Review

:
Megakaryoblast ʹ earliest cell in megakaryocytic series
Promegakaryocyte ʹ largest cell in BM
Megakaryocyte ʹ once mature, cytoplasm breaks off & becomes platelets
*1
st
3 stages are not normally found in circulation
*sometimes, megakaryocytes produce platelets in the lungs

Platelets/Thrombocytes
fragments of cytoplasm of megakaryocyte
newly released p bigger, more active & more effective hemostatically

Morphology:
y Size: 2-5Q
y No nucleus
y Anticoagulated blood: round, oval or rod-shaped
y Capillary blood: irregular borders (due to filopodia/hairlike
projections)
r of irreg is assoc w/ relative time bet. pricking & smearing

Distinctive Areas of a Wright-Stained Blood Smear:
1. Granulomere/Chromomere ʹ central area filled w/ purplish granules
2. Hyalomere ʹ pale blue cytoplasm

Lifespan: 8-11 days in circulating blood
24 hrs outside the body (extracted blood)
Stored blood in blood banks is deficient of viable platelets
*Platelet concentrate is prepared fr fresh blood
Functions:
1. Maintaining integrity of BVs ʹ ͞leak-free͟; fill gaps
2. Hemostasis
a. adhere to injured BVs
b. aggregate @ site of injury forming 1r platelet plug
c. release biochemicals important in hemostasis
release serotonin & thromboxane A2 (for vasoconstriction) &
ADP (for clumping)
d. Source of Platelet factor 3 ʹ for prodxn of throboplastin
3. Initiate Clot Retraction ʹ mediated by Thrombosthenin (contractile
CHON produced by platelets); process by w/c serum is expressed fr the
clot

PLATELET COUNT
*More difficult to do compared to RBC & WBC cuz:
1. very small
2. disintegrate easily when expose to air
3. tendency to stick/clump together
4. adhere on to glass surfaces/any foreign body
5. difficult to differentiate fr dust, dirt & bacteria
6. not well distributed in circulation

Thrombocytosis ʹ ј in no. of platelets above normal
Thrombocytopenia ʹ љ in no. of platelets below normal
Nr condition: represents only 2/3 of total platelet mass; 1/3 found in spleen

PHYSIOLOGIC VARIATION OF PLATELET COUNT
Spleen
platelet reservoir in man
release of splenic pool can be caused by:
o admin of epinephrine
o variety of stresses: excitement, hypoxia, strenuous exercise, ј
altitude
y Neonates (1
st
4 days): slightly љ than adults
y Menstruating: q PC shortly before & during 1
st
day
y Arterial blood: slightly ј platelet count than venous blood
y Venous blood: slightly ј platelet count than peripheral blood
y Nr absent in lymph/other body fluids

SPECIMEN
Venous blood
o EDTA vacutainer tube
o satisfactory: 5 hrs @ RT; 24 hrs @ 4°C
o siliconized plain vacutainer tube (red-stoppered) or plastic syringe
y transfer immediately into tube containing EDTA
Capillary blood
o Fingertip & heel are recommended but not the earlobe (fine hair
favors adhesion of platelets)
o Free flow of blood is ideal
y if flow is poor, massage away from the puncture site
y don͛t squeeze
y if necessary do another puncture
o Collect blood for platelet count 1st before doing the other tests
o Manual CBC count: Hgb, Hct, WBC differential count

I. INDIRECT METHOD
y Platelets & RBCs are counted simultaneously in a blood smear
y PC/µL or PC/L is calc based on RBC ct obtained using hemacytometer
y results are less reliable: RBC factor is 50

A. Dameshek Method (Wet Method)
o Diluent: Rees-Ecker diluent
y 3.8 g Na Citrate (prevents clumping of platelets)
y 0.2 ml 40% formaldehyde (preservative)
y 0.1 g brilliant cresyl blue (dye)
y 100 ml dH2O
Filtered before use to remove any debris present
Doesn͛t lyse RBC & WBC (platelet is 1/5 ʹ 1/10 the size of RBC)
Dilution is stable only for 30mins

Special steps in procedure:
1. After finger puncture, wipe 1
st
drop of blood then place a large drop
of diluent over the punctured site - to avoid exposure to air &
disintegration of platelets
Ratio = 1:5 (blood to diluent)
2. Transfer a portion on a cover glass & invert on a slide
3. Allow to stand for 15mins
4. examine under OIO (diaphragmpartly closed) & count platelets & RBCs
until 1000 RBCs are recorded; platelets are lilac colored, tiny,
glistening

Calculation: Platelet countȀɊL ൌ ͓ of platelets x
ோ஻஼Ȁஜ௅
ଵ଴଴଴

NV: 500, 000 ʹ 900, 000/µL or 500 ʹ 900x10
9
/L

Wet mount - RBCs tend to concentrate @ the edges of coverslip, so
counting is done in central area (falsely ј ratio of platelets
to RBC)
Modified Dameshek
uses a siliconized medicine dropper in diluting blood

B. Fonio Method (Dry Method)
o diluent: 14% Magnesium Sulfate (MgSO4)
Doesn͛t lyse RBCs
Special steps in procedure:
Ratio = 1:3 (blood to diluent)
1. transfer mixture on 1 end of a clean slide
2. make a wedge smear w/ a spreader slide
3. air dry the smear
4. stain w/ Wright͛s stain
5. examine under OIO & count platelets & RBCs until 1000 RBCs are
recorded (do the counting @ 1/5 ʹ 1/3 part from end of smear)
NV: 250, 000 ʹ 500, 000/µL or 250 ʹ 500x10
9
/L


Advantages:
y easier to count platelets & RBCs
y size & shape of platelets can be observed

C. Olef͛s Method
o Same principle, but cumbersome procedure
o NV: 437, 000 ʹ 586, 000/µL or 437 ʹ 586x10
9
/L

To Counter Check results:
Examine a well prepared blood smear stained w/ Wright͛s stain
1 platelet/OIF Thrombocytopenia
5-20 platelets/OIF or 1 platelet/10-30 RBCs Nr or adequate
>25 platelets w/ clumps/OIF Thrombocytosis

II. DIRECT METHOD
y employs dilution of blood using RBC/WBC pipet w/ the use of a
hemacytometer

A. Brecker & Cronkite Method
y reference method
y Phase-contrast microscopy (w/ green or gray filter)
Appearance of platelets:
o Green filter: Dark
o Gray filter: Pink/purple
y Flat-bottomed hemacytometer (focus can be easily adjusted)
y no. 1 or ½ thin coverslip (thick coverslip retards refraction of light)
y 2 RBC pipets (classic method); 1 RBC pipet (modern method)
y Diluent: 1% Ammonium Oxalate
stable for 8 hrs
stock bottle (brown) & refrigerated
Amt needed for the day is filtered before use
Adv: lyse RBCs, but not WBCs & platelets
y Dilution = 1:100
Procedure:
a. moisten the inner wall of each RBC pipet w/ diluent
aspirate diluents into the bulb & expel excess fluid (because
platelets can adhere to glass surfaces)
b. prepare fingertip for puncture
c. wipe off the 1
st
drop of blood
d. fill end pipet w/ blood up to 1 mark, then diluent up to 101 mark to
make 1:100 dilution
e. mix blood & diluting fluid using the pipet shaker
f. discard 1
st
few drops & charge the hemacytometer using a different
pipet for each chamber
g. place the hemacytometer in a Petri Dish w/ wet cotton ball or wet
filter paper (to prevent evaporation) & allow to stand for 15mins
h. examine under HPO of phase-contrast microscope
platelets seen as sm., glistening, oval/round w/ irregular borders
i. count platelets in 10 R squares on each counting chamber
total of 20 R squares
allowable difference bet each chamber is ±10 platelets
o if >10, repeat mixing and charging to countercheck the test
o if no. of platelets in 20 R squares is <100, add more squares
until 100 platelets are recorded
o if no. of platelets in 50 R squares isu50, repeat procedure
w/ 1:10 or 1:20 dilution using the WBC pipet

Calculation: Platelet count ൌ
noǤ of platelets
noǤof R squaies
ൈ iecipiocal of uilution x ʹͷͲ
Factoi ʹͷͲ ൌ
ͳ
ͳͲ

ͳ
ʹͷ


NV: 150, 000 ʹ 400, 000/µL or 150 ʹ 400x10
9
/L



B. Rees-Ecker Method
y progenitor of Brecker & Cronkite
y ordinary L/M
y Diluent: Rees-Ecker diluent
same as Dameshek method
y Only 1 RBC pipet is needed
y Dilution = 1:200
Procedure: (only the differences from B&C)
a. draw blood up to 0.5 mark, then diluent up to 101 mark
b. place the hemacytometer in a Petri Dish w/ wet cotton ball (to
prevent evaporation) & allow to stand for 10mins
c. examine under HPO of L/M
d. count platelets in 25 R squares on each counting chamber (a total
of 50 R squares)

Calculation: Platelet count ൌ
noǤ of platelets
noǤof R squaies
ൈ iecipiocal of uilution x ʹͷͲ
oi
Platelet count ൌ noǤ of platelets ൈ ͳͲͲͲ
NV: 150, 000 ʹ 400, 000/µL or 150 ʹ 400x10
9
/L

C. Guy & Leakes Method
y modifies Rees-Ecker Method
y Diluent: same as Rees-Ecker but uses crystal violet as dye
y Ordinary L/M
y 1 RBC pipet
y Diluent to 1 mark, Blood to 0.5 mark, Diluent again to 101 mark
(moistening of pipet is no longer necessary)
y Dilution = 1:200
y Platelets are counted in 25 R squares only
y Calculation & NV: same as Brecker & Cronkite
y Correction factor: 2000

III. ELECTRONIC METHOD
1) Electrical Impedance
- Coulter Thrombocounter
- Celloscope 401
2) Light Scattering
- Autocounter

NV: same as Brecker & Cronkite
Pseudothrombocytopenia may occur when giant platelets are
present
confirm the count w/ Brecker & Cronkite Method

QUANTITATIVE PLATELET DISORDERS
A. THROMBOCYTOSIS
1. Myeloproliferative syndrome
y Polycythemia vera
y Thrombocythemia
- platelet count as ј as 1,000,000/µl
2. Post Splenectomy
3. After admin of epinephrine
4. After blood loss (including surgery)
5. Accompanying BM recovery
y After cytotoxic chemotherapy
y After treatment of Vit B12
B. THROMBOCYTOPENIA
1. due to љ platelet production
y Aplastic anemia
y Paroxysmal nocturnal hemoglobinuria
y Leukemia (acute, chronic)
y Metastatic lymphoma or carcinoma
y Folate & Vit B12 deficiency
y Cytotoxic & immunosuppressive chemotherapy
y Viral infections (dengue)
y Drug-induced (Quinine, Quinidine, Penicillin & Sulfa drugs)
y Bacterial infections (Diphtheria, Typhoid fever)
2. Due to Platelet Sequestration
y Massive splenomegaly
y Liver diseases
y Portal hypertension
y Lymphomas
3. Due to Immune Destruction of platelet
y Autoimmune thrombocytopenia
- presence of anti-platelet IgG
- formerly ITP (idiopathic thrombocytopenic purpura)
4. After Massive Blood Transfusion
- dilution of circulating platelets w/ banked blood
- takes 3-4 days of platelet count to return to normal

000 – 400. then diluent up to 101 b.000/µl b.• Flat-bottomed hemacytometer (focus can be easily adjusted) • no. Quinidine.of R squares × reciprocal of dilution x 250 Calculation: III. prepare fingertip for puncture Dilution = 1:200 Procedure: (only the differences from B&C) a. • • 1. Penicillin & Sulfa drugs) Bacterial infections (Diphtheria.5 mark. 000/µL or 150 – 400x10 /L 9 – – 1) Coulter Thrombocounter Celloscope 401 Light Scattering – Autocounter • • • A.wipe off the 1 drop of blood c. of plateletsno. place the hemacytometer in a Petri Dish w/ wet cotton ball or wet filter paper (to prevent evaporation) & allow to stand for 15mins g.discard 1 few drops & charge the hemacytometer st 101 mark to make 1:100 dilution using a different pipet for each chamber f. glistening. 1 or ½ thin coverslip (thick coverslip retards refraction of light) • 2 RBC pipets (classic method). 000 – 400.Due to Platelet Sequestration • Massive splenomegaly • Liver diseases • Portal hypertension • Lymphomas 1. THROMBOCYTOPENIA 1. of platelets After treatment of Vit B12 A. add more squares until 100 platelets are recorded • 1. due to ↓ platelet production • Aplastic anemia Paroxysmal nocturnal hemoglobinuria Leukemia (acute. Diluent again to 101 mark (moistening of pipet is no longer necessary) Dilution = 1:200 Platelets are counted in 25 R squares only Calculation & NV: same as Brecker & Cronkite Correction factor: 2000 ○ if >10. but not WBCs & platelets Dilution = 1:100 Procedure: a. then diluent up to st Post Splenectomy After admin of epinephrine After blood loss (including surgery) Accompanying BM recovery After cytotoxic chemotherapy d.Guy & Leakes Method modifies Rees-Ecker Method Diluent: same as Rees-Ecker but uses crystal violet as dye Ordinary L/M 1 RBC pipet Diluent to 1 mark. chronic) Metastatic lymphoma or carcinoma Folate & Vit B12 deficiency Cytotoxic & immunosuppressive chemotherapy Viral infections (dengue) Drug-induced (Quinine. 1 RBC pipet (modern method) • Diluent: 1% Ammonium Oxalate stable for 8 hrs stock bottle (brown) & refrigerated Amt needed for the day is filtered before use Factor 250= 110×125 NV: 150. ELECTRONIC METHOD 1) Electrical Impedance . repeat mixing and charging to countercheck the test ○ if no. of platelets in 20 R squares is <100.mix blood & diluting fluid using the pipet shaker e.moisten the inner wall of each RBC pipet w/ diluent aspirate diluents into the bulb & expel excess fluid (because platelets can adhere to glass surfaces) a.of R squares × reciprocal of dilution x 250 Calculation: or Platelet count = no. • • • • • • • • • Myeloproliferative syndrome Polycythemia vera Thrombocythemia – platelet count as ↑ as 1. 4. 2. of plateletsno.fill end pipet w/ blood up to 1 mark. THROMBOCYTOSIS • Adv: lyse RBCs.examine under HPO of phase-contrast microscope platelets seen as sm.Rees-Ecker Method progenitor of Brecker & Cronkite • ordinary L/M • Diluent: Rees-Ecker diluent same as Dameshek method Only 1 RBC pipet is needed NV: same as Brecker & Cronkite Pseudothrombocytopenia may occur when giant platelets are present confirm the count w/ Brecker & Cronkite Method QUANTITATIVE PLATELET DISORDERS A. Blood to 0.examine under HPO of L/M d. 3.Due to Immune Destruction of platelet • Autoimmune thrombocytopenia – presence of anti-platelet IgG – formerly ITP (idiopathic thrombocytopenic purpura) 1.count platelets in 10 R squares on each counting chamber total of 20 R squares allowable difference bet each chamber is ±10 platelets • • • • • • • • • A.5 mark.. of platelets in 50 R squares is ≥ 50.place the hemacytometer in a Petri Dish w/ wet cotton c.000. Typhoid fever) × 1000 irregular borders NV: 150. 000/µL or 150 – 400x109/L a.draw blood up to 0. repeat procedure w/ 1:10 or 1:20 dilution using the WBC pipet Platelet count = no. oval/round w/ Platelet count = no.After Massive Blood Transfusion – dilution of circulating platelets w/ banked blood – takes 3-4 days of platelet count to return to normal ○ if no.count platelets in 25 R squares on each counting chamber (a total of 50 R squares) ball (to prevent evaporation) & allow to stand for 10mins mark 1.