Diagnostic Procedure for

Thrombocyte Disorders

Angelic Trina I. Escoto, RMT

 Outline

• Platelets
• Stages of development
• Platelet Structure
• Platelet Functions
• Thrombocyte Disorders
• Diagnostic Procedures
Introduction
• Thrombocytes
• 1000-2000 plts/megakaryocyte
• 5 days maturation, 9 days life span (8-10 days)
• 2-4um,150 to 450 x 109/L (T)
• Anuclear,discoid shape, light blue cytoplasm,evenly dispersed fine red purple
granules, irregular sphere with spiny pseudopods
• Thrombopoietin
• stimulates production and maturation
• Cytokines:erythropoietin,IL-3, GM-CSF
• ↑ megakaryocyte size
• ↑ maturational stage
•
Stages of development
Platelet structure
Platelet structure
• Electron microscope examination
• Glycocalyx: fluffy coat of cell membrane
• with plasma proteins and CHO molecules, glycoprotein
receptors
• Mediates membrane contact reactions

• Cytoplasmic membrane
• surface connecting system
• sponge-like portion
• transfer of granule products & phagocytosis
Platelet structure
• Microfilaments and microtubules
• Cytoskeleton
• maintains circulating discoid shape of platelets
• positioning of organelles
• makes up contractile system (sol gel zone)
• Granules
• Alpha Granules
• PF4,beta-thromboglobulin, platelet-derived growth factor, Platelet
fibrinogen,fibronectin,vWF, thrombospondin
• Dense/Delta Granules
• serotonin,ADP,ATP, calcium
• Lysosomes
• Other Cytoplasmic constituents
• Contractile proteins
• actomyosin (thrombastenin), filamin, myosin
• Glycogen
• Enzymes
• glycolytic and hexose pathway
• metabolic rate (10x )
Figure 23.6 Platelet with its inventory of granular content. ADP, adenosine
diphosphate; ATP, adenosine triphosphate; FV, factor V; GPIb, platelet surface
glycoprotein Ib; PDGF, platelet-derived growth factor; VEGF, vascular endothelial
growth factor; PF4, platelet factor 4; TGF-β, transforming growth factor-β; vWf, von
Willebrand factor.
Platelet Functions

• Platelet adhesion
• Platelet aggregation
• Platelet Secretion
Overall Functions

• <10% of plts for normal vascular integrity

• Series of events during blood vessel injury
• adhesion to the injured vessel, shape change, aggregation, and secretion.
• Platelet activation : series of biochemical reactions
• Agonists
• nucleotide, lipids , a structural protein , and a proteolytic enzyme
Platelet Functions

Adhesion
• Spread of pseudopods along the surface
• within 1 to 2 mins after a break in the endothelium
• release of ADP
• glycoprotein receptors(gp IIb/IIIA )
• platelet cohesion: adhesive molecules
• 50 000/plt
Platelet Functions
Aggregation
• Gold standard test
• Bridges formed
• Fibrinogen+calcium+thrombin= platelet aggregation
• Fibrin= thrombus
• release of thromboxane A2, serotonin, epinephrine
• Prostaglandin E (PG E), adenosine, and nonsteroidal anti-
inflammatory agents (e.g., aspirin)
Thrombocyte Disorders
Quantitative platelet disorder
Quantitative Platelet Disorders
• Thrombocytopenia
• increased number of petechia,hemorrhages, prolonged
bleeding and impaired CRT
• low number/decreased amount of platelets in the circulating
blood
• decreased platelet production
• dilutional loss
• increased platelet destruction
• Autoimmune activity
• Increased platelet aggregation (DIC)
• Mechanical Destruction
• Septicemia
Quantitative Platelet Disorders
• Decreased Platelet Production
• defective bone marrow
• Hereditary thrombocytopenia
• Vit.B12/ Folate deficiency (megaloblastic anemia)
• Di guiglielmo's syndrome (erythroleukemia)
• Proximal nocturnal hemoglobinuria (PNH)

• decreased number of megakaryocyte

• congenital disorders (fanconi's anemia)
• acquired disorders (radiation,alcohol)
• marrow replacement by malignant cells (leukemia,lymphoma)
Quantitative Platelet Disorders
• Distribution and Dilutional Loss
• Splenic Pooling (splenomegaly)
• Hypothermia (vascular shunting)
• Transfusion

• Increased Platelet Destruction

• Combined consumption
• Toxicity due to snake venoms
• tissue injury
• obstetric complications
• neoplasms
• microbial infections
• intravascular hemolysis
Quantitative Platelet Disorders
• Increased Platelet disorders
• Isolated Consumption of Platelets
• Thrombotic Thrombocytopenic Purpura(TTP)
• Hemolytic Uremic Syndrome
• Vasculitis (SLE)
• Disseminated intravascular Coagulation (DIC)
• Immune destruction of platelets
• Idiopathic thrombocytopenic Purpura (ITP)
• Posttranfusion purpura
• Isoimmune neonatal purpura
• Drug induced antibody formation
• thrombocytopenia associated with HIV (T-HIV)
TTP
• Plt aggregate deposition in Renal & Cerebral vessels
(vascular wall dysfunction)

• Clinical Diagnosis
• associated with microangiopathic hemolytic
anemia,thrombocytopenia,neurologic
symptoms,fever, renal disease
• Lab.Diagnosis
• PT=normal, APTT=normal,fibrinogen level= nmal,
FDP= normal (occ.positive)
TTP
• Pathophysiology
• vascular endothelial cell damage
• increase in platelet-aggregating factor
• platelet aggregating factor inhibitor
• degradation/decrease in prostacyclin (PGI2)
• degradation/absence of plasminogen activator
ITP
• autoimmune disorder
• increase MPV,decreased platelet count, increased
BM plt production,increased marrow
megakaryocyte, BT=normal, plt-associated IgG
• Acute ITP
• Chronic ITP
HIT
• Risk from thrombosis w/o heparin therapy is greater
than the risk of bleeding from HIT.
• Direct platelet-aggregating effect,immune destruction
by antiplatelet antibodies
• Lab.diagnosis
• Platelet aggregometer :Normal
• increased aggregation with addition of Heparin
T-HIV
• severe but rarely hemorrhagic
Similarities w/ ITP Differences ITP

Abundant megakaryocytes greater levels of bound antibody

Occ.Giant platelets involvement of immune complexes

Immune origin

No splenomegaly
Quantitative Platelet Disorders
• Thrombocytosis
• characterized by increase in circulating platelet counts
• >450 000/mm3
• Essential Thrombocytosis
• Secondary thrombocytosis
Quantitative Platelet Disorders
• Essential Thrombocytosis
• Primary bone marrow disorder
• clonal proliferation
• associated with
• Hodgkin's lymphoma
• Polycythemia vera
• Myelofibrosis
• Chronic Myelogenous Leukemia
• Thrombocythemia
Quantitative Platelet Disorders
• Secondary Thrombocytosis
• Iron deficiency anemia
• Chronic Inflammatory Disease
• Splenectomy-associated thrombocytosis
• Rebound thrombocytosis
Qualitative Platelet
disorder
Qualitative Platelet Abnormalities
• Surface membrane defects
• Glanzmann thrombasthenia
• Bernard Soulier Syndrome
• Abnormalities in the granular fraction of the platelet
• Defects in dense granules
• Hermansky Pudlak Syndrome (HPS)
• Chediak Higashi syndrome (CHS)
• Wisktt Aldrich syndrome (WAS)
• Alpha granule deficiencies
• Storage Pool Disease (Grey Platelet Syndrome)
Qualitative Platelet Disorder
• Deficiencies of thromboxane generation
• Genetic Deficiency
• Thromboxane Receptor problems

• Acquired disorders of platelet function

• After Cardiopulmonary bypass
• DIC
• Lupus like anticoagulant
• Heparin Induced Thrombocytopenia
• Hemolytic Uremic Syndrome
• Drug Induced Platelet Abnormalities
Surface Membrane Defects
• Glanzmann thrombasthenia
• homozygous autosomal recessive disorder,one of two genes
(GPIIb/GPIIIa) ,found in chromosome 17 ,is affected
• symptoms
• Bruise,nose bleeds,bleeding of gums,heavy menorrhagia
• Lab.diagnosis
• Plt.count and Morphology=normal
• Bleeding time=prolonged
• Plt aggregometer=no aggregation (vWF only)
Glanzmannz Thrombasthenia
•Clinical Variants
• Type I
• severe deficiency of <5% of the glycoprotein complex
• Type II
• mild to moderate deficiency, 5% to 20% remains
• Type III
• qualitative dysfunction

•Prognosis and Treatment

• lifelong disorder
• BM transplant
• Platelet transfusions
Surface Membrane Defects
• Bernard Soulier Syndrome
• (Giant platelet syndrome)
• inherited quanti/qualitative defect of GPIb/IX complex
• GP Ib alpha-short arm of Chromosome 17
• GP IB beta-long arm of chromosome 22
• GP IX&GP V- long arm of chromosome 3
• Clinical symptoms
• epistaxis,gingival/cutaneous bleeding,hemorrhage
• Laboratory Diagnosis
• Ivy BT-prolonged, giant plts,plt count very low-low
normal,reduced aggregation with ristocetin
BSS
• Clinical Variants
• Type A
• Classic BSS, GP Ib beta gene mutation
• Type B
• localized to the GPIb alpha gene on chromosome 17,22,or
GP IX gene on chrosome 3
• Type C
• GP V gene lesions
• Acquired BSS
• pxs with myelodysplasia,AML (M6)
BSS
•Prognosis and Treatment
• Supportive management
• Iron Deficiency Anemia
• Splenectomy
• Blood & plateletsTransfusion
• Gene Therapy
 Abnormalities in the granular fraction of the
platelet
• Defects in dense granules
• Hermansky-Pudlak syndrome (HPS)
• decreased numbers of plt dense granules
• HPS1 gene on chromosome 10q23
• production&control of melanosomes,plt dense bodies,
lysosomes function
• Symptoms
• albinism,prolonged BT, ceroid pigment accumulation in
lysosomal organelles, bruising,nose bleeds,prolonged
bleeding(wounds,menstruation,dental procedures)
• Lab diagnosis
• electron microscope observation of platelets to find absence of dense
granules
Defects in dense granules
• Chediak-Higashi syndrome (CHS)
• autosomal recessive genetic disorder with recurrent
infections in combination with ocular,neurological and
skin manifestations

• Pathogenesis
• decreased pigmentation of hair and eyes
• photophobia,Nystagmus,abnormal susceptibility to
infection,neutrophils defect
(chemotaxis,degranulation,bactericidal activity)
• motor,sensory, and cranial nerve defects
CHS
• Lab.diagnosis
• CBC-neutropenia,plts-normal,large
eosinophils,peroxidase-positive inclusion bodies
(myeloblasts &promyelocytes)
• BT-prolonged,Coagulation screening-normal,PBS-WBC
w/ darkly stained giant granulation,decreased plt
aggregation

• Prognosis and treatment

• Vit. C-improve phagocytic function,
• Accelerated phase: corticosteroids,vincristine,cyclophosphamide
• BM transplantation
Defects in dense granules
• Wiskott-Aldrich syndrome (WAS)
• rare X-linked immunodeficiency disorder
• deletion of WAS protein gene(WASp)
• immunodeficiency; low B & T cell, IgM deficiency
• Characteristics
• eczema,recurrent infections,predisposition for secondary
leukemia/lymphoma
• Lab Diagnosis
• thrombocytopenia,Low MPV,weak antibody & CD43
response,low CD8 count
• Prognosis and treatment
• antibiotics,immune globulin,splenectomy,BM transplant
frrom compatible sibling
Alpha-granules deficiencies
• Grey Platelet Syndrome (GPS)
• congenital, autosomal dominant
• Characteristics
• mild epistaxis,easy bruising,long lasting
hemorrhages
• Lab.diagnosis
• decrease or absence of platelet alpha
granules,large platelets with few granules (grey
appearance w/ Wright-giemsa stain),decreased
serotonin,impaired aggregation studies (excep
thrombin &arachidonic acid)
• low MPV, prolonged BT
GPS
•Prognosis
• mild/not life threathening
• platelet transfusion in severe thrombocytopenia
Deficiencies of thromboxane generation
• genetic deficiency of cyclooxygenase enzyme
• impaired secretion and secondary aggregation similar
to storage pool disease
• acquired functional and structural modification
of arachidonic acid/pathway enzymes
• dysfunction/deficiency of thromboxane receptors
• Lab.diagnosis
• impaired platelet aggregation
• Avoid antiplatelet drugs and hormonal therapy
Acquired disorders of platelet function
• secondary to the following conditions
• Cardiopulmonary bypass
• DIC
• Lupus-like anticoagulant
• HIT
• HUS
• Drug induced plt abnormalities
Diagnostic Procedures for
Thrombocytic Disorders
I.Bleeding Time
• time it takes a standard wound to stop bleeding
• dependent on vasocontriction, platelet plug, coagulation
• Ivys Method
• 2-3 standardized punctures are made on the forearm under
constant pressure
• 40 mmHg
• 1-7 minutes
I.Bleeding Time
• Duke's Method
• Standardized puncture is made on earlobe
• 1-3 minutes
I.Bleeding Time
• Aspirin Tolerance Test
• Aspirin inhibits thromboxane A2
• BT is prolonged with aspirin intake
• 4 tablets (prolonged BT)
• 2 tablets (prolonged PT = von Willebrand disease)
II. Capillary fragility test
• measurement of the stability of capillaries
• Rumpel leede test
• 100mmHg for 5 minutes
• Quicks Test
• inflate between the systolic and diastolic pressure for 5 minutes
• < 100 mmHg
• Gothlins Test
• Both arms inflated at 35 mmHg for 15 minutes
III.Clot Retraction Time
• Principle:
• A normal blood clot retracts and in so doing gives out serum. The
degree of the reaction is measured by amount of the serum
expressed.

• Test the adherance and aggregation function of platelets

• % clot retraction = volume of serum expressed/ volume of whole

blood X 100
III. Clot Retraction Time
• Hirschboeck Method or Castor Oil Method
• observation of dimpling phenomenon or extrusion of serum on the
surface of a drop of clotted blood in a slide

• Normal Value: 15 -45 minutes

III. Clot Retraction Time
• MacFarlanes Method
• blood is allowed to clot in a graduated tube containing an
applicator stick and the incubation period. The clot
attached to the stick is then removed and the serum
extracted from the clot is measured. Retraction is
expressed in terms of serum obtained from the % of the
oiginal volume of blood.
III. Clot Retraction Time
DISEASES
Thrombocyte Deficiencies
• Thrombocytopenia
• Thrombasthenia
Fibrinogen Deficiencies
• afibrinogenemia
• Hypofibrinogenemia
Delayed Clotting
• Hemophilloid State
• Presence of Circulating Anticoagulants
CLOT CHARACTERISTICS
clot non retractile or retracts poorly
blood does not clot
clot is small
increased red cell fall out
slow clotting with slow sedimentation of
red cells
IV. Platelet Aggregation Test
• Platelet Aggregometry
• PRP is placed in the test well of the platelet aggregometer. An
aggregating reagent is added to the PRP, then the OD of the
sample is monitored. As the platelets in the plasma clump, the
plasma becomes clearer and light transmittance through the
specimen increases. These changes in OD are recorded by the
instrument in the form of a graph . The aggregometer also
calculates the slope of the aggregation curve and the amount (%)
of platelet aggregation.
V. Platelet Adhesiveness Test
• Salzman Method
• A platelet count is performed on two blood specimen.
• The number of platelets in the blood, collected through the glass
bead collecting system will be lower than the number obtained by
routine venipuncture. This is because platelets have adhered to the
column of glass beads due to their adhesive characteristics. The
results of this procedure are expressed as the percentage of platelets
retained in the glass bead column.
 V. Platelet Adhesiveness Test

% platelet Adhesiveness = platelet count w/o - platelet count w/ beads X 100

/ platelet count w/o beads

Normal Value: 26% to 60% platelet adhesiveness

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