brief period of equilibrium, as an
alternating current is applied across the
electrodes, a monolayer of platelets forms on
the exposed portions of electrodes, resulting in
stable impedance value

Specimen
1. Whole blood

Centrifugation is not required

Reduced potential plt. activation and loss of plt

subpopulations
2. PRP (Platelet Rich Plasma)

Light transmittance aggregommetry (PAP-8E)

200, 000- 300, 000 uL

Citrated blood

Spin for 30 mins at 50g (g-force)

Materials should be plastic

Stopper is maintained (for pH)

Specimen Collection and Transport for Whole
Blood Aggregometry

1. Anticoagulant
o 105 mmol/L to 109 mmol/L (3.2%) dehydrate
form of trisodium citrate
o 25 ug/mL hidurin
o Mix 3-6x gently with end over end inversion by
hand
2. Needle Gauge
o 19 and 21
3. Mode of Transpotation
o Hand carried (avoid pneumatic tube system)
o Avoid traumatic handling
o Transport and maintain at RT (20-250C/ 68770F)
o Avoid exposure to severe cold or heat
o Remain capped to minimize changes in the pH
o Test within 3 hours
Specimen platelet concentration determines the need
for dilution. For all agonists except ADP, whole blood plt
conc. of 100- 1000x109/L should be tested undiluted
(For ADP not greater than 225 x109/L)
Agonist Used
1. Adenosine diphosphate (ADP)
o Most commonly used agonist
o Commonly used at 5mol/L to 20mol/L
o Binds P2Y1 and P2Y12
o Induced biphasic aggregation

Primary aggregation: shape change with

formation of microaggregates

Secondary aggregation: formation of final

platelet aggregates after release of
platelets
o In whole blood, small amounts of ADP released
by red blood cells during sampling may cause a
desensitizing effect
o Reagent ADP is stored at -200C, reconstituted
with NSS
o Secretion in response to 5mol/L is diminished
in:

Platelet membrane disorders

Eicosanoid synthesis pathway enzyme

deficiency

Storage pool disorders

Aspirin, NSAID or Clopidogrel therapy

2. Thrombin Receptor Activating Peptide-6
(TRAP-6)
o

For MEA analysis, TRAP-6 is used in a final

concentration of 32mol/L to assess
aggregation independent of platelet inhibition
by aspirin or clopidogrel

3. Thrombin
o
o
o
o

Most potent and physiologically one of the

most important platelet agonists
Binds to GPIb
Cleaves PAR1 & PAR 2 (Platelet Activatable
Receptors)
Cleaves GP Ib and GP V

o
o
o
o
o
o

Results in full secretion and aggregation

Detected by firefly luciferin-luciferase
luminescence assay
Reagent thrombin is stored at -200C,
reconstituted with NSS
Heparin blocks thrombin generation indirectly
by accelerating the inhibition of various
coagulation proteins by Antithrombin
Direct Thrombin Inhibitors (DTIs) such as
hirudin directly inhibit thrombin activity by
binding to the active site of enzyme
DTIs inhibit thrombin bound to fibrin whereas
the heparin/antithrombin complex can only
inhibit circulating thrombin

4. Arachidonic Acid
o Commonly used at 0.5mmol/L to 1.0mmol/L to
induce monophasic aggregometry
o Assesses viability of eicosanoid synthesis
pathway
o Aggregation is independent of membrane
integrity
o It is useful in detecting aspirin related defects
o Aspirin and similar drugs inhibit the production
of thromboxane A2, which in turn, inhibits
aggregation and ATP secretion when tested
with low concentrations (0.5mmol/L) in whole
blood
o Reagent is stored at -200C in the dark, diluted
with bovine-albumin
5. Collagen (Type 1 Fibrillar)
o Is commonly used at 1g/mL to 5g/mL
o Binds GPIa/IIa and GP VI
o A loss of collagen induced aggregation may
indicate

Membrane abnormality

Storage pool disorder

Release defect

o
o

Presence of aspirin
A lag phase of up to one minute is typically
seen with this agonist
The platelets of patients taking aspirin may
demonstrate reduced aggregation to collagen
at 1g/mL to 2g/mL, but aggregate normally
at 5g/mL
Reagent is stored at 1-60C, no dilution, cant be
frozen

6. Epinephrine
o Binds platelet to adregenic receptors
o Activate platelet same as ADP
o Cant induce aggregation in storage pool
disorder/ eicosanoid synthesis pathway defects
o Is not recommended as a standard agonist for
whole blood testing clinically, as fewer than
50% of subjects respond
o Reagent is stored at 1-60C, reconstituted with
d.water
o Modulates the interaction between plasma von
Willebrand factor (vWF) and platelet membrane
glycoprotein Ib (GPIb), the vWF receptor on
platelets
o The agonist is commonly used two
concentration ranges, high dose and low dose
PHYSIOLOGIC VARIATION IN PLATELETS
o
o
o

Birth: (84-478 x109/L)

After first week of life: normal value
Women: in menstruation
(plt, WBC, neutrophil) than men