o serotonin, ADP & ATP

PLATELETS
o Destroyed by RES
Lab. Exercises:
 Developed by a pluripotent stem cells that has been
 Platelet Count
influence by colony stimulating factor produce:
o Hemocytometer
macrophages, T lymphocytes, fibroblasts, stimulated
o Blood smear endothelial cells.
 Platelet Function Tests Platelet Structure and Biochemistry
o Bleeding Time  Four Functional Platelet Zones
o Capillary Fragility Test 1. Peripheral zone
o Clot Retraction Time - Associated by platelet adhesion and aggregation
 Coagulation Studies 2. Sol gel zone
o Clotting Time (Slide and Tube) - Provide cytoskeletal system for platelets and contact
o PT when the platelets are stimulated.
o APTT 3. Organelle zone
2 Phases: - Contain 3 types of granules: lysosomes, alpha, dense
1. Primary Coagulation/ Hemostasis – the platelet are 4. Membrane system
involved in the formation of clotting. - Contains dense tubular system which the enzymatic
2. Secondary Hemostasis/Coagulation- involved are the system for the production of the prostaglandins
coagulation studies particularly the coagulation factors. synthesis is found.
General Consideration Platelet Ultrastructure
 Bizzozero (1882)
- Recognized the platelet as a cell structure, different
from the RBC & WBC.
- 1970 the scientist recognized the relationship of this
cell in hemostasis and thrombosis
 Thrombocytes/ Platelet
- Small cells, Disk-shaped cells (0.3 to 3.0 μm)
 Involved in Hemostasis
 Normal Value – 150,000 – 400,000/cumm
 Makes 14,000 trips through the blood stream during the
Life span – 7 to 10 days (after 10 days they engulf
macrophages in the spleen)
 Platelet Count
o Wright’s stained blood smear  Glycocalyx
o Haemocytometer - Outer membrane surface, rich in glycoprotein that
o Electronic cell counter serves as a membrane receptor:
Platelet Development o Gp Ib – receptor for von willebrand factor
 Synthesized in the bone marrow (coagulation factor that is release by the endothelial
 Stimulated by the hormone TPO (Thrombocytopoietin) cells whenever there is damage to the vessel wall),
 Thrombopoietin is generated in the kidneys, partly also ristocetin is also present. For primary hemostasis.
in the spleen and liver. o Gp IIb & Gp IIIa
 Parent cells – megakaryocytes ( large cells found in the - Receptors for Von willebrand factor and fibrinogen ,
bone marrow, does not undergo cellular division but and expose by the stimulation of thrombin and
rather they undergo the process endomitosis/ adenosine diphosphate. For primary hemostasis.
endoreduplication) (80 to 150 μm) o Gp Va
 Endoreduplication / endomytosis – creates a cell with - Receptor for thrombin, for secondary hemostasis.
multi-lobe nucleus  Microtubule and microfilament
 Each megakaryocytes produces about 2,000 platelets - Provide an active means of platelet contraction in
 80% - circulation order to squeeze out the contents of cytoplasmic
 20% - spleen granules within the platelets.
 Granules: alpha & dense granules - secreted during - Microtubules- support the normal discoid shape of
platelet release reaction and contain many bio- chemical the platelets
active components:
 - Microfilaments/ thrombosthenin – contain an actin the local inflammation and vasodilation which
the is closely related to microtubules. increases the blood.

 Open canalicular system Platelet Kinetics

- Provides a direct communication between the Extra-  Megakaryopoiesis – production of platelets, occurs in
cellular and Intra- cellular compartments endomitosis ( nuclear splitting without cell division)
 Dense tubular system  Stages of Megakaryocytic maturation
- Forms a circle within the microtubules, consider as a o Megakaryoblasts
site of arachidonic acid metabolism. - Single megakaryoblasts nucleus may contain 2-64
- Also a calcium sequestering pump that maintains times the normal number of chromosomes.
platelet cytoplasmic calcium levels. - Nuclear chromatin is densely staining and more
 Mitochondria compact in the later stages
- Responsible for energy production - Nucleoli are small in the developmental stages
 Glycogen granules o Promegakaryocytes
- Provide energy substrate - Larger, have more cytoplasms
 Alpha granules - Nucleus becomes increasingly lobulated and spread
- Contain a contact promoting factors including: out in a horseshoe shape
platelet fibrinogen, PDGF (platelet derived growth - Red pink color
factor), von Willebrand factor, beta o Granular megakaryocytes
thromboglobulin, fibronectin, platelet factor 4 - Characterized by spreading of nuclear lobes,
 Dense granules spreading also of the pink granules throughout the
- Contains non-protein factors: ADP,ATP serotonin (5- cytoplasm
hydroxytryptamine), calcium o Mature megakaryocytes
Platelet Function - Compact nucleus, basophilia of the cytoplasm
 Start of the platelet function when there is completely disappear
Damaged subendothelium – releases factors that - Produces 2,00-7,000 platelets
activate the platelet. - Platelet fields – cluster of pink granules in the
5 Factors: cytoplasm. Produce by the invagination of the
1. Collagen surface membrane separating the cytoplasm into
2. Fibronectin individual platelets ( are shed in the megakaryocyte
3. Von Willebrand factor cytoplasm into the marrow sinuses and released into
4. Thrombin the peripheral circulation)
5. Adenosine diphosphate - Maturation time: megakaryoblasts to become a
 Tissue platelet activators – change platelet shape. (from mature megakaryocytes- 5 days
discoid to spherical shape). - if undergo bone marrow examination it is seen that
- The dense and alpha granules undergo internal there are 5-10 megakaryocyte per lpf. Normal
contraction and centralization. marrow – 15M of mature megakaryocyte. 5-10 per
- Calcium dependent lpf
 Plug formation –consider as the secondary aggregation.
stimulated by thrombin and TXA2 (thromboxane a2)
- Thromboxane a2- inhibit adenylate cyclase (form
the CAMP) there is also inhibition in the formation of
cyclic adenosine monophosphate.
- Thrombin- cleave the fibrinogen to fibrin (to stabilize
the platelet plug)
 Growth-limiting factors of the platelet aggregate
- Blood flow – washes away the coagulation
promoting factor
- Release of the prostaglandins : prostacyclin by the
surrounding vascular tissue
 Granular release
- Refers to substances from the dense granules: Platelet Count
serotonin, prostaglandins, lysosomes which causes  Direct Platelet Count
 1. Dilution using blood diluting pipette (RBC pipette)
o Use .5 mark of blood. Dilute up to 101
mark.
o Wipe only the sides of the pipette but not
the tip.
o Mix in a figure of 8 for 3-4 mins to mix the
blood and diluting fluid.
o Discard 3-5 drops to expel the pure diluting
fluid.
o During charging it is important that the
hemocytometer is clean.
 Indirect platelet count  The enzyme that the arachidonic acid be converted into
- Counting in the blood smear, not definite for prostaglandins is the cyclooxygenase
measuring platelet count. For checking only.  The enzyme that the arachidonic acid be converted into
How to check if the machine is giving the correct result? leukotriens is the lipooxygenase
- Use the indirect platelet count
Procedure in how to perform indirect platelet count:
1. Make a blood smear
2. Look for proper field to were to count the platelets
3. Check the distance of the RBC. At least 2 cells apart. No
RBC overlapping
Arachidonic Acid Pathway
o Comes from the cell membrane (plasma membrane) :
lipid bilayer
o Lipid bilayer is composed of: protein 55% lipid 42%,
Carbohydrates 3%
o Lipids comprises : phospholipid 25%, cholesterol 13%,  Leukotriens B4 is a chemotaxis
other lipids sphingolipids 4%, especially in nerve cells  Leukotriens C4, D4,E4 are broncho constrictor
o Phospholipids form the arachidonic acid  Cyclooxegenase inhibit by aspirin and non-steriodal
o Each phospholipid molecule has a head and two tails  Platelets are the only cells in your body that possess
 Polyunsaturated omega-6 fatty acid thromboxane synthase
 Chemically, it’s carboxylic acid (20 carbons)  Thromboxane is bronchoconstrictor, vasoconstrictor,
 Present in the phospholipid portion of the lipid- bilayer platelet aggregator
plasma membrane Thrombocytosis
 Inflammatory mediator-> vasodilation &  Normal platelet count= 150,000 -400,000 per microliter
vasoconstriction(blood coagulation)  Thrombocytopenia – dec. # of platelets
 Freed from phospholipid via phospholipase A2 (inhibited  Thrombocytosis- inc. # of platelets
by steroids) 2 types:
 Not an essential fatty acid, unless linoleic acid is deficient 1. Primary (essential) – one of the myeloproliferatice
Aristotle’s deductive reasoning: disorders “elderly".
Premises: 2. Secondary (reactive) – 100% of cases in kids, 80% of
1. If arachidonic acid is pro-inflammatory cases in adults. More common
2. Steroids prevent arachidonic acid formation
Conclusion:
 Steroids are anti-inflammatory
  ITP- immune thrombocytopenic purpura
 DIC- disseminated intravascular coagulopathy
 TTP- thrombotic thrombocytopenic purpura
 HUS- hemolytic uremic syndrome
 HELLP- help syndrome which is hemolysis elevated liver
enzymes and low platelets which means
thrombocytopenia
 SEL- systemic lupus erythematosus
 APS- antiphospholipid antibody syndrome

Symptoms of thrombocytosis:
 Asymtomatic
 Primary thrombocytosis: erythromelalgia,
thrombosis,bleeding
How to diagnose:
 Peripheral smear
 Bone marrow biopsy
How to treat:
 No symptoms  no treatment
 Secondsry thrombocytosis: aspirin (low dose)
 Primary thrombocytosis with symptoms:
o Aspirin
o Hydroxyurea(cytoreducing agent)
o Anegrelide
 JAK2  Ruxolitinib (tyrosine kinase inhibitor)
Thrombocytopenia
 Decrease number of platelets
 Causes: Pseudothrombocytopenia,
Truethrombocytopenia
  Blood sample is collected either by finger prick or in a
tube with anticoagulant EDTA
Equipments & Reagents:
1. Haemocytometer – improved Neubauer’s counting
chamber
2. Compound microscope
3. Cover slips – thicker than those for conventional
microscopy. Distance bet. the bottom of the chamber
and the cover is 0.1mm
4. Pricking apparatus
5. Spirit swab
Platelet Pathophysiology 6. Diluting Fluids
o The platelets are tiny and should be made
visible by staining
o Fluids prevents platelet clumping
7. Diluent
2 Kinds:
1. Fluids that lyse RBCs
- Only platelets are seen
- Ammonium oxalate
2. Isotonic to RBCs
- Formol citrate
- Platelets are counted in bet. RBCs

Platelet Count by Improved Neubauer Counting Chamber

 Platelets are the smallest of all blood cells
 Important to monitor platelet count in order to diagnose
or screen various medical conditions that may affect the
process of blood clotting, leading to bleeding
 A low count may be because of inadequate production or
peripheral destruction of platelets
 Commonest causes of low platelet counts are viral
Microscopic Appearance
infections like dengue fever.
 Monitoring platelets on a continuous basis over the span
of the fever and beyond is very vital.
 Cell counters give a fairly accurate count
 Platelet counting on cell counters is often flagged due to
several reasons
 Count >10lakhs points towards a neoplastic process
 In adults, the normal platelet count ranges from 1.4-4.5
larkhs platelets per microliter of blood
Dilution
2kinds:
1. 1:100 : standard dilution
Count low – create errors
- 1.98 ml of platelet diluting fluid
+ 20µl blood
2. 1:20
- 50µl of blood + 950µl of diluent solution
 Sample: whole blood EDTA sample or capillary blood Precautions:
 1. Platelet count should be performed at the earliest - Filter paper
2. If blood is collected by a skin puncture, remove the first - Sphygmomanometer
drop of blood free-flowing blood PROCEDURE:
3. If clumps of platelets are seen in the hemocytometer the A. IVY METHOD
procedure should be repeated a) identification of the patient
4. A stained peripheral blood smear should be examined b) ask if the patient is taking over the counter
and the platelet estimate determined to confrim the medication (aspirin) in the last 7-10 day
hemacytometer platelet count. If a discrepancy exists c) assemble all the required materials
repeat test. d) wash hands, put on gloves
e) place patient’s arm, volar surface facing up
1. Apply pressure cuff on the patient’s upper
forearm (above the elbow) and inflate to a pressure of
BLEEDING TIME
40 mmHg. Maintain this pressure all throughout the
- Perform to measure the time required for platelets entire procedure.
to form a plug (strong enough to stop the bleeding 2. Disinfect an area (approximately three finger-
from incision) widths below the bend in the elbow) on the volar surface
- Measure the time it takes for the standard puncture of the arm with 70% alcohol. Wipe dry with sterile gauze.
 Length of bleeding time is increased when: 3. Hold the skin tightly by grasping the underside
1. Platelet counts are low arm firmly and make skin incision avoiding any
2. Platelet disorder affecting the platelet adhesion to subcutaneous veins.
form organelles 4. Start timer.
3. Taking aspirin or medication including herbs 5. Blot the drop of blood from the puncture site
 Test results also can be affected : every 30 second interval. The filter paper should not
4. Temperature of patients skin touch the wound at any time.
5. Vascularity 6. Stop timer as soon as bleeding stops.
6. phlebotomist technique 7. Release the pressure cuff. Ignore oozing.
 A screening test, abnormal results followed by another 8. Record the bleeding time result.
test 9. Label the center of the filter paper with the
 A pre-surgery work up name of the patient, date, and bleeding time result.
PRINCIPLE 10. Paste or staple the filter paper to the result
Capillaries subjected to a small, clean incision bleed until page.
the defect is plugged by aggregating platelets. If blood 11. Discontinue the test at the end of 15 minutes in
accumulates over the incision, coagulation occurs, and the those individuals who show no sign of cessation of
overlying fibrin prevents additional bleeding. bleeding at this time.
Materials and Equipment: B. DUKE METHOD
IVY METHOD 1. Disinfect the site of puncture (most preferred
- Sphygmomanometer ring finger or middle finger)) with 70% alcohol and allow
- Lancet to air dry.
- 70% alcohol 2. Puncture to a depth of 3 mm with a sterile,
- Cotton disposable blood lancet.
- Stopwatch 3. Immediately start the timer as soon as the first
- Filter paper drop of blood appears.
DUKE METHOD 4. Blot the drop of blood with filter paper every 30
- Lancet second interval making sure that the filter paper does
- Glass slides not touch the wound.
- 70% alcohol 5. Stop the timer as soon as bleeding stops.
- Cotton 6. Ignore oozing.
- Stopwatch 7. Record the bleeding time result.
- Filter paper 8. Label the center of the filter paper with the
TEMPLATE METHOD name of the patient, date, and bleeding time result.
- Template device 9. Paste or staple the filter paper to the result
- 70% alcohol page.
- Cotton
- Stopwatch
 10. Discontinue the test at the end of 15 minutes in
those individuals who show no sign of cessation of
bleeding at this time.
REPORTING RESULTS
Bleeding time values depends on location and orientation of cut
and on particular method and device used.
A. Ivy Method – 2.5 – 7 minutes
B. Duke Method – 1 – 3 minutes (However,
normal persons may occasionally give values up to 5 minutes)
C. Template Method
Adults – 2 – 8 minutes
Children – 1.30 – 8.99 minutes
Newborn – 0.85 – 1.65 minutes
NOTES
• There is occasional scarring when the bleeding time test
is administered using the template method. Patients, parents or
guardians should be advised of this possibility, especially in cases
of a positive clinical history of keloid scar formation. Potential
scarring may be reduced by approximating the skin edges with a
non-allergenic wound closure strip for 24 hours. If there is ooze
from the incision as may be encountered in severe primary
hemostatic disorders, then a pressure-type dressing should be
used in conjunction with the wound closure strip.
• A newborn bleeding time incision is best performed over
the lateral aspect, volar surface of the forearm approximately
midway between the antecubital crease and the wrist. The size
and shape of the newborn’s arm and the physiology and the
vasculature of the newborn require an incision perpendicular to
the antecubital crease.