Megakaryocyte- Platelet System: Structures and Function

Platelets

4 maturation stages

Maturation sequence of megakaryoblast takes about 5 days

Platelets are produced directly from the megakaryocyte cytoplasm

As the megakaryocyte matures, clusters of granules aggregate to form platelets

2/3 Circulation

1/3 Spleen

Thrombopoietin

Influence the maturation of platelet

Produced by the liver and kidney

Platelet:

Maturation Period: 5 days

Lifespan 8-11 days

Megakaryocyte maturation

Megakaryoblast

Promegakaryocyte

Megakaryocyte

Metamegakaryocye

1. Megakaryoblast
James Wright
Thrombopoietin
Megakaryocytic cells undergo endomitosis
Earliest recognizable stage of maturation
Blunt protrusions from its cytoplasmic membrane and contains a multitude of
polyribosomes and clear vacuoles with diameters as large as 0.2 um
Central area mitochondria and primitive endoplasmic reticulum
Nucleus has prominent nucleoli
Golgi complex occupies the area surrounding the nucleus
Cytoplasm alpha-granules and centrioles
Normally are found only in bone marrow (1 to 4 per 1000 nucleated cells)
Few may be found in the lungs
Less than 1% of the nucleated cells within the bone marrow
Identified by light microscopy using a Romanowski-stained marrow specimen
15 to 50 um diameter
Single, centrally located nucleus or multiple round and oval nuclei containing
several nucleoli and distinct but fine, delicate chromatin strands
Cytoplasm stains a diffuse blue, indicating absent of specific granules
Irregular in shape
Start of endomitosis
20-50 micrometer
Blue cytoplasm
N:C ratio is about 10:1
Fine chromatin
2. Promegakaryocyte
Increases in volume
20um to 80 um
Retains its blunt protrusions
Cytoplasm is rich in polyribosomes
Number of nuclear lobe begins to increase
Barely detectable margination of the chromatin around the nuclear membrane
Thought to be capable of protein synthesis
Demarcating membrane system forms the invagination of the plasma
membrane

Outer cell membrane and the demarcating membranes have structural similarities,
suggesting that the DMS functions as the future membrane system of the
metamegakaryocyte
Larger
Nucleus has usually undergone one or two divisions
Bluish-stained granules around the periphery of the nuclei
Distinct marginal zone with blunt cytoplasmic protrusions that stain a dark blue and
often contain small colorless globules
20-60 micrometer
Less basophilic cytoplasm
Chromatin becomes coarse
Irregularly shaped nucleus, may show light lobulation
N:C ratio is 4:1 to 7:1

3. Megakaryocyte
Maturation stage following the Promegakaryocyte
Round and expanded in volume
Multiple nuclei and even, peripheral margins
Numerous small, uniformly distributed granules with a reddish blue hue
Chromatin pattern is linear and course, with distinct spaces between the strands
Begins to contain all of the structural constituents of a megakaryocyte
Cytoplasm is devoid of specific organelles other than polyribosomes and numerous
mitochondria located in the central area of the cell
Incomplete endoplasmic reticular system
Known as the stage that does not ordinarily produce platelets
Megakaryocytes with at least four nuclei can produce platelets
3.a. Granular Megakaryocyte
3.b. Mature Megakaryocyte
40-120 micrometer
Cytoplasm contains coarse clumps of granules aggregating into little bundles, which
bud off from the periphery to become platelets
Multiple nuclei are present
No nucleoli is visible
N:C ratio is less than 1:1
With aggregate of granules which bud off to become platelets
4. Metamegakaryocyte
Fourth stage of maturation
Very large cell, many times the size of the mature granulocyte
With a decreased nuclear-cytoplasmic ratio compared with the immature stages of
development
Nucleus is multi-lobed and ploidy ranges from 4N to 16N when not stressed to
produce more platelets
o Ploidy levels can be as high as 64 N under abnormal conditions

Mature Megakaryocyte Platelet shedding

Metamegakaryocyte disintegrated cells surrounded by platelets
Platelet precursors - cytoplasmic appearance (reliable characteristic in
differentiation)
Aggregation of granular material into masses that are separated by relatively clear
areas that represent the DMS or vesicles
On electron microscope, is seen to contain predominantly polyribosomes, with
occasional mitochondria
The remainder of the cytoplasm contains the extensive DMS, which created the
future platelet fields
o Each future platelet contains mitochondria and cytoplasmic granules
Problems in identification of Maturation stage
Emphasis should be placed on the cytoplasmic appearance rather than the number of
nuclei r the chromatin structure
The reason is occasionally, a megakaryocytic cell with only one or two nuclei forms
platelets
Problems in identification of cells in the megakaryocytic series
Megakaryocytes and metamegakaryocytes are usually identified easily by their large size
in comparison with other bone marrow cells and their 4N or greater ploidy
Osteoclasts are confused with the megakaryocyte
Osteoblast, reticulum cells and abnormal multinucleated erythroblast, multinucleated
plasma cell and red cell, various tumor cells, and Reed Sternberg cells
Morphologic differentiation of megakaryocytic cell series

MATURATION
STAGE

CYTOPLAS
MIC
GRANULES

CYTOPLASMI
C TAGS

NUCLEAR FEATURES

THROMBOC
YTES
VISIBLE?

Megakaryoblas
t

Absent

Present

Single nucleus, fine

chromatin structure,
nucleoli

No

Promegakaryo
cyte

Few

Present

Double nucleus

No

Megakaryocyte

Numerous

Usually
absent

Two or more nuclei

No

metamegakary
ocyte

Aggregate
d

Absent

Two or more nuclei

Yes

Initiation of platelet formation

DMS becomes dilated, and the individual platelets become apparent
On the completion of this demarcation, and following the appropriate stimulus,
megakaryocytes extend portions of their cytoplasm through the basement membrane and
between the endothelial cells of the marrow sinusoids
1000 to 4000 platelets

Platelet shedding
To facilitate the release of platelets in the bone marrow sinus, cytoplasmic pseudopodia of
the megakaryocytes protrude through the extravascular side of the endothelium making
into the bone marrow sinus
Megakaryocytic Micro tubular system
Within the megakaryoblast and megakaryocyte, there are a series of microtubules that
converge on centrioles adjacent to the nucleus
The microtubules in the immature megakaryocyte display a well-organized dispersement,
whereas those of the mature megakaryocyte display a random dispersement

Megakaryocytic filaments
ACTIN AND MYOSIN

Megakaryocytic protein synthesis

Fibrinogen
Factor VIII:Ag
Myosin
Actin
Fibronectin platelet factor
Platelet factor 4
Glycoprotein IIb-IIIa

DIFFERENTIATING FEATURES OF CELLS CONFUSED WITH MEGAKARYOBLASTS

MEGAKARYOBLAST
Single, centrally located nucleus with one or more nucleoli; nuclear chromatin
strands distinct; marginal cytoplasmic extensions represent early platelet formation
N:C = 10:1; cytoplasm is blue and non-granular
PLASMA CELL
Single, round eccentric nucleus with nucleolus
Cytoplasm is blue, bubbly, and non-granular
Cytoplasm is more abundant than megakaryoblasts
Cytoplasm contains prominent light zone adjacent to nucleus
Generally smaller than osteoblast
TISSUE NEUTROPHIL
Nuclear para-chromatin more distinct than Megakaryoblasts
Nucleoli usually distinct
Chromatin is more coarse and cytoplasm more abundant (N:C = 1:1) than
Megakaryoblasts
Cytoplasm stains light blue
Cytoplasmic granules vary in number and usually stain reddish purple
Cell is generally bizarre in shape with blunt cytoplasmic pseudopods

OSTEOCLAST
Very large cell
Multinucleated
Nuclei are separate and not visibly connected number of nuclei may be uneven
Cytoplasm has pink background with blue granules and frayed edges
TUMOR CELLS
Nuclei have variable color and chromatin clumping
Nucleoli common
Cells are variable in size and shape
Cytoplasmic borders may be difficult to distinguish, cells tend to clump
Cytoplasm color is variable
REED STERNBERG CELL
Nuclear lobes are often mirror images
Nucleoli are more prominent than Megakaryoblasts
Cytoplasm color is variable

DIFFERENTIATING FEATURES OF CELLS CONFUSED WITH PROMEGAKARYOCYTES,

MEGAKARYOCYTES, AND METAMEGAKARYOCYTES
PROMEGAKARYOCYTE
Double centrally located nucleus
Nuclei connected by strands or superimposed
Cytoplasm is blue and scant but often has extensions that stain blue and have
rounded contours with a homogeneous or bubbly appearance
Bluish granules may appear near nucleus
Osteoblast
Single, round eccentric nucleus with nucleolus
Cytoplasm is blue, bubbly, and non-granular
Cytoplasm contains prominent light zone separated from nucleus
Generally larger than plasma cell
MULTI-NUCLEATED RBC
RBC cytoplasm polychromatophilic or pink and non-granular; nuclear chromatin
clumpy; cell is smaller than pro- and meta- megakaryocyte
TUMOR CELL
Nuclei have variable color and chromatin clumping nucleoli are common
Cells are variable in size and shape
Cytoplasmic borders may be difficult to distinguish
Cells tend to clump
Cytoplasmic color is variable
MULTINUCLEATED PLASMA CELL
Foamy cytoplasm stains dark blue
Nuclei may be double or triple but never more
OSTEOCLAST
Very large cell
Multinucleated
Nuclei separate and not visibly connected
Number of nuclei may be uneven
Cytoplasm has pink background with blue granules and frayed edges
MEGAKARYOCYTE
Very large cell with two or more nuclei
Number of nuclei is always even
Nuclei connected by strands or superimposed
Cytoplasm reddish-blue with granules
Very similar to metamegakaryocyte except that platelet budding is not yet
detectable
METAMEGAKARYOCYTE
Very large cell with multiple nuclei
Number of nuclei is always even
Abundant granular cytoplasm (granules stain pinkish-red) with evident platelet
budding
PLATELET STRUCTURE
4 um
Appear as dense blue to purple particles with granules that stain with graded intensity
during Romanowsky stain preparation
Electron microscope
Peripheral zone
Sol-gel zone
Organelle zone
Membranous System
60% protein, 30% lipids, 8% carbohydrates, various minerals, water and nucleotides

1. Peripheral zone
Glycocalyx
Plasma membrane
Submembrane area
Under hemostatic conditions, discoid-shaped
A. Glycocalyx
Relatively smooth and contains pore-like indentation that open communication channels
into the platelet cytoplasm, providing a distinct connection between the inside of the
platelet and surroundings
10-50 nanometer
Ia, Ib, Ic, Iia, IIb, III, IV and V
Platelet adhesion and aggregation
Provides a surface to which some coagulation factors may adhere, including factors I, V,
VIII, X, XI, XII, and XIII (all of these are absorbed selectively onto platelet to facilitate
assembly of the prothrombinase complex consisting of factors Va, Xa, and calcium
B. Plasma membrane
Serves as the physical and chemical battier between the intracellular and extracellular
constituents of the platelet
Sodium/potassium ATPase ionic pump maintains a transmembrane ionic gradient
Phosphatidylserin, phosphatidylcholine, and phosphatidylinositol required for fatty acid
metabolism
Factor VIII
C. Submembrane Area
Identified as a specific area because the organelles within the inner matrix of unaltered
never come in contact with the internal side of the platelet cell wall, but appear instead to
be separated from it by the submembranous area
2. Sol-gel Zone
Consists of a circumferential microtubule system and randomly arranged microfilaments
that form an intraplatelet matrix
Serves a stable gel component to regulate the arrangement of the internal organelles and
microtubular system within the resting platelet body
Influences communication of the organelles with the platelets external surroundings
-actinin and actin-binding protein transmembrane protein
Linking the outer membrane to the microfilaments in the sol-gen area
May help to explain how the inner microfilaments influence platelet contraction by
modulating the cytoskeleton of the outer layers of the platelet
2. A. microfilaments
Actin and myosin - ACTOMYOSIN or Thrombostenin
Provide the contractile force after activation that directs the organelles toward the
center of the cell with control and direction from the microtubules
Clot retraction
Projected outward, forming pseudopodia
50nm
2. B. Microtubules
Extend around the platelet perimeter
Contributes significantly to the cytoskeletal support system
Consists of tubulin maintains the shape of the platelet
When platelets are stimulated and lose their discoid form, the circumferential band of
microtubules disappears, but on the reestablishment of the discoid shape of the
platelets, such as by treatment with cooling and biochemical agents such as
prostaglandin (PG) I2, the microtubules reappear
3. Organelle Zone
Constitute the major portion of the platelet cytoplasm
Electron-dense granules
Alpha-granules
Peroxisomes
Lysosomes
Mitochondria
3.a. Dense granules
250-350 nm
Classified as dense because of their appearance by electron microscopy
Adenosine
Guanosine
Diphosphates
Calcium
Magnesium
Serotonin
ADP probably the most important component, binds to specific receptors and
initiates platelet aggregation
Short-lived effect

Rapidly degraded to adenosine which, in turn, inhibits platelet function by

enhancing cAMP levels
3.b. Alpha granules
Spherical and somewhat larger than the dense granule
300 500 nm
Coagulation factors
Permeability factors influences vessel wall permeability and tone
Cationic proteins
Platelet-derived growth factors stimulates smooth-muscle cell growth and
proliferation
Platelet factor 4
Beta-thromboglobulin BIND HEPARIN
Fibrinogen
Factor V probably not synthesized by the platelet, maybe acquired by platelet
absorption from the plasma or through pinocytosis
In thrombocytopenia, the vessel wall becomes leaky, indicating the important
contribution of platelets in keeping vessels intact
3.C. Lysosomes
Platelet vesicles that contain a number of acid hydrolases
May digest materials that the platelet endocytosis
It is possible that lysosome acid hydrolases are released at the site of the forming
thrombus or platelet plug to digest cellular debris and foreign material or that they
contribute to clot lysis

4. Platelet Membrane System

Canalicular system (surface-connecting canalicular system)
Opens to the platelets external environment is evidenced by the presence of
indented pores on the platelets surface and by cross-sections illustrating
interconnecting tubules reaching from the outer membrane to the intercytosol
components of the cell
Serve as a delivery routs for substances ingested by the platelet
System for release of granules
Dense tubular system randomly dispersed in the platelet cytosol and appear to be
close to the circumferential band of microtubules.
Influences the microtubules supporting the discoid platelet shape
Platelets pump calcium out of the cytosol through the dense tubules
System site for arachidonic acid metabolism
PLATELET LIFE SPAN AND TURNOVER RATE
The rate of platelet release from megakaryocytes is equivalent to the rate of platelet
removal from the circulation
Platelet Turnover net rate of production
35 x 109 / L 4.3 x 109 / L per day
Normal platelets injected into thrombocytopenic recipients 5 to 10 days
Normal platelets injected into normal recipients 2 to 9 days
These estimates unfortunately influenced by the method of life span calculation
Indium and chromium analysed the radiolabeled recovery curves by various statistical
approaches
Circulating platelets: young and old
30% - spleen

PLATELET FUNCTION
Adhesion
Activation
Platelet Release Reaction / Secretion
Aggregation
1. Adhesion
Platelet adherence to exposed subendothelial surface and the subendothelial
network of collagen fibrils, fibrinectin, and basement membranes
The degree of platelet response to such injury is, in part, influenced by the
extend and depth of vessel wall injury.
Stimuli that cause endothelial desquamation only expose platelets to the
basement membrane, onto which they will adhere and spread
Adhesion occurs in the presence of vWF
13- Hydroxyoctadeocadienoic acid (13-HODE) - endothelial cell-derived
chemorepellant
o Released from endothelial cells into the vascular tissues immediately
underlying the endothelium
o Inhibits platelet adhesion to that surface
Collagen types I and III synthesized by smooth muscle cells

Promote deeper regains of the vessel wall and promote platelet adhesion
and facilitate aggregation and release
o Collagen types IV and V synthesized by endothelial cells
o Promote platelet adhesion but do not cause platelet aggregation except
under specific conditions
A. von Willebrand Disease (vWF deficiency)
o Needed for normal platelet adhesion
B. Bernard-Soulier Disease
o Glycoprotein Ib deficiency (platelet receptor for vWF)
o Giant platelet syndrome
2. Activation
Morphologic and functional changes in platelets
Agonists: substances that initiate activation (E.g., Arachidonic acid)
Arachidonic acid ---cyclooxygenase--- Thromboxane A2
Aspirin: 3 days deferral, inhibits cyclooxygenase, thus prolonged BT
3. Platelet Release Reaction
Release granules
ALPHA GRANULES: Platelet factor, Platelet derived growth factor, Fibrinogen,
Factor V, von Willebrand Factor, Thrombospondin, Beta-thromboglobulin,
Fibronectin and Albumin
DENSE GRANULES: Calcium, ADP, Serotonin, ATP, Pyrophosphate, Magnesium
Serotonin: Vasoconstriction
ADP: Stimulate Platelet Aggregation
Release Disorders (Storage Pool Defects)
ALPHA GRANULE DEFICIENCIES
Gray Platelet Syndrome appear gray on Romanowsky-stained blood film
Quebec Disorder (Factor V Deficiency) deficiency of factor V-binding protein
multimerin
DENSE GRANULE DEFICIENCIES
Hermansky-Pudlak
Chdiak-Higashi Syndrome
Wiskott-Aldrich Syndrome
o

4.

Platelet Aggregation
Induced by stimuli such as ADP, thrombin, TxA2, collagen, and epinephrine
Preceded by a change in platelet shape
Characterized morphologically by pseudopod formation attributable in part to the outward
projection of the microfilaments.
Independent mediators of platelet aggregation
ADP
Thrombin
TxA2
ADP released from the dense granules in response to collagen, epinephrine, or TxA2
stimulation
Causes secondary irreversible platelet aggregation
Dependent on the presence of IIb-IIIa
GLANZMANNS THROMBASTHENIA
GP IIb-IIIa complex deficiency
Platelet Aggregation Test:
Normal Ristocetin
Abnormal Epinephrine, Collagen, ADP
Thombastenic platelets do not aggregate with ADP, thrombin, collagen, epinephrine,
or arachidonic acid as normal platelets do
When thrombin and collagen are used together, thrombastenic platelets will
undergo the release reaction

Influence of platelets in contact activation

When platelet-poor plasma is recalcified, the plasma clotting time is much longer than the
clotting time of recalcified platelet-rich plasma
The plasma clotting time of platelet-rich plasma is further shortened when ADP is added in
a concentration that itself does not facilitate platelet aggregation

This ADP effect has been termed platelet contact-product forming activity and
represents the ability of platelets to initiate intrinsic coagulation in the presence of
ADP by activating factor XII absorbed on the platelet surface
Collagen-induced activation of platelets also shortens the plasma clotting time by a
mechanism that bypasses the requirement of factor XII

Coagulation Activities of Platelets

Platelet provide a membrane surface on which several clotting factors can bind in order to
activate factors X and II
Stimulated platelets secrete a number of clotting factors such as fibrinogen, factor Va,
factor VIII (VIII:vWF), factor XI and factor XII
As a result of these activities, the prothrombinase complex is formed, leading to
fibrin clot formation
Interaction of Platelets with coagulation factors
Binding of VIII:vWF to platelets by a specific protein receptor is a prerequisite for in vitro
platelet aggregation induces by a substance called ristocetin, which is used routinely to
evaluate platelet function
Platelet factor V and plasma factor V appear to be immunologically identical. Factor V is
most likely acquired by the platelet in the circulation
Factor V is most likely acquired by the platelet in the circulation
Factor V and Factor Va bind avidly to the platelet, and this binding does not require platelet
activation
Factor X activation by platelets may permit a more rapid formation of thrombin that
conventional coagulation pathways
Interaction of platelets with vitamin K-dependent clotting factors
Although factors II, VII, IX, and X can bind to negatively charged phospholipids, their ability
to bind with high affinity to platelets in the absence of factor V has not been demonstrated
Factors IIa and Xa are the only vitamin-K dependent factors that can bind specifically to
platelets
In the plasma, the activation of factor X by factor IXa and cofactor VIIIa is dependent on
the availability of the platelet coagulant phospholipids. The phospholipid reduces the
concentration of factor X required to enable its activation by factors VIII and IX
Prothrombinase complex
Consists of factor Xa bound to its receptor in the presence of calcium on the platelet
surface
Binding of prothrombin to the prothrombinase complex is calcium dependent
The relative rate of conversion of prothrombin to thrombin by the prothrombinase
complex on the platelet surface is 300,000 times the rate achieved by factor Xa
alone
Role of Platelets in Conversion of Prothrombin to Thrombin
When platelets are activated in vitro with suboptimal concentrations of collagen or
thrombin, there is an increase in both the rate and the amount of thrombin generated
compared with using gel-filtered, unstimulated platelets as a surface for the activation of
prothrombin to thrombin by factor Xa
Platelet activation with either collagen of thrombin causes the appearance of
phosphatidylserine on the platelet surface.
Phospahtidylserine increases the interaction of factor V/Va and prothrombin with
platelets
QUANTITATIVE PLATELET DISORDERS

150 400 x 109 / L

Decreased (Thrombocytopenia)

Increased (Thrombocytosis or Thrombocythemia)

THROMBOCYTOPENIA

Common clinical disorder and results from one or any combination of the following
mechanisms:

Production defects

Accelerated loss or destruction

Splenic sequestration

A. Decreased Production

Pathophysiology

Thrombocytopenia diminishes the effectiveness of hemostasis, and bleeding may result. The
severity of bleeding is related to the degree of thrombocytopenia. Hemostatic defects are said to
be more severe when the platelet count falls rapidly rather than gradually
Clinical Presentation

Small-vessel bleeding (petechiae < 3 mm, purpura 1 cm, or ecchymoses > 3 cm)

Skin and mucous membranes of the nose and mouth and the gastrointestinal,
urinary, and respiratory tracts - common sites

Significant bleeding: <60 X 109 / L

20 to 60 X 109/ L: Minor bruising, menorrhagia, and postoperative or post-traumatic

bleeding

< 20 x 109 / L: Spontaneous bleeding into the skin and mucous membranes

Central nervous system (CNS) - most serious site

Laboratory Findings

Decreased platelet count

Template bleeding time is not increased when platelet counts are >100 X 109 / L

<100 X 109 / L, the degree to which the bleeding time is prolonged is inversely
proportional to the decrease in the platelet count

< 60 X -109 / L - clot retraction usually is abnormal

Platelet aggregation studies require platelet-rich plasma , reproducible results are possible
on PRP suspensions containing at least 50 X 109/L
Etiologies

Aplastic anemia - generalized bone marrow suppression

Acquired aplastic anemia - toxic chemical or physical agents, ionizing radiation and
chemotherapeutic agents, patients with Fanconi syndrome, viral infections

Selective suppression of the megakaryocyte - associated with chlorothiazide (a

diuretic) and other drugs

Although the mechanism by which thiazides and related compounds induce

thrombocytopenia is unclear, it is not considered to be immunologic but rather
related to drug toxicity

Thrombocytopenia

with

absent

radius

(TAR)

syndrome

inherited

disorder

of

megakaryocyte production

Episodes of thrombocytopenia in the neonate, absent or decreased and abnormal

bone-marrow megakaryocytes, congenital deformities of the arm, and early death
usually secondary to hemorrhage

Thrombopoietin - stimulates megakaryocyte maturation and platelet production

Myelophthisic process - space-occupying lesion in the bone marrow such as metastatic

tumor, fibrosis, or leukemia

Ineffective thrombopoiesis - associated with:

Ethanol abuse with and without malnutrition secondary to ethanol interruption of

megakaryocyte differentiation

Megaloblastic states - secondary to impaired DNA synthesis caused by folate or

vitamin B12 deficiency

Severe iron-deficiency anemia - iron is essential for the enzyme systems involved in
platelet protein synthesis

Paroxysmal nocturnal hemoglobinuria (PNH) - possess a membrane defect that

results in abnormal sensitivity to complement and various antibodies

Some viral infections

Certain inherited anomalies

May-Hegglin anomaly - thought to result from ineffective thrombopoiesis and increased

platelet destruction.

Wiskott-Aldrich syndrome (WAS) - defect in the production or release of platelets

Immune deficiencies, hemorrhage, eczema, and extreme susceptibility to

infections

Bernard-Soulier syndrome - thrombocytopenia and giant platelets with impaired function

B. DILUTIONAL LOSS

Extensive blood transfusion often is accompanied by thrombocytopenia, the degree of which

is directly proportional to the number of units transfused

C.1. NON-IMMUNE DESTRUCTION

May result from:

Exposure of platelets to non-endothelial surfaces, activation of the coagulation

process, or platelet consumption by endovascular injury

Artificial surfaces such as cardiovascular prosthetic devices, prosthetic vascular

grafts, and dialysis membranes

Extensive thermal injury

Drugs

IgE-sensitized mast cells

DIC

Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura

(TTP)

clinically

and

pathologically

similar

disorders

characterized

by

microangiopathic hemolytic anemia secondary to platelet aggregates or platelet fibrin

thrombi in small blood vessels
Pathophysiology

TTP - affects many organs

HUS - predominantly involves the afferent arterioles and glomeruli of the kidneys

Two mechanisms are offered for the platelet thrombus formation: intravascular aggregation,
and endothelial injury.
Clinical Presentation

HUS:

Vomiting and diarrhea

Brief febrile disease

Pale and slightly jaundiced and may present with purpura and bleeding from the
mucous membranes

Platelet thrombi in the kidneys lead to renal failure

Most frequently in children under 8 years of age but has been reported in renal
transplant recipients, postpartum women, and women taking oral contraceptives

TTP:

Ages of 10 and 40

Peak incidence in the third decade of life

Women being affected more frequently than men.

Fever, pallor, petechiae, neurologic manifestations, and renal disease are the chief
clinical

Mortality rate:

TTP 25%

HUS 5%

Laboratory Findings

Peripheral blood:

Anemia

Reticulocytosis

Polymorphonuclear leukocytosis

Thrombocytopenia

Numerous fragmented erythrocytes

Increased variation of red cell size

Intravascular Hemolysis:

Hemoglobinemia

Decreased haptoglobin levels

Markedly increased lactate dehydrogenase (LD)

Mildly elevated bilirubin levels

Hemosiderinuria

Hemoglobinuria

Urine contains:

Casts

Erythrocytes

Leukocytes

Large quantities of albumin

Serum levels of creatinine and urea nitrogen are increased because of reduced glomerular
filtration.

Fibrinogen levels, prothrombin time, and activated partial thromboplastin time usually are
normal; however, fibrin degradation products may be increased slightly. These test results
may be helpful in differentiating HUS or TTP from DIC, in which the blood film morphology
may be similar.

Current

tests

for

circulating

soluble

immune

complexes

or

platelet-associated

immunoglobulin (PAIg) are inconclusive, and yield various results. They are not readily
available in the routine clinical laboratory
Treatment

The cause of the thrombotic microangiopathy remains uncertain; therefore, treatment tends
to be based on the practical experience of the clinician. In addition, no single therapeutic
approach is likely to be effective in all patients because of the heterogeneity of
pathogenesis in these disorders.

Glucocorticoids and inhibitors of platelet function - reduce platelet aggregation

Exchange plasmapheresis, whole-blood exchange transfusion, or plasma infusion

Platelet transfusions are avoided because rapid clinical deterioration and life threatening
hemorrhaging are likely to result

The response to therapy usually is monitored by serum LD assays and platelet

counts.

An increase in the platelet count and a decrease in the serum LD level within 12 to 48
hours

C.2. Immune Platelet Destruction

Associated with increased levels of immunoglobulins (usually lgG) or complement on the

platelet surface

Antigenic determinants:

Major histocompatibility complex (HLA)

ABH(O) blood group system

Platelet-specific alloantigens such as P1A1

Immune thrombocytopenias may be caused by alloantibodies (isoimmune), autoantibodies

(autoimmune), or drugs

Immune thrombocytopenia purpura (ITP) is grouped with the autoimmune disorders,

although the etiology of its platelet-associated immunoglobulin is unknown
1. Post-transfusion (Isoimmune) Purpura

Rare complication characterized by sudden, profound, and selflimited thrombocytopenia

(less than 20 X 109/ L)

Bleeding occurs 2 to 12 days after the transfusion of products containing platelet antigens,
with the time of recovery ranging from 5 to 60 days.

Most cases have been reported in women who lacked the platelet-specific antigen P1A1 and
who had been sensitized by pregnancy or transfusions. This antigen is located on platelet
membrane glycoprotein Ill and is present in 97% of the general population. The antibody
produced shows PIA1 specificity and fixes complement in some cases, although not in others.
The mechanism responsible for the destruction of the patients P1 A1 negative platelets is
unknown. After recovery, transfusion with blood containing the antigen does not always restimulate antibody production or cause thrombocytopenia, but recurrences have been
reported years after the initial episode.

Plasmapheresis appears to be most effective in resolving bleeding complications, with

an increase in the platelet count within 48 hours

2. Neonatal Isoimmune Thrombocytopenia

Transplacental passage of maternal IgG antibodies directed against fetal platelet antigens
inherited from the father and absent on the mothers platelets. Sensitization to the platelet
antigen by previous pregnancies or transfusions appears unimportant. The disorder is
analogous to Rh hemolytic disease. The infants, born of hematologically normal women,
may have a severe generalized petechial rash or may appear normal at birth and develop
symptoms of thrombocytopenia within 2 or 3 days. Usually, all hematologic parameters are
normal except the platelet count, which may be 30 X 109/ L or less.

The treatment of choice is transfusion with maternal platelets washed with ABO
compatible plasma

In the maternal platelets, the infant receives platelets that are nonreactive with the
alloantibody, regardless of antigen specificity.

3. Patients Refractory to Platelet Transfusion

Patients refractory to platelet transfusion, such as those with aplastic anemia or acute
leukemia who have received long-term platelet support, may have developed platelet
alloimmunization

Characterized by failure to achieve adequate increments in the circulating platelet

count after transfusion

The alloantibodies produced in about two-thirds of the cases are directed

against HLA antigens

Leukocyte-poor platelet concentrates - may alleviate the problem for some recipients

For life threatening situations, two additional treatments are proposed:

Extensive platelet infusion - temporarily reduces the level of offending alloantibody

by adsorption, allowing the excess platelets to halt bleeding

Plasmapheresis - reduces the alloantibody level prior to platelet transfusion.

Primary Autoimmune Thrombocytopenia

Includes immune thrombocytopenic purpura (ITP)

In the past, ITP was the acronym for idiopathic

No longer considered strictly idiopathic but rather immune. It is too early to

rename the condition autoimmune thrombocytopenic purpura;" however, because
it is not known whether the platelet-associated immunoglobulin is an autoantibody
directed against the platelet, an immune complex bound to the platelet surface, or an
antibody directed against a non-autologous antigen adsorbed to the platelet

ACUTE ITP

Most common cause of thrombocytopenia in children

Self-limited disorder affecting boys and girls equally, most frequently those between the ages
of 2 and 6 years

This acquired disorder occurs predominantly in the winter and spring months after a viral
illness such as measles, chickenpox, rubella, or infectious mononucleosis.

The platelet count usually returns to normal spontaneously within 6 months, but
approximately 10% of the cases become chronic. \

CHRONIC ITP

Occurs primarily in adults

Women are affected more frequently than men at a ratio of 2 to 4:1.

The onset is insidious, and the disease may last for years, almost never remitting
spontaneously

Pathophysiology
Because acute ITP frequently follows a viral illness, the immune system may be responding to viral
antigen adsorbed onto the platelet surface or to immune complexes bound to the platelet. The
increased PA IgG levels are consistent with either hypothesis.

The PA IgG of chronic ITP is thought to be an antiplatelet antibody produced by splenic

cells. Studies suggest an abnormality of suppressor T cells. The antibody binds to both
autologous and homologous normal platelets, as well as to megakaryocytes but not to
platelets from patients with Glanzmanns thrombasthenia, which lack glycoproteins IIb and
IIIa. The antiplatelet antibody present in the serum of about 95% of patients with chronic
ITP is lgG, either alone or in combination with lgA or IgM. Serum from the remaining 5% of
patients only demonstrates lgM antibodies

Clinical Presentation

Symptoms of thrombocytopenia, including petechiae, ecchymoses,

and mucosal

bleeding, usually appear abruptly in patients with acute ITP.

The clinical history of chronic ITP is longer and includes easy bruising and abnormal
menstrual bleeding. If mucosal bleeding occurs over long periods, iron deficiency may
develop.

Blood blisters in the mouth, hematuria, epistaxis, hematemesis, and melena are
associated with more severe cases of chronic ITP

Laboratory Findings

The peripheral blood displays isolated thrombocytopenia, with platelet counts generally
less than 50 X 109/ L and an increased percentage of megathrombocytes, whereas bone
marrow megakaryocytes are normal or increased in number

Thrombocytopenia causes a prolonged bleeding time and abnormal clot retraction.

Megathrombocytes represent young, metabolically active platelets and may explain why some

patients with ITP have less bleeding than would be expected for the degree of
thrombocytopenia.

Antibody bound to platelets is demonstrable in more than 90% of the cases of ITP

Treatment

The purpose of therapy is to raise the platelet count to a level that will maintain
hemostasis

Acute ITP often recover spontaneously

Chronic ITP may be treated with splenectomy to remove a major site of platelet
storage and destruction and antibody synthesis or with immunosuppressants such as
corticosteroids.

The management of ITP in pregnancy requires special consideration, because the IgG
antibody from the mother is transported across the placenta to the fetus, causing neonatal
thrombocytopenia. Postnatal thrombocytopenia created by this mechanism usually is selflimited, with platelet counts returning to normal within 1 to 2 months. Intracranial
hemorrhage may occur in the thrombocytopenic infant secondary to trauma to the
head during vaginal delivery; therefore, low platelet counts on blood obtained from a fetal
scalp vein early in labor may indicate need for a caesarean section

Secondary Autoimmune Thrombocytopenia

About 5% to 10% of patients with chronic lymphocytic leukemia and a smaller

percentage

of

patients

with

other

lymphoproliferative

disorders

develop

immune

thrombocytopenia

Thrombocytopenia also is noted in 14% to 26% of patients with systemic lupus

erythematosus (SLE)

Splenomegaly is usual, the bone marrow has a larger than normal number of
megakaryocytes, and increased levels of PA IgG frequently are demonstrated

Immune thrombocytopenia appears to be a manifestation of the acquired immunodeficiency

syndrome (AIDS)

Bone marrow megakaryocytes are adequate to increased

PA IgG is increased

and patients are seropositive for antibodies to the causative retrovirus, human
immunodeficiency virus (HIV)

Unexplained thrombocytopenia in otherwise healthy homosexual men may be an early

manifestation of AIDS
Drug-Induced Immune Thrombocytopenia

The great majority of cases of immune thrombocytopenia are caused by a few drugs, notably
quinidine, quinine, gold salts, sulfonamides and their derivatives, chloroquine, and
rifampicin. The role of the immune system in some drug-associated thrombocytopenias,
such as that involving heparin, is debatable.46

The diagnosis of drug-induced immune thrombocytopenia is based primarily on the clinical

history of drug ingestion and the exclusion of other causes. The clinical history should
document thrombocytopenia while the patient is receiving the drug and normalization of
the platelet count after the drug is discontinued.

Reproducible, sensitive, and specific in vitro tests for drug-induced thrombocytopenia are not
widely available.
Pathophysiology

Drug-induced immune thrombocytopenias appear to be analogous to the drug-induced

immune hemolytic anemias

The antibody is directed against either a drug platelet complex or a drug-plasma protein
complex

that

then

binds

to

the

platelet. Drug-induced

synthesis

of

platelet

autoantibody is a third possible mechanism, similar to that of methyldopa (Aldomet)induced autoimmune hemolytic anemia, but there is no direct evidence for this. The drug
may bind to the platelet membrane, with the antibody then attaching to the drug alone, or
the drug may expose neoantigenic sites on the platelet membrane, with antibody binding to
these sites. The altered platelets are then removed from the circulation by the
reticuloendothelial system.

Drugs may also act as haptens, binding to a plasma protein and inducing antibodies that
bind to the drug, forming an immune complex. The complexes settle on the platelets
(innocent by-standers), which are then removed by the reticuloendothelial system.
Clinical Presentation

Most patients with drug-induced immune thrombocytopenia seek medical attention because
of the sudden onset of petechiae, ecchymoses, blood-tilled blisters in the mouth, and
mucosal bleeding

The severity of the purpura may be disproportionate to the degree of thrombocytopenia,

suggesting that the antibody also damages endothelium or induces a platelet function defect.

Most patients experience spontaneous recovery, with the platelet count returning to normal
limits 7 to 10 days after the drug is discontinued, although in some patients,
thrombocytopenia persists for 3 to 4 weeks.
Laboratory Findings

Similar
Therapy

Discontinue the drug suspected of triggering the immune thrombocytopenia

Corticosteroids may be administered because of their positive effects on vascular integrity.

D. SEQUESTRATION

The spleen may be responsible for thrombocytopenia either by increased phagocytosis and
destruction of damaged platelets or by increased sequestration of normal, undamaged
platelets.

30% to 45% - normal amount of platelets in the spleen

50% to 90% - of the platelets may be sequestered

The splenic pool consists of a larger proportion of megathrombocytes, which are

younger and possibly more effective in hemostasis than the remainder of the
circulating platelets

Hypersplenism may be the chief mechanism of thrombocytopenia in Gaucher disease,

sarcoidosis, and Felty syndrome

THROMBOCYTOSIS

Abnormal increase in the number of circulating platelets as a result of physiologic or

pathologic processes

A transient increase in the platelet count occurs with mobilization of the splenic platelet pool
during physiologic stress such as vigorous exercise or epinephrine infusion
Primary

Primary thrombocytosis (uncontrolled proliferation of platelets) is characteristic of the

myeloproliferative disorders

Polycythemia vera (PV)

Myelofibrosis with multiple myeloma

Essential Thrombocythemia (ET)

Chronic Granulocytic Leukemia (CGL) STEININGER, (CML for other references)

Megakaryocyte number and volume are increased in these disorders in spite of

the high circulating numbers, suggesting autonomous proliferation

Secondary (Reactive)

Broad spectrum of acute and chronic illnesses elicit an increase in platelet production

Platelet count usually is not as great as that seen in primary thrombocytosis

Hemorrhage or thrombotic episodes are less likely to occur because the

thrombocytosis usually is transient.

Iron deficiency may be associated with thrombocytosis. It is believed that iron normally is
involved in regulating thrombopoiesis by inhibiting Thrombopoietin, thereby inhibiting
platelet production. When iron deficiency develops, this control mechanism is decreased, and
thrombocytosis occurs.

An increased platelet count is a common feature of rapid blood regeneration; therefore,

thrombocytosis is observed with hemolytic anemias and acute blood loss. In addition,
increased platelet counts may be seen after cessation of cytotoxic drugs, withdrawal from
alcohol,

treatment

of

megaloblastic

anemia,

or

recovery

from

postoperative

thrombocytopenia

A modest elevation in the platelet count usually follows any major surgical procedure and is
secondary to tissue damage.

Splenectomy is followed by thrombocytosis, with platelet counts of 1000 to 2000 X 109/ L

not uncommon

An increase in the platelet count is noted 1 to 10 days after splenectomy and peaks 1 to 3
weeks later. Normally, the thrombocytosis subsides over weeks to months without ill
effects
LABORATORY EVALUATION OF PLATELETS

ESTIMATES FROM PERIPHERAL BLOOD FILMS

140 to 440 x 109 / L (Steininger); 150-400 x 109 / L (Other references)

10 to 40 red cells per platelet in normal peripheral blood

Oil immersion field containing 100 red cells should have been 3 and 10 platelets, whereas
a field containing 200 red cells should have between 5 and 20 platelets

1. MANUAL PLATELET COUNTS

2 methods: utilize a 1:100 or 1:200 dilution of blood applied to a Neubauer

hemocytometer chamber
A. Tocantins method - Rees-Ecker diluent employs a citrate-formaldehyde buffer with
brilliant cresyl blue (also used as a supravital stain for Hb H) as a platelet stain for light
microscopy

Diluent fixes and preserves red blood cells as well as platelets to prevent their
disintegration

B. Phase-contrast microscopy method - uses a phase microscope and 1% ammonium

oxalate diluting fluid which lyses red cells and allows platelets to form pseudopods
Specimen Requirements

Venous blood collected with EDTA

Reagents and Equipment

Neubauer hemocytometer and coverslip Rees-Ecker Method

Special thin, flat-bottom counting chamber Phase Method

Red blood cell diluting pipets or the unopette platelet-counting system is used

DILUENT FOR REES-ECKER METHOD:

3.8 g of sodium citrate, 0.22 mL of neutral formaldehyde (38%), and 0.05 g of brilliant
cresyl blue in approximately 50 mL of distilled water. Bring to 100 mL with distilled
water and filter

DILUENT FOR PHASE METHOD:

Filtered 1% ammonium oxalate in distilled water

A standard light microscope or phase microscope with a long working distance phase
condenser and phase objective is needed
Procedure

Depending on laboratory preference, platelets may be counted either in 1/5 of a mm2 or 1

mm2 in the center of the chamber

Both sides of the chamber should be mounted and counted and the results averaged
o

With the Rees-Ecker method, platelets appear as highly refractile, round bodies that
are approximately 1/10 the size of the surrounding red cells

With the phase-contrast method, red cells are lysed, leaving only platelets and
leukocytes, which can be distinguished easily

Calculations

Reported per L but may be reported per uL (equivalent to mm3)

Number of platelets counted is multiplied by the dilution factor (100 or 200), by a chamber
depth-correction factor (10) because the depth of the chamber is 0.1 mm, and by an area
correction factor depending on whether 1 mm2 or 1/5 mm2 was counted (1 or 5)

For example, if 1 mm2 is counted and the dilution is 1:100, the number of platelets counted is
multiplied by 1000 (100 X 10 X 1) to find the number of platelets per mm3 or ML. Multiplying
this result by 106 gives the result per liter (L)
Reference Range

140 to 440 X 109/ L (Steininger);

Comments and Sources of Error

Rigid standardization of technique and quality control is extremely important in manual

platelet counting

Results should be checked periodically against peripheral blood estimates, automated

counts, or both.

The phase microscope must be checked periodically according to manufacturer

recommendations to ensure that the phase objective and annulus condenser are aligned
correctly

Special care should be taken to keep the chambers free of dirt or debris that might be
mistaken for platelets

Ethyl alcohol (95% v/v) and a lint-free cloth or lens paper are recommended for cleaning.

Platelet clumping or discrepancies between the two chambers of 20% or more necessitates
repeat counts

When the platelet count is low, a larger volume on each side may need to be counted to
improve accuracy and precision

For very low counts (i.e., less than 50 X 109/L), a 1:20 dilution should be made in a
white cell diluting pipet and a new dilution factor used in the calculation

The coefficient of variation (discrepancies in duplicate counts on the same patient) for
this method typically is 10% for the phase-contrast method and 16% to 25% for
the Rees-Ecker method

2. AUTOMATED PLATELET COUNTS

Optical methods (detection of the degree of light scattering) or electrical methods (change
in the electrical resistance or capacitance across a circuit)

Varying the suspending diluent or the threshold settings of the circuitry allows separate
counting of blood cells of different sizes

Automatic platelet counters may sample whole blood and discriminate platelets from red cells
on the basis of their size; sample whole blood, lyse the red cells, and discriminate between
platelets and leukocytes on the basis of their size; or sample platelet-rich plasma (PRP) and
calculate platelet counts on the basis of sample dilution, volume counted, and hematocrit of
original sample
Quality Control

Once an instrument has been standardized or calibrated, suspensions of fixed human

platelets, which are available from several suppliers, may be used as day-to-day controls

Controls are usually run at the beginning of each batch of platelet counts and may be
interspersed between patient samples when batches of more than 15 samples are run

Records of quality control data must be examined periodically to detect subtle instrumental
or reagent problems

Samples from each batch should be screened for accuracy by quick estimates from the
peripheral blood film

Where the estimates and automated counts do not agree, results should be obtained by the
phase-contrast method

Histograms of platelet populations provide a further means of quality control

Comments and Sources of Error

Depending on the particular instrument, the coefficient of variation is 1% 5%

The key to reliable platelet counts by these methods is good quality control

SEVERAL SOURCES OF ERROR:

Falsely high platelet counts:

Contamination of reagents with particles or bacteria

Carry over from a prior sample with a high platelet count

Particles that should not be counted but are registered as platelets, such as
fragments of red or white blood cells, insufficiently lysed red cells, or red cells
with inclusion bodies (with methods that lyse red cells)

Falsely low counts:

Platelet agglutinins

Platelet satellitism

Exceptionally large platelets may not be counted if upper as well as lower

thresholds are used

Each of these sources of error can be eliminated with a good quality control regime,
including careful review of histograms generated by automated instruments and review
of the peripheral blood film

Reference Range

140 to 440 X 109/ L (Steininger)

PLATELET ADHESION

Platelet adhesion to a vessel wound with exposed subendothelial collagen or to glass beads in
vitro requires the presence of plasma von Willebrand factor (VIII:vWF), platelets capable of
interacting with VWF (platelets with glycoprotein Ib receptors), and platelet capacity to
aggregate

Bleeding Time preferred test

Two other tests have been in clinical use:

glass bead column

measures the number of platelets that have adhered to a standardized skin wound

1. BLEEDING TIME
Principle

Time it takes for a standard wound to stop bleeding

Key to precision is standardization of the wound

This standardization was a problem with the tests described earlier by Duke (fingertip
and earlobe) and Ivy (forearm)

Modifications of the Ivy method using a template such as described by Mielke

et al or the commercially available devices that make a standard wounds are
attempts to solve this problem

These tests require standardization of venous and capillary pressure with the
use of a blood pressure cuff inflated to 40 mm Hg for the duration of the test

Procedure for the Ivy Method

1. Inspect the volar surface of forearm carefully for a site that is free of visible vessels (e.g.,
veins or capillary beds) and hair.
Clean the site with alcohol sponges and allow it to dry thoroughly.
2. Position the sphygmomanometer about the upper arm (do not place over clothing), and
inflate it to 40 mm Hg. Maintain this pressure throughout the procedure.
3. Hold the template or commercial device firmly and position it on the skin without exerting
excessive pressure: avoid indenting skin surface.
Incisions should be made parallel to the elbow crease
Start the stopwatch as soon as blood appears in the wound. Blot the blood from the wound
at 30-second intervals with the edge of filter paper. Care must be taken not to touch the
incision. The test is complete when blood ceases to stain the filter paper. If a device with two
blades is used for duplicate testing, the times for the two incisions to stop bleeding should
agree within 1 minute and are averaged for the final result.
Reference Range

2 to 9 minutes (Steininger); 3 to 6 minutes (other references)

Comments and Sources of Error

The volar skin must be absolutely dry before proceeding

The template device must be held firmly so an adequate incision of the proper depth is made

Incision site should not be touched directly by the filter paper

The arm must not be held such that traction is put on the skin about the incision site

Some normal individuals are more susceptible to the effects of aspirin-containing

medication

Patients should be questioned regarding medications they are taking

Should not have taken any aspirin or aspirin-containing products during the week
preceding testing

Medications of the non-steroidal anti-inflammatory class, such as ibuprofen, tolmetin

sodium, and naproxen, should be discontinued 24 hours before the test

Patients with mild qualitative platelet disorders and a significant bleeding history may
manifest variability in their bleeding times and occasionally will have a normal result

To ensure consistency in test performance and results, the procedure should be reviewed
periodically with all personnel who perform it

The bleeding time is prolonged in thrombocytopenia, von Willebrand disease, acquired

and inherited platelet disorders, afibrinogenemia, severe hypofibrinogenemia, and
some vascular disorders

2. GLASS BEAD RETENTION TEST

Principle
When blood is passed through a glass bead column, normal platelets that have access to normal
vWF will adhere and aggregate to the beads such that the effluent from the column will have a
much lower platelet count than the starting sample
Reference Range
The percentage of platelets retained in the glass bead column should be approximately 70% or
greater
3. PLATELET ADHESIVENESS IN VIVO

Serial platelet counts on blood exuding from a forearm incision

Normally, the platelet counts decrease because of platelet adhesion to the wound. These
counts are compared with a venous blood platelet count as a control to calculate percent
platelet adhesiveness

Range for the percentage of platelet adhesiveness is 15% to 45%

CLOT RETRACTION

The principle of the test is that within 1 hour after whole blood is allowed to clot in a
clean glass tube at 37C, the clot will begin to shrink and retract from the walls of the tube

Retraction process is maximal at 24 hours, by which time the clot occupies about
half of the original blood volume

This process depends on normal numbers of contractile platelets, the

presence of calcium and ATP, and a normal concentration of fibrinogen.
The interaction of platelets with fibrinogen and fibrin must also be normal for
clot retraction to occur.

Clot retraction is abnormal with thrombocytopenia, low or abnormal fibrinogens,

paraproteinemias (multiple myeloma, macroglobulinemia) that interfere with fibrin
formation, and Glanzmann's thrombasthenia in which the platelet is incapable of
interacting with fibrin

PLATELET FACTOR ASSAYS

1. PLATELET FACTOR 3 AVAILABILITY
Principle

Platelets facilitate the ability of plasma to clot by providing a surface to which certain
clotting factors bind, thereby enhancing their reaction rates. This facilitative platelet capacity
in clot formation is synonymous with platelet factor 3 (PF3)

Platelet factor 3 is a functional concept rather than a discrete molecular substance, as is

usually implied by the word factor.

When platelets are incubated with kaolin and epinephrine, they are stimulated to provide
PF3 activity. Thus, the clotting time of PRP incubated with kaolin and epinephrine is
considerably shorter than that of platelet-poor plasma (PPP)

The test compares the clotting time of the patients PRP with that obtained for a group of
normal individuals.
Specimen Requirements

3.2% sodium citrate (3.2% to 3.8% is acceptable)

Reagents and Equipment

0.030 M CaCl2.

Kaolin, 1.5% suspension (1.5 g/IOO mL) in Owrens Veronal buffer, pH 7.35 (11.75 g of
sodium diethylbarbituate and 14.67 g of NaCl in 1570 mL distilled water to Which 430 mL of
0.1 N HCl is added).

Epinephrine (109 uM): a 121000 vial of epinephrine for injection (1 mg/mL) is diluted 50-fold
in Tris saline buffer pH 7.4. This solution contains 20 ug/mL of 109 uM epinephrine (Note:
this is the same stock solution of epinephrine used for platelet aggregometry)

Commercially available normal control plasma or pooled PPP (minimum 20 donors)

Procedure and Quality Control

1. Platelet-rich and platelet-poor plasma are prepared. Plasmas should be kept at room
temperature in plastic tubes.
2. Perform a platelet count on patients PRP and dilute with patient PPP to a final concentration of
50 X 109/ L
(Note: if patient has a prolonged PTT because of a factor deficiency, dilute PRP with normal
control PPP)
3. Prepare the following in duplicate:

a. 0.1 mL of control PPP + 0.1 mL of kaolin suspension + 0.01 mL of epinephrine stock.

b. 0.1 mL of patient PPP + 0.1 mL of kaolin suspension + 0.01 mL of epinephrine stock.

c. 0.1 mL of patient plasma with platelets (50 X 109/ L) + 0.1 mL of kaolin suspension + 0.01
mL of epinephrine stock.

4. Incubate these three mixtures at 37C for 10 minutes with occasional shaking

5. Add 0.1 ml. of CaC12 solution, and measure the clotting time with a stopwatch. The
endpoint is the first visible clumping of the kaolin. '

6. The clotting time of mixture c should be considerably shorter than that of mixtures a or b
and is a reflection of PF3 availability. The clotting times of mixtures at and b should be close;
these results serve as controls for a patient coagulation factor defect. If b is longer than a by
10 seconds or greater, or if the PTT of the patient is abnormal, patients PRP should be
diluted with normal control PPP (see step 2). The clotting time of each is noted.
Reference Range

37 to 51 seconds for PRP adjusted to 50 x 109 / L

Clotting times longer than that reference range are reported semi-quantitatively
Comments

Platelet factor availability is decreased in acquired or congenital defects of platelet secretion

or release

In some cases, impaired function of factors V and Xa has been demonstrated

2. ASSAYS OF PLATELET FACTOR 4 AND BETA-THROMBOGLOBULIN

Heparin-binding proteins found in platelet alpha-granules

Their levels in vivo are an indicator of the presence of ongoing platelet activation in a variety
of disease states such as myocardial infarction, venous thrombosis, diabetes,
inflammatory states, and myeloproliferative disorders
Procedure

There are several commercial RIA kits available for PF4 and beta-thromboglobulin
measurement in plasma or from alpha-granule release.
Comments

The clinical value of PF4 and beta-thromboglobulin assays is still under investigation. At
present, they are considered research procedures.

PLATELET AGGREGATION
1. PLATELET AGGREGOMETRY
Principle

Aggregating agents added to a stirred suspension of PRP induce a shape change and
aggregation of platelets. As a result, the PRP changes from a turbid suspension to one that
transmits more light as the aggregates are formed. Thus, the process of platelet aggregation
can be followed by a platelet aggregometer that measures and records a change in light
transmission.
Special Patient Requirements

Should be fasting

Fat-free meal

Should not be taking aspirin or non-steroidal anti-inflammatory drugs.

Specimen Requirements

Venous blood

9 parts blood to 1 part 3.8% sodium citrate

Platelet-rich plasma

Centrifuged at room temperature at 200 X g for 10 minutes

The supernatant PRP is withdrawn gently, taking care not to aspirate any red cells, and
transferred to another plastic tube, stoppered, and kept at room temperature

Testing must be completed within 2 hours

Platelet-poor plasma is obtained from both specimens by further centrifuging the remaining
PRP and red cells in the tubes at 2500 to 3000 X g for 30 minutes

The PPPs should be kept at room temperature until use

As a consequence of processing of the PRP, platelets sometimes exhibit a platelet shock

phenomenon and will be poorly or not responsive to ADP, collagen, and epinephrine for as
long as 30 minutes. If a poor response is obtained with one of these reagents, the test is
repeated 30 minutes later

This problem may also be avoided by either waiting 30 minutes before initiating
testing or by performing tests with these agents well into the testing session after
recording the results with other stimuli, such as ristocetin

AGGREGATING AGENTS
1. ADP (125 uM)

10 mg is diluted with 196 mL of saline and stored frozen in 1.0-mL aliquots

20 uL of 125 MM ADP added to 0.5 mL of PRP will result in a final concentration of 5

MM ADP

A 20-uL sample of a 1:2 dilution of the stock solution (125 uM ADP added to 0.5 mL of
PRP) will result in 2.5 MM ADP

Other appropriate dilutions may be made for lower concentrations of ADP.

2. Epinephrine (109 uM)

A 1:1000 vial of epinephrine for injection is diluted 30-fold in Tris-saline buffer pH 7.4
for a stock solution - stored frozen in 1.0-mL aliquots

A 50-uL sample of stock solution in 0.5 mL of PRP gives a final concentration of 10 uM

epinephrine; 25 uL in 0.5 mL of PRP yields a 5 uM final concentration of epinephrine.

3. Collagen

Acid-soluble collagen reagent is reconstituted with distilled water to yield a stock

solution of 50 ug/mL

A 50-uL sample of stock solution added to 0.5 mL of PRP gives a final concentration of
5 ug of collagen/mL.

4. Thrombin (human)

Lyophilized human thrombin is reconstituted in saline to a final concentration of 7.8

units/mL and stored frozen in 1.0-mL aliquots until use

A 20 uL aliquot of the stock solution added to 0.5 mL of PRP results in a final

concentration of0.3 units of thrombin/mL.

5. Ristocetin

Ristocetin reagent is diluted in saline to a final concentration of 20 mg/ mL. This is

Stable at 4C for 2 weeks. A 40 uL sample of stock solution added to 0.5 mL of PRP
results in a final concentration of 1.5 mg of ristocetin/ mL; 25 uL in 0.5 mL of PRP
produces a concentration of 1.0 mg/mL

6. Arachidonic acid is a clear viscous oil at room temperature and is not readily soluble in
concentrated aqueous solvents. A 50 mM stock is made by thoroughly mixing 657 uL of 70%
ethanol with the contents of a 10-mg vial of arachidonic acid. This stock is stable for 2 to 3
weeks if kept at -20C in a sealed vial under N2 atmosphere entered only with a Hamilton
micropipette. If these conditions are not available, the stock solution should be made fresh each
day. A 10-uL portion of stock solution added to 0.49 mL of PRP results in a final concentration of
1.0 mM. Lower concentrations can be made with a pre-dilution in ethanol.
Procedure
1. Perform platelet counts on patient and control PRP; adjust the platelet counts to 200 to 300 X
109/ L by diluting with their respective aliquots of PPP. Keep samples stoppered at room
temperature
2. Warm 0.5-mL aliquots of PRP in appropriate cuvettes to 37C for several minutes as needed for
testing
3. Adjust the 100% transmittance of the platelet aggregometer using patient PPP
4. Place warmed PRP samples in the aggregometer and adjust the baseline reading after at least 1
minute
5. After stirring PRP for 2 minutes with small magnetic stir bars, forcefully add the appropriate
volume of aggregating agent to ensure adequate mixing
6. Record the aggregation curve for 3 to 5 minutes or until no further change is noted
7. Repeat steps 2 through 6 for each aggregating agent with patient and control PRP

Comments

All testing should be completed within 2 hours of blood collection.

Should not come in contact with any type of glassware

Should not refrigerate specimens

While awaiting testing, platelets are viable longer if they are kept at room temperature;
however, aggregation occurs best at 37C

Most aggregation instruments rotate the stir bars at 1200 rpm

Multichannel instruments are synchronized to ensure comparability

Instruments should be checked periodically with a strobe light to determine the actual
stirring rate. Stir bar characteristics can also be a source of variability; if two bars are used,
they should be identical
Comments

Extremes of hematocrit will affect the concentration of citrate anticoagulant in the plasma. A
9-mL blood sample requires more anticoagulant if the hematocrit is low and less if the
hematocrit is high

Low levels of calcium are necessary for platelet aggregation; therefore, if a sample is overanticoagulated with citrate (which binds calcium), it will appear less responsive and
will require higher agent concentrations to produce a response

The issue of pH is complex. The pH optima of the various aggregating agents are different:

ADP and epinephrine responses are best between pH 7.7 and 8.0

Ristocetin responses are best at pH 7.3 and decline sharply above pH 7.7

Collagen has a broad optimal range, between 7.0 and 8.0; pH 7.7 therefore is a
reasonable compromise

Comments

Unfortunately, PRP has weak buffering capacity, and CO 2 will diffuse out, causing a
rise-in PRP pH

Such a rise increases the chelation of calcium by citrate, and the

responsiveness to aggregating agents will decline. Stoppering PRP tubes will
retard the CO2 loss, as will keeping the PRP under an oil layer or in a 5% CO2filled chamber. Attention to and monitoring of these pH effects will improve test
reliability

Comments

At low concentrations of ADP or epinephrine, an initial (primary) response is seen, followed by

disaggregation and a return of the reading to baseline

At optimal reagent concentrations, two aggregation waves are seen; the primary response
is followed by a brief plateau, and then a much larger secondary response occurs during
which platelets aggregate irreversibly

At higher concentrations of ADP and epinephrine, the stimulus is strong enough to obliterate
the primary response, and a single wave of aggregation is seen

ADP and epinephrine that give this important two-wave response will differ from laboratory
to laboratory
o

The concentrations suggested in the reagent section are simply guidelines

It is also important to note that approximately 30% of the normal population does not
respond to epinephrine, presumably because of a lack of surface membrane receptors
responsible for platelet sensitivity to epinephrine. Another 30% of normal subjects have
only a single wave of aggregation in response to ADP regardless of concentration.

Thrombin and ristocetin aggregation curves also may yield a two-wave response,
depending on the concentration of the stimulus

Collagen produces a single wave of aggregation after a lag

The factors responsible for this response involve the polymerization of acid-soluble
collagen to the fibrillar form in plasma, adhesion of platelets to the collagen fibril,
platelet release of ADP, and the response of other platelets to released ADP

An abnormal response can be secondary to Glanzmann's thrombasthenia or to a defect in

platelet release or storage pool ADP.

As is the case with ADP and epinephrine, using a concentration of collagen that is too high
can cover up a subtle release or storage pool defect. Only thrombasthenic platelets will not
respond to excessively high concentrations of collagen, thus to increase the sensitivity of
collagen aggregation, each laboratory should determine and use the lowest continuation of
collagen that still stimulates aggregation in a large number of normals.

The response to arachidonic acid is typically a two-wave response.

Abnormal responses are most commonly a perfect flat line and are easy to detect

A problem with the platelet cyclooxygenase enzyme, thromboxane A production, or

thrombasthenia are rare causes of an abnormal response

The most common cause of an abnormal response is aspirin. Other medications discussed in
the bleeding time section will also cause an abnormal response to arachidonic acid

Interpretation of platelet aggregation tests first involves comparison of the patient's curves
with the corresponding curves of a normal control. Minor differences in the slope (rate) of
aggregation and in the extent (plateau) of aggregation of as much as 25% are not significant.

ASSAY OF VON WILLEBRAND FACTOR

o

Used to measure VIIIR:RCo, the factor VIII-related activity required for platelet aggregation
with ristocetin. Historically, von Willebrands disease was thought to be a combined defect of
the factor VIII molecules and platelets

VON WILLEBRAND FACTOR ASSAY

Principle
Agglutination of fixed platelets in response to ristocetin depends on the presence vWF in plasma.
The rate or percentage of agglutination is proportional to the amount of VWF present. Thus, a
standard curve of the amount of VWF versus platelet agglutination can be constructed, and the
amount of VWF in an unknown can be determined by reference to this standard curve.

Specimen Requirements

3.2% sodium citrate

Reagents and Equipment

Platelet aggregometer

Ristocetin, 20 mg/ mL in saline

Phosphate-buffered saline (PBS), pH 7.3 Bovine serum albumin (BSA), 4 g/100 mL of PBS

Pooled normal PPP or commercial lyophilized control plasma assayed for VWF

Commercial formalin-fixed platelet suspension adjusted to 400 to 500 x 109/L

Plastic tubes and pipets

Procedure
1. Platelet poor plasma is prepared from the patient specimen by centrifugation at 2500 x g for 20 to
30 minutes. Normal PPP is diluted in two-fold series from 1:2 to 1:16 using BSA. Patient PPP is
diluted 1:2 and 1:4 with BSA
2. A 100% transmittance blank is prepared with PPP.
3. To aggregometer cuvette is added 0.4 mL of fixed platelet suspension and 0.1 mL of normal PPP
with stir bar.
4. The sample is placed in the aggregometer (no incubation necessary with fixed platelets), the
setting is adjusted to the baseline, and readings are recorded {or at least 1 minute.

5. Ristocetin (50 uL) is added; if necessary, the baseline is readjusted after maximum deflection.
Generally, a transient decrease in light transmission occurs after ristocetin is added. If it is not
possible to re-zero the aggregometer after adding ristocetin to the sample, one should start again at
step 3 with a fresh sample of fixed platelets and PPP. The 0% baseline is adjusted to 15% or 20% to
ensure that the ristocetin-induced trough can be seen on the recorder. When the curve appears to
have returned to original baseline, this is adjusted to read 0% transmittance.
6. The change in the percent transmittance is recorded until no further agglutination occurs.
7. Steps 3 through 6 are repeated for each normal PPP dilution to construct a standard curve.
8. Steps 3 through 6 are repeated for patient PPP samples: undiluted and diluted 1:2 and 1:4.

Calculations
A standard curve of the percent of vWF versus either the slope of agglutination or the percent
agglutination (curve plateau) is constructed. Undiluted normal PPP or lyophilized control plasma is
considered 100% VWF, the 1:2 dilution 50%, and so forth. The results from the patient samples are
converted to percentages of VWF according to where they fall Within the standard curve and are
corrected for the dilution factor. Results falling along the standard curve are averaged. If commercial
standard plasma is used for the standard curve, the patient results can be corrected relative to the
standard. For example, if the reference plasma contains 105% vWF, the standard curve is
Constructed using 100% as the maximum. The final results of the patient from the standard curve
are then multiplied by 1.05 to relate them to the reference plasma. Another acceptable practice is to
use normal pooled PPP for the standard curve and to test dilutions of reference (standard) plasma
just as the patient samples are tested. Patient results can then be corrected with regard to the
reference plasma. For example, if our 105% vWF reference plasma is 115% in our assay, the patient
results would be adjusted by a factor of105/115 = 0.91 in order to relate them to the reference
plasma.
Reference Range

45% to 140%
Comments

Once reconstituted, the lyophilized formalin-fixed platelets are not stable indefinitely. They
can give satisfactory results the next day if refrigerated, but generally, they should not be
used after 24 hours. There are protocols for in-house fixation of platelets, but the
commercial preparations are easier to use and give consistent results. Patients with severe
von Willebrands disease will have essentially no vWF. Those with variants of the disease
have typically low values, but these differ greatly and, depending on the testing
circumstances, may be normal (e.g., in pregnancy, during estrogen use, after trauma or
surgery).

Comments

Patients with Bernard-Soulier disease and those with von Willebrands disease both have PRP
that does not respond to ristocetin in the platelet aggregometry profile. In the VWF assay,
Bernard-Soulier patients have normal results, and those with von Willebrands disease will
have abnormal findings.

ADDITIONAL NOTES:
LABORATORY TESTS FOR PRIMARY HEMOSTASIS
1. Platelet Count
-

NV: 150,000-400,00/uL

THROMBOCYTOSIS PCV, Idiopathic thrombocytopenia, CML, Splenectomy

THROMBOCYTOPENIA Thrombocytopenic purpura, Aplastic anemia, Acute

leukemia, Pernicious anemia, Gauchers dse, and sometimes following
chemotherapy.
NOTE: Platelet estimate in Wedge Smear 8-20 platelet/field, platelet estimate
factor 20, 000
PLATELET ESTIMATE
0-49,000/uL
50,000 99,000/uL
100,000 149, 000/uL

RERPORTING
Marked decrease
Mod. Decrease
Slight Decrease

150,000 199,000/uL
200,000 400,000/uL
401,000 599,000/uL
600,000 800,00/uL
>800,000/uL

Low normal
Normal
Slight increase
Moderate increase
Marked increase

NORMAL PLT CT + PROLONGED BT

Qualitative Plt. Abnomality
Primary Vasculat Abnormality
von Willebrands Disease
LOW PLT CT + NORMAL BT
Autoimmune Thrombocytopenia
LOW PLT CT + VERY PROLONGED BT
Simultaneous Quantitative and Qualitative Platelet Deficiency
SIGNIFICANT PLATELET LEVELS
a. <100,000/uL abnormally low
b. 30,000 50,000 bleeding possible w Trauma
c. <30,000 Spontaneous bleeding possible
d. <5,000/uL Severe spontaneous Bleeding
DETERMINATION
A. DIRECT platelets are counted w a hemocytometer, uses RBC, platelet diluting fluid and
counting chamber
1. REES & ECKER METHOD
-

Sodium citrate buffer with BCB

Diluting fluid is same as Damasheks except for sucrose

Platelet/cumm = plt ct x 20 x 200 x 4

2. GUY & LEAKE METHOD

-

Sodium oxalate, HCHO, CV

May do simulataneous RBC ct.

Platelet/cumm = Plt ct x 10 x 200 x 1

3. BRECKER-CONKITE METHOD
-

No stain

Uses 1% ammonium oxalate

Uses automatic disposable pipette

4. Unopette

B. INDIRECT platelets are counted in relation to 1000 RBC on smear. 3-8 plt/100RBC
1. DAMASHEKS
-

BCB, NaCl, dist. H20, Formalin, and Sucrose

NV: 500,000 900,000/cumm

Uses the Wrights stain and 14% MgSO4

NV: 250,000 500,000/cumm

Uses BCB

NV: 457,000 580,000/cumm

2. FONIOS

3. OLEFS

2. PLATELET AGGREGATION