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PLATELET PRODUCTION, STRUCTURE AND

FUNTION

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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

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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

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“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
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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
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Uses monoclonal antibody that will bamd to a
single receptor

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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

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CFU- GEMM

Megakaryoblast

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Terminal Megakaryocyte Differentiation

Megakaryocyte

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Platelets

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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)

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

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. Mpl – receptor for TPO V-mpl. 5. 3. meg maturation & plt release IL 11  Enhances endomitosis. 7.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. 3.Macrophage Inhibitors of Megakaryocyte Growth 1. more active metabolically and more effective hemostatically Dwarf Micromegakaryocytes . 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. 2.% 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 α gran & DMS) Basophilic Granular (granules adjacent to nucleus) GP Ib vWF adhesion receptor(CD4 2) Mpl Can’t 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. Hormones & Cytokines of Megakaryopoiesis 1. 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.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. 6. 3. 7. 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. 6. Largest cell in the BM No visible nucleoli Endomitosis is complete Irregular shape Regulated by TPO Full ploidy     4. 4.detected by immunostaining in fully developed megakaryocyte Polyploid. TPO/ Trombopoietin  23% homology with EPO  mRNA for TPO is found in the kidney (primary source). stains alpha granule phosphates 5. 8.

fibrinogen & transports them to storage organelles (endocytosis) 2. 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.- Circulating. PERIPHERAL ZONE 1. 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.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. Plasma membrane  Selectively permeable  Made up of:  Cholesterol o Maintains fluidity o Controls passage o For stability  Fatty acids  Carbohydrates 3. Glycocalyx  20. 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. other blood cells  Surrounds are rich in protein  Rich in glycoprotein and proteoglycans  Support surface glycosaminoglycans. Submembranous area  Provides phospholipids (where clot formation happens) . ec. Stroke: clot in the brain (blood flow is blockedIn the supra.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. oligosaccharides & glycolipids  Absorbs albumin. 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. non granular. centrally located Hyalomere: surrounds chromomere.30 nm  Platelet membrane outer surface  Adhesive  Negative surface charge  Repels other plts. 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 βthromboglobu lin PF4 PDGF Delta granules ADP ATP Serotonin Ca and Mg Lysosomes A. 2.

e.derived growth factor (PDGF)  Involved in repair of damaged blood vessels  Stimulates mitosis in vascular smooth muscle cells 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. Dense core granules/ Delta granules  2-7 per platelet  “bull’s eye”  Stains black (opaque) with osmium-dye TEM  Released directly in the plasma  Locus of stored. neurophil. extension of psuedopods. g. secretion of granular contents  Platelet myosin C.promotes plt adhesion β-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. c. f.  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. injured endothelial cells that leads to expression of circulating factors Factor V (Multimerin) and VII binding protein  Labile factors  Factor V. Alpha Granules  50-80 per platelet      As the plasma activates.cofactor in fibrin clot formation  Factor VII binding protein. ORGANELLE ZONE 1. b. → actin → filamentous & contractile  Desmin and Vimetin o Intermediate microfilament that connects actin and tubules maintaining platelet shape o Controls platelet shape change. d. 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 α granule contents  Large & light gray in wright-stained films Alpha granules protein content: a. 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 . SOL-GEL ZONE 1. 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. 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. 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).

Lysosomes  Stains positive with:  Arylsulfatase  β-glucuronidase  Acid phosphatase  Catalase  Digest vessel wall matrix components during in vivo organization  Digest autophagic debris HEMOSTASIS - Components of Hemostasis A. Bernard. 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. ADP (Adenosine Diphosphate)  Supports neighboring plt. D. Coagulation cascade 2. TF-bearing cells 3. 2. Fibrinolytic proteins & inhibitors Function of Hemostasis - MEMBRANOUS SYSTEM 1. Glanzmann’s Disease  Lacks GPIIb/IIIa receptor c. Platelets B. 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. E. 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. Vascular intima 2. Aggregation  Receptors: P2Y.Soulier Syndrome  Defect in GPIb receptors b. 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. Cellular 1. Thrombocytopenia Von Willebrand disease Other platelet disorder a. Y. Surface Connected Canalicular System  Alpha granules Heme: blood. coagulation enzyme activation Limit blood loss resulting from injury Maintain intravascular blood fluidity Promote revascularization of thrombosed vessels after injury Factors Affecting hemostasis: 1. platelet aggregation. c. 3. D-dimer) . 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. d. Biochemical 1. b. 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.

elastic structural protein  Binds and activates platelets  Upon stimulation by collagen: . Endothelial Cell Synthesizes: 1. short lived Blood vessel contracts to seal the wound ↓ Platelets fill the open space to form a plug Secondary Primary hemostasis Large wounds Trauma.activates coagulation system Basement membrane collagen Collagen of matrix Elastin Fibronectin Laminin Vitronectin Thrombospondin Procoagulants Properties of Vascular Intima 1. Non hemolytic function  Tissue barrier from  Collagen. 2. surgery (exposed tissue factor) Platelets Coagulation cascade Delayed.promotes plt activation & adhesion  TF. Neutrophil & Macrophage 3. FVII activated ↓ Extrinsic pathway ↓ Fibroblast (scar tissue) takes part in the healing PRIMARY HEMOSTASIS B.       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. monocytes) Rapid. unbroken surface  Damaged endothelial cells secrete vWF & P-selectin (WBC & platelet binding) b. once platelet is activated. Tunica adventitia  collagen fibers Tunica media  smooth muscles & elastic fibers Tunica intima  endothelium that lines the lumen of all vessels a. Thrombomodulin  Protein that activates the protein-C pathway (digests Factor V & Factor VIIIa) hindering the intrinsic pathway of coagulation  Inhibitor of thrombin formation 6. TFPI (Tissue Factor Pathway Inhibitor)  Inactivated Factor VIIa  Controls TF or extrinsic coagulation pathway 5. 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. Endothelial cell  Innermost vascular lining  Rhomboid and contagious  Should be smooth. Nitric Oxide  Counteracts vasoconstriction & maintains healthy arterioles  Also secreted by Smooth Muscles. neutro. it will recruit other platelets  Thrombin: responsible for hydrolyzing fibrinogen Blood Vessel Layers 1. Vasoconstriction  Constricts by mechanical or chemical stimulus  Mimimizes blood flow on injured site Collagen  Flexible. 2. Heparan sulfate  Intimal glycosaminoglycan  Retards coagulation by activating antithrombin3  Heparin prevents propagation of thrombin that causes coronary thrombosis. long term TF exposed. 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. Prostacyclin (PGI2)  Synthesized through eicosanoid pathway  Inhibits/ prevents activation of platelets  Prevents platelet aggregation in healthy blood vessels  Vasodilator 2. 3.

platelet surfaces Internal Ca++ reaches a threshold Reversible Secrete growth factors (PDGF) Involvement of vWF → GPIb (receptor site) Requirements for Adhesion 1. - 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. Normal fibrinogen concentration. Secretion of TPA (Tissue Plasminogen Activator)  Binds fibrin.000-20. Normal secretion of granules ADHESION Binding of non platelet to platelet Platelets roll and cling to non. GPIb vWF  Stored in the alpha granules & weible palade bodies of EC Result - Fibrinolytic Properties of Vascular Intima 1. 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 2.000. 4.   3. 6. - each other granule’s 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.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. fibrinogen and psuedopod formation Requires redistribution of P-selectin to the surface membrane . Functional plasma vWF (vWF isn’t necessary in arterioles for adhesion) 1. 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. 5. activates nearby fibrin  Secreted by EC  Converts of plasminogen to form plasmin  Plasmin digests thrombus & restores blood flow 2.

- Activation by contact w/ agonist (promoter)  ADP  TXA2 (produced by alpha granules) Phases of Aggregation: a. - 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 . 3. b.

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. 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.Role in Hemostasis Promote coagulation Substance Source Comments on principal function HMWK/fitzgerald factor Fibrinogen Factor V Factor VII. Simplate Method Procedure        Select site at the volar area of the arm (muscular) Place sphygmomanometer Simplate/ surgicutt (5 mm wide. 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. BLEEDING TIME A. 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 . 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. 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.

000 uL (150400x109/L) Thrombocytosis Thrombocytopenia Polycythemia vera Thrombocytopenia .400.000 rpm Recollect specimen on blue top (citrated blood) PBS Decreased Platelet Count a.800.000 uL 150. PLATELET COUNT Reference Range 150. 000.000. 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. 000.000 uL 600.000 uL 401.000 uL >800. Even cellular distribution o No overlapping o Space should be approximately ½ 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. 2.**Above platelet count of 100x109/L should fall within the reference range C.199.400.149. run specimen on machine for 10.000 uL 200. b.599.99.000. c.000.000 uL Marked decrease Moderate decrease Slight decrease Low normal Normal Slight increase Moderate increase Marked increase Quality Assurance 1.000 uL 50. 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: ¿ platelets∈10 fields x 100 x 14. 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. 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. 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.000 uL 100. 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 Wright’s stain b.000. Platelets from skin puncture: irregular in shape.000.000/uL) - Vortex for 2 mins.000 100 **answer must be compared with the result obtained from the instrument.

000 uL Methods for Platelet Count <5.Idiopathic thrombocythemia CML Splenectomy purpura Aplastic anemia Acute leukemia Gaucher’s disease Chemotherapy and radiation Significant Platelet Levels <150. 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. 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.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. 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. Prolonged BT o Quality platelet abnormality  Aspirin  GpIIb/IIIa deficiency x dilution factor  von Willebrand syndrome  Primary vascular abnormality 2.50. d. Normal BT o Autoimmune thrombocytopenia 3.000 uL 30. Light Transmittance Aggregometry As a plt aggregates form.H2O Fonio’s: 14% Magnesium sulphate Olef’s Abnormally low Possible bleeding with trauma Possible spontaneous bleeding Severe spontaneous bleeding 1. 000 RBC x platelet counted Dameckek: BCB. Research Applications o Anti-platelet drugs o Effect of aspirin/ clopidogrel o Monitoring transfusions Types of Aggregometry A. formalin. aggregation and secretion Instrument designed for measuring platelet aggregation Importance of Platelet Function Monitoring 1. more light passes through the PRP and the tracing begins to move towards 100% light transmission . Normal PC. sucrose. Decrease PC. Hematology o Assessment of platelet disorders 3.000 uL A. sodium citrate 2.000. 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 ( ) 1.000 RBCs in the blood smear platelet count= o o o RBC count 1.000 uL <30. formalin. Decreace PC. Very Prolonged BT o Quantitative + qualitative platelet dysfunction AGGREGOMETRY - Assess platelet adhesion. d. BCB. Indirect Method o Platelets are counted in relation to 1. crystal violet. Na Citrate. round/oval. Light Microscopy o Reese-Ecker  Sodium Citrate.

of 100.300. Thrombin Receptor Activating Peptide-6 (TRAP-6) o For MEA analysis. small amounts of ADP released by red blood cells during sampling may cause a desensitizing effect o Reagent ADP is stored at -200C.- 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. resulting in stable impedance value Specimen 1.1000x109/L should be tested undiluted (For ADP not greater than 225 x109/L) Agonist Used 1. 000. whole blood plt conc. NSAID or Clopidogrel therapy 2. 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 Ibα and GP V . as an alternating current is applied across the electrodes. TRAP-6 is used in a final concentration of 32µmol/L to assess aggregation independent of platelet inhibition by aspirin or clopidogrel 3. Adenosine diphosphate (ADP) o Most commonly used agonist o Commonly used at 5µmol/L to 20µmol/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. reconstituted with NSS o Secretion in response to 5µmol/L is diminished in:  Platelet membrane disorders  Eicosanoid synthesis pathway enzyme deficiency  Storage pool disorders  Aspirin.2%) dehydrate form of trisodium citrate o 25 ug/mL hidurin o Mix 3-6x gently with end over end inversion by hand 2. - 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. activation and loss of plt subpopulations 2. 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. For all agonists except ADP. 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. PRP (Platelet Rich Plasma)  Light transmittance aggregommetry (PAP-8E)  200. 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. Anticoagulant o 105 mmol/L to 109 mmol/L (3. a monolayer of platelets forms on the exposed portions of electrodes. Whole blood  Centrifugation is not required  Reduced potential plt. Needle Gauge o 19 and 21 3.

Epinephrine o Binds platelet to adregenic receptors o Activate platelet same as ADP o Can’t induce aggregation in storage pool disorder/ eicosanoid synthesis pathway defects o Is not recommended as a standard agonist for whole blood testing clinically. inhibits aggregation and ATP secretion when tested with low concentrations (0.5mmol/L to 1. reconstituted with d. the vWF receptor on platelets o The agonist is commonly used two concentration ranges. as fewer than 50% of subjects respond o Reagent is stored at 1-60C.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. Arachidonic Acid o Commonly used at 0.0mmol/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. Collagen (Type 1 Fibrillar) o Is commonly used at 1µg/mL to 5µg/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 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 1µg/mL to 2µg/mL. no dilution. WBC.5mmol/L) in whole blood o Reagent is stored at -200C in the dark.water o Modulates the interaction between plasma von Willebrand factor (vWF) and platelet membrane glycoprotein Ibα (GPIbα). 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. neutrophil) than men . 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. diluted with bovine-albumin 5. can’t be frozen 6. which in turn. but aggregate normally at 5µg/mL Reagent is stored at 1-60C.