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Transcribed

GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

Platelet Maturation Sequence

LECTURE 1
HEMOSTASIS

*TO STOP THE BLEEDING

Process that retains the blood within the vascular


system during periods of injury, localizes the
reactions involved to the site of injury, and repairs
and re-establishes blood flow through the injured
vessel

* MEGAKARYOBLAST unnucleated blood cells; aka Cytoplasmic Fragments of

Hemostasis is checked if the patient will be having a minor or major


surgery
Possible INCREASE bleeding & clotting time
ask if the patient is taking antiplatelet/anticoagulation medicine
because it will INCREASE in the formation of blood clot
Hemophilia A continues bleeding INCREASE

Megakaryocyte.
* MEGAKARYOCYTE largest cell in the Bone marrow
* THROMBOPOIETIN Hormone needed for the maturation & production of platelets;
produced by the Liver & Kidneys
* The LARGER the NUCLEUS the MORE IMMATURE
* To identify look for the CHROMATIN PATTERN

A system in dynamic balance that when tipped by


deficiencies (congenital or acquired) of the
procoagulant portion or excesses of the fibrinolytic
portion or uncontrolled activation of the procoagulant
portion, the result is excessive clot formation or
persistence of clot (thrombosis)

GENERAL FEATURES:
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

Megakaryoblast

20 50m diameter

Blue cytoplasm

N/C ratio is about 10:1

Multiple nucleoli

Fine chromatin

2.

Promegakaryocyte

20 60m

Less basophilic cytoplasm

Chromatin becomes coarse

Irregularly shaped

N/C ratio 4:1 7:1

3.

Mature Megakaryocyte (Megakaryocyte)

40-120m diameter

Cytoplasm contains coarse clumps of


granules aggregating into little bundles,
which bud off from the periphery to
become platelets

Multiple nuclei

N/C ratio is less than 1:1

ENDOMITOSIS: Mature
megakaryocytes form by cell division
without cytoplasmic division

Approx. 2,000-4,000 platelets in 1


megakaryocyte

4.

Metamegakaryocyte

Disintegrated cell surrounded by platelet

*Visible platelet; ready to move to


circulation

5.

Platelet / Thrombocyte

1-4m diameter

Light blue to purple, very granular

Life span: 8-11 days

2/3 platelets are in the circulation

1/3 are in the spleen

Either Primary or Secondary Hemostasis

Stages of Hemostasis
1. Primary Hemostasis *CHECK FOR THE BLEEDING TIME
Involve the blood vessels and the
platelet formation of platelet plug

1.

* Unstable, temporarily arrest the bleeding

1.1 Vascular system


a) Arteries
b) Veins
c) Capillaries
1.2 Platelets
a) How are platelets formed?
b) Where do platelets come from?

CARRIES BLOOD FROM THE HEART TO THE CIRCULATION

CARRIES BLOOD TO THE HEART

EXCHANGE OF GASES

STEPS IN PRIMARY
HEMOSTASIS
a) Adhesion
b) Activation
c) Release (secretion)
d) Aggregation

Adhesive it adheres in the circulation (blood vessels)


Activation (agonist) arachidonic acid transform thrombocytic in the presence of
cyclooxygenase to have a vasoconstriction
Release Alpha & dense granules
Aggregation clumping to form platelet clot to Temporary arrest bleeding

2. Secondary hemostasis*CHECK FOR THE CLOTTING TIME


Involves the interaction of
coagulation factors
Formation of fibrin clot
Involves the coagulation factors

*Permanently arrest the bleeding

* INCREASE IN BLEEDING TIME BUT NORMAL IN CLOTTING TIME MEANS PROBLEM COULD BE DUE TO
INCREASE PLATELET COUNT OR THERE IS A PROBLEM IN THE VASCULAR SYSTEM.
*

* INCREASE IN COAGULATION FACTOR BUT NORMAL IN BLEEDING TIME CAN BE SPECIFY WHAT
COAGULATION FACTOR IS THE PROBLEM so there will be no #or$ in clotting time

*FIRST HEMOSTATIC RESPONSE OF BLOOD VESSEL IN CASE OF INJURY IS VASOCONSTRICTION DUE


TO SECRETION OF SEROTONIN AND THROMBOXANE A 2 (derived from platelets & Epithelial cells. So without
the functions of platelets there will be No Secretion of Serotonin & Thromboxane A 2)
* VASODILATION widening of the blood vessels; incase of inflammation this is the first response

RV: 150,000 400,000/mm or


9
150-400 x 10

SPLENOMEGALY Enlargement of spleen


DECREASE Plt. Count
* SPLENECTOMY Removal of Spleen
INCREASE plt. Count
* Harmful because platelet is very adhesive & it can
aggregate
* VERY GRANULAR because of the ALPHA & DENSE Granules

Transcribed GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

Morphologic differentiation of Megakaryocyte Cell


Series
Matura&on
Stage

Cytoplasmic
granules

Cytoplasmic
tags

Nuclear
features

Thrombocytes
visible

Absent

Present

Single nucleus,
ne chroma9n,
neucleoli

No

Few

Present

Double nucleus

No

Megakaryocyte

Numerous

Usually absent

Two or more
nuclei

No

Metamegakaryocyte

Aggregated

Absent

Four or more
nuclei

yes

Megakaryoblast

Promegakaryocyte

FORMATION OF PRIMARY HOMEOSTATIC PLUG


1. Adhesion
Platelet adherence or expose sub
endothelial surface / collagen (in vivo)
Occurs in the presence of Von
Willebrands factor Adherence
a. Von Willebrands disease
Due to lack of Von
Willebrands factor

* functions as the adhesion of Platelets; INCREASE platelet count but abnormal collagen

b. Bernard Soulier disease /


Giant platelet syndrome
Deficient in
glycoprotein1b acts
as Von Willebrand
factor

PLATELET STRUCTURE
Composed of 60% protein, 30% lipid, 8%
carbohydrate, various minerals, water and
nucleotides
Divided anatomically into four areas:
peripheral zone, sol-gel zone, organelle and
membranous system
* in periphery
1. Peripheral zone
Responsible for platelet
adhesion and aggregation
Composition:
a) Glycocalyx function
as the outer surface
b) Plasma membrane
consist of 30 or more
Glycoprotein
c) Submembranous
area

2. Activation
Morphologic and function changes in
platelets
Agonists: substance that stimulate
activation; act as Arachidonic acid
TXA2 stimulate platelet secretion;
for vasoconstriction
Aspirin inhibits the cyclooxygenase;
it increases in bleeding time.
3. Secretion
Release of granules

2. Sol-gel zone
a. Microfilaments: actin and
myosin *responsible for clot retraction
b. Microtubules consists of
tubulin that maintains the
shape of platelet
(discoidamoeboid)

a.

Alpha granules
Platelet factor, platelet
derived growth factor
(PDGF)
Thrombospondin
B-thromoglobulin
Fibrinogen
Factor V
WVF
Albumin
Fibronectin

b.

Dense granules
CAS (CAPAS):
Calcium
ADP (stimulates aggregation)
Pyrophosphate
ATP
Serotonin

* We NEED CLOT RETRACTION so there will be permanent stability and NO


Clogging of platelets

3. Organelle zone
a. Alpha, dense granules
b. Mitochondria, lysosomal
granules

4. Membranous system
a. Dense tubular system

9 Site of Arachidonic Acid metabolism in the membranous

b.

system

b.

Open canalicular system

9 functions for the release of granules

(surface connecting system)


Membrane with open cananicular and
tubular systems for increased surface are and
rapid release of clotting factors

Dense Granule deficiencies


Hermansky-Pudlak
Chediak-higashi
Wiskott-Aldrich
Syndrome (WAS)

*WAS-triad of Immunodeficiency, thrombocythemia


& Eczema; categorized as ALPHA Deficiency
2

Release Disorders (storage pool defects)


a. Alpha Granules
Gray platelet syndrome

Transcribed GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

SUMMARY OF MOST IMPORTANT SUBSTANCES


SECRETED BY PLATELETS AND THEIR ROLE IN
HEMOSTASIS
Role in hemostasis

Substance

Source

HMWK

Promote
COAGULATION

Fibrinogen
Factor V

Alpha granules

Factor VIII: vWF

ADP
Calcium
Platelet factor 4
Thrombospondin
Serotonin
Promote
Thromboxane A2
VASOCONSTRICTION
precursors
Promote
AGGREGATION

Promote VASCULAR
REPAIRS

Dense bodies
Alpha granules
Dense bodies
Membrane
phospholipids

Platelet-derived
growth factor
Beta thromboglobulin
Alpha granules
Plasminogen

Other system
affected

Alpha - antiplasmin
C1 esterase inhibitor

Comments on
principal function

Contact activation of
intrinsic coagulation
pathway
Converted to fibrin
for clot formation
Assists platelet
adhesion to
subendothelium to
provide coagulation
surface

Disease associated with platelet count:


Patient with dengue fever
Factors associated with increase in platelet count:
Polycythemia vera
Thrombocythemia
After Splenectomy
Among patient with Tuberculosis

Promote platelet
aggregation

* POLYCYTHEMIA more than >6.5 million/mm RBC


REMEDY: Therapeutic phlebotomy

Promotes
vasoconstriction at
injury site
Promotes smooth
muscle growth for
vessel repair
Chemotactic for
fibroblasts to help in
vessel repair
Precursor to plasmin,
which induces clot
lysis
Plasmin inhibitor;
inhibits clot lysis
Complement system
inhibitor

Factors associated with decrease in platelet count:


Pernicious anemia
Aplastic anemia
Lesions involving Bone marrow
Acute leukemia
Idiopathic * No known CAUSE
Platelets are difficult to read because of the following:

They adhere on foreign surfaces

They easily disintegrated

They are unevenly distributed

They are hard to distinguished from debris

*HMWK High Molecular Weight Kininogen


st
*PLASMIN 1 substance responsible for FIBRINOLYSIS
*PLASMINOGEN not activated plasmin
*Quebeck platelet syndrome disorder (specific deficiency is Factor V) - Alpha
Granules Deficiency

* COAGULATION OF CHOICE for platelet counting is EDTA


* DO NOT Use Glass Tube because it adheres the platelets on the surface and it will cause
Falsely decrease platelet count.

4. Aggregation
Platelet attachment to each other
Requires fibrinogen and Calcium
Glanzmann thrombosthenia
deficiency of glycoprotein IIb IIIa
complex (receptor for fibrinogen)

3 General Methods of Platelet count


1. Direct method
2. Indirect method
3. Automated method
DIRECT METHOD for Platelet Counting
1. Light Microscopy * uses THOMA pipet
Rees and Eckers method, dilution fluid
R&E is made up of:
Brilliant Cresyl blue
Na Citrate
Distilled water

LABORATORY 2
PLATELETS STUDIES
Irregular in size and irregular shape
Derive from megakaryocyte
Non-nucleated because they derived from the
cytoplasmic extension of megakaryocyte
Size: 1-4 cubic micra;
Life Span: 8-11days
Thrombocytosis increase in platelet associated
with disease
Thrombocythemia increase in platelet count
but without disease
3
N.V: 200,000 400,000/mm or
3
150,000 400,000/mm (Henrys)

Functions:
Plays a very important role in
hemostasis
For blood coagulation and clot retraction

Procedure:
We need to wet the inner wall of
the platelet
Suck the blood up to 0.5 mark

Formula:
. 10 200
= /3
4

NV: 140,000 340,000 /mm

Transcribed GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

2. 2

nd

Light Microscopy
Guy and Leakes Method
Diluting fluid is made up of:
Crystal violet
Na oxalate
Distilled water
Formalin

INDIRECT METHOD *obsolete

Platelet is done in relation to 1,000 RBC

This method is not reliable

2 popular method:
1. Damasheks method
2. Fonios method
1) Damasheks method
Diluting fluid, made up of:
Brilliant Cresyl blue
Na citrate
Distilled water
Formalin
Sucrose solution

Procedure:
Suck blood up to 0.5 mark
Suck dil. Fluid up to 1 mark
Suck again dil. Fluid up to 101
Mix then charge to counting
chamber in (central primary
square)

Formula:
/3 = Platelet counted x 10 x 200 x 1

NV: 140,000 340,000 /mm

/3 =

NV: 500,000 900,000

2) Fonios method
Diluting fluid, made up of:
14% Mg2SO4 [Magnesium sulfate]

NV: 250,000 500,000

AUTOMATED METHOD

Most accurate method in platelet count

2 methods:
1. Semi-automated
2. Fully automated

* WEDGE SMEAR METHOD - Approximate # of platelet count


ex. 8-20 plt/field
Platelet = Adequate *
If approx.. # or complete blood count with value

1) Semi-automated
Counted by means of electronic particle
counter

* Oil immersion objective to look for the platelet per fields

2) Fully-automated
Counted by means of optical particle
under the principle of dark field
microscopy or electronic particle
aperture

*Factor used is the Wedge smear


*Example
st
1 field = 8
nd
2 =7
rd
3 = 11
th
4 =5
th
5 = 13
44

44
= 8.8 20,000 = 176,000/3
5

*20,000 is the FACTOR used to multiply


*5 total # of platelet field
*2,000 = WBC factor; objective used is HPO



1,000

(Same with Light Microscope)

3. Phase microscopy
Unopette Method
Nygards
Van Allen
Tocantins
Brecher Cronkite method
nd
2 most accurate method
Uses 1% NH4 C2O4 [Ammonium oxalate]
diluting fluid
3
NV: 150,000 450,000/mm

Formula:

Transcribed GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

B. PLATELET DISORDERS

LECTURE 2

I.

QUANTITATIVE abnormal platelet count

a) Thrombocytopenia

b) Thrombocytosis

5. Pseudoxanthoma elasticum
6. Senile Purpura
7. Scurvy
8. Henoch - Schonlein Purpura

Autonomous marked increase platelet


count, associated with
thrombotic/hemorrhagic complications

Reactive modified(moderate) increase,


asymptomatic
Immediately after the blood loss (hemorrhage) and
after Splenectomy (after the removal of spleen)

It has a lot of platelets but functionally abnormal


Seen in patient with Myeloproliferative disorder (in the
bone marrow it produce a lot of blood cells
abnormally) Seen in MD:
CML chronic myelogenous leukemia
MMM myelofibrosis myeloid metaplasia
ET essential Thrombocythemia
PV Polycythemia vera
This one has 1million/mm3 platelet count

QUALITATIVE

* cannot adhere

von Willebrand Disease (vWD)


9 Lack of vWF for normal platelet
adhesion
9 Platelet aggregation test:
o Normal platelet aggregation with
Epinephrine, Collagen & ADP
(ECA)
o Abnormal: Ristocetin
Bernard soulier
9 Deficiency in Glycoprotein 1b (gp1b)
Acts as receptor for vWF]

9
9

2) PLATELET
AGGREGATION

3) PLATELET
SECRETION/
STORAGE POOL
DEFECT

b) ACQUIRED
1) Uremia

fragility

It affects the Bleeding time


Elastic fibers in small arteries are

calcified and structurally abnormal

Immunologic damage to endothelial

cells

Platelet aggregation test:


o Normal: platelet aggregation
with Ristocetin (substance test)
o Abnormal: ECA
Gray platelet
9 Deficiency in Alpha Granules
Hermansky-Pudlak
Wiskott-Aldrich
Def. in Dense Granules
Chediak-Higashi

Accumulation of platelet disorder


*Accumulation of toxic metabolites
increase of UREA in blood

2) Paraproteinemias

3) Acute Myeloblastic
Leukemia (AML)
4) Myeloproliferative
disorders

5) Drugs

Degradation of collagen and elastin


Deficiency in vitamin C or Ascorbic acid

for fibrinogen) Fibrinogen needed for the normal


platelet aggregation

*Dense granules check for deficiency in CAPAS (Calcium,


ADP, Pyrophosphate, ATP and Serotonin)

Both condition with increase vascular

Giant platelet syndrome


Platelet aggregation test:
o Normal aggregation with ECA
o Abnormal: Ristocetin
9 > 20m/diameter
9 1-4 m Normal size of platelets
Glanzmanns thrombosthenia
9 Deficiency in glycoprotein 2b 3a
(gpIIb-IIIa) complex (fx as platelet receptor
9

disorder
The blood vessel walls are thin and lack
smooth muscle
Tumor composed of blood vessel

* Kasaback (buhol-buhol na veins)

*Decrease platelet counts

II.
a) HEREDITARY
1) PLATELET
ADHESION

Most common inherited vascular

* Kasaback buhol-buhol na ugat

9
9

A. VASCULAR DISORDERS

*Blood donationwhat if you are taking aspirin, stop taking of


aspirin for 3days prior to blood donation, should have a
Normal platelet counts 150-400,000/L

3. Ehlers Danlos syndrome


4. Marfans syndrome

Dilution loss of platelet count

9 *Extensive blood transfusion often is


accompanied by thrombocytopenia, the
degree of which is directly proportional to
the number of units transfused.

DISORDERS OF PRIMARY HEMOSTASIS

# Thrombocytopenic Purpura destruction or


Idiopathic/immune thrombocytopenic Purpura (ITP);
auto antibodies for platelets to your own platelets
Increase sequestration by the speen. Platelet leads to
Splenomegaly

*Seen in Mass Blood Transfusion (MBT)

2. Congenital hemangiomata
(Kasaback-Merritt syndrome)

Decreased survival time due to platelet


destruction

xx iron supplement should not taking by MEN because it can cause Hardening of their organs

*ex. Disseminated Intravascular Coagulation (DIC);


9 Problem in Mass consumption of platelets; formation
of clot

*Dengue patient nosebleed, bleeding gums due to due to deficiency in


Vitamin C/Ascorbic Acid [it helps in strengthening the collagen]

*to differentiate Hematuria from Hemoglobinuria you have to centrifuge the pinkish blood sample of
urine.
*Hemoglobin (after centrifugation) remain pinkish color; RBC is covered by hemoglobin;
*Hematuria --supernatant sediment clotted sample; RBC Sediment

*Uremia presence of UREA in blood; Ureaend product of PROTEIN metabolism

*Melenausually problem in Cancer in Upper GastroIntestinal tract (old blood)



Lower GastroIntestinal tract (fresh blood)


*Possible conditions assoc. with BLACK Stool
--Physiologic condition (No disease): taking of Iron containing diet/supplement (Fe sulfate)

1. Hereditary hemorrhagic
telangiectasia (Osler-WeberRendu disease)

Decreased platelet production


*Possible condition ex. Aplastic Anemia; decrease bone
marrow cells including RBCs & WBCs
*Pancytothemia-decreased in all forms of blood cells
*Aplastic Anemia could be Acquired AA (ex. Fanconis Anemia)
due to too much exposure to: Radiation, Chemicals like
BENZENE (acquired in pesticides),
Antibiotic (Chloramphenicol)-highly affect the bone marrow that
may lead to AA; antibiotic of last resort; causes the depression of
Bone marrow that may lead to AA.

Basic terminology for clinical findings in bleeding


disorders
1. Petichiae
Purplish red pinpoint hemorrhagic
*usually detected during tourniquet test
spots in the skin caused by loss of
*determine possible infection from
Dengue virus
capillary ability to withstand
*done in house, observation seen after
5min.
normal blood pressure and trauma
2. Purpura
Hemorrhage of blood into small
*Larger than Petichiae
areas of skin mucous membranes,
and other tissues
3. Ecchymosis
Form of purpura in which blood
escapes into large areas of skin
and mucous membranes, but not
into deep tissues
4. Epistaxis
Nosebleed
5. Hemarthosis
Leakage of blood into joint cavities
6. Hematemesis
Swelling or tumor in the tissues or
a body cavity that contains clotted
blood
7. Hematoma
Swelling or tumor in the tissues or
a body cavity that contains clotted
blood
8. Hematuria
RBC in urine
9. Hemoglobinuria Hemoglobin in urine
10. Melena
Stool containing dark red or black
blood
11. Menorrhagia
Excessive menstrual bleeding

Abnormal protein condition characterized by


coating of platelet membrane with abnormal
protein
Condition is characterized by the presence of
abnormal megakaryocytes
*To much increase in platelets; abnormal
functionally
CML Chronic Myeloid Leukemia
MMM Myelofibrosis Myeloid
Metaplasia
ET Essential Thrombocythemia
PV Polycythemia vera
Aspirin
9 Inhibits cyclooxygenase that inhibits
the conversion of Arachidonic acid to
Thromboxane A2

Transcribed GEN CAMATO

HEMOSTASIS
HEMATOLOGY 2

2. Platelet aggregation

LABORATORY 2

In vitro test to determine the ability of platelets


to aggregate with certain agonist:
a. Epinephrine
b. Collagen
c. ADP
d. Ristocetin

Platelet Rich Plasma (PRP) + Agonist = Optical


Density (O.D) monitored
*Citrated blood sample for coagulation studies

LABORATORY TEST FOR PRIMARY HEMOSTASIS

1. Platelet count
o
o

NV: 150,000 400,000/L


9
150 400 x 10 /L
Thrombocytosis
Polycythemia vera
Idiopathic Thrombocythemia
Chronic Myleoid Leukemia (CML)
Splenectomy
Thrombocytopenia
Thrombocytopenia Purpura
Aplastic anemia
Acute leukemia
Pernicious anemia
Gauchers disease
Sometimes following
chemotherapy and radiation

3. Platelet adhesiveness (Salzmann method)


o
o
o
o
o
o
o

DETERMINATION (manual platelet count)


A. Direct
1) Reese Ecker
Na Citrate
HCHO (formaldehyde)
BCB (Brilliant Cresyl Blue)

Detects in vitro adhesion


Measures the ability of platelets to adhere in glass
surfaces
Decrease vWD
Plate count #1: routine procedure, collection (EDTA)
PC #2: collected, through glass bead collecting
system
(!"!!!"!)
% =
100
!"!
NV: 26-60% (if decrease indicates vWD)

4. Clot Retraction Time (CRT)*to check if able to RETRACT


o

Proportion to platelet count


a. Castor oil / Hirschboeck
9 NV: 15-45mins *should have retraction after
9 Formation of dimpling/droplet like serum
on the surface of blood drop

2) Guy and Leakes


Na oxalate
Formaldehyde
CV (Crystal violet)

b.

Stefanini *for Research purposes


9 3-5mL blood (37C)
9 Read after 1 / 2 / 16 / 18 / 24 hours
9 *Normal begins within 1 hour, complete
within 18-24 hours

3) Wedge smear
Platelet estimate (OIF):
8-12 plts/field (Factor:20,000)

c.

MacFarlane
9 5mL of blood at 37C (1hr)
!"#.!"#$%
9 % =
100
!"
9 NV: 44-67%

4) Automated
Particles 2-20fl

5. Capillary resistance test


o
o
o

SMEAR: PLATELET ESTIMATES


Platelet estimate
0 49,000/L
50,000 99,000/L
100,000 149,000/L
150,000 199,000/L
200,000 400,000/L
401,000 599,000/L
600,000 800,000/ L
Above 800,000/L

Report platelet estimate as:


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

Measures capillaries to resist pressure


Correlated with the degree of thrombocytopenia
100mmHg(average), 5mins, after 15-30min., count
petechiae *uses sphygmomanometer (get Blood pressure)
Grade
Petichiae
1+
0-10 (few)
2+
10-20 (many)
3+
20-50 (multiple on hand and arm)
4+
>50 (confluent on hand and arm)

6. Bleeding Time
o
o
o
o

Associated Condition
Normal platelet count + prolonged BT

Low platelet count + normal BT


Low platelet count + very prolonged BT

Qualitative platelet abnormality


Primary vascular abnormality
Von Willebrands syndrome
Autoimmune thrombocytopenia
Simultaneous quantitative and
qualitative platelet deficiency

* Dukes every after 30sec. blot using Filter paper


to Report: # of blot in the filter paper
(ex. 2min & 30 sec)

In vivo measure of primary hemostasis


NV: 2-4 minutes
Dukes method finger tip or earlobe
Ivys method blood pressure cuff at 40mmHg
Puncture on forearm