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Review: SPECIMEN Advantages:

Megakaryoblast – earliest cell in megakaryocytic series Venous blood  easier to count platelets & RBCs
Promegakaryocyte – largest cell in BM o EDTA vacutainer tube  size & shape of platelets can be observed
Megakaryocyte – once mature, cytoplasm breaks off & becomes platelets o satisfactory: 5 hrs @ RT; 24 hrs @ 4°C
*1st 3 stages are not normally found in circulation o siliconized plain vacutainer tube (red-stoppered) or plastic syringe C. Olef’s Method
*sometimes, megakaryocytes produce platelets in the lungs  transfer immediately into tube containing EDTA o Same principle, but cumbersome procedure
Capillary blood o NV: 437, 000 – 586, 000/µL or 437 – 586x109/L
Platelets/Thrombocytes o Fingertip & heel are recommended but not the earlobe (fine hair
 fragments of cytoplasm of megakaryocyte favors adhesion of platelets) To Counter Check results:
 newly released  bigger, more active & more effective hemostatically o Free flow of blood is ideal Examine a well prepared blood smear stained w/ Wright’s stain
 if flow is poor, massage away from the puncture site 1 platelet/OIF Thrombocytopenia
Morphology:  don’t squeeze 5-20 platelets/OIF or 1 platelet/10-30 RBCs N or adequate
 Size: 2-5  if necessary do another puncture >25 platelets w/ clumps/OIF Thrombocytosis
 No nucleus o Collect blood for platelet count 1st before doing the other tests
 Anticoagulated blood: round, oval or rod-shaped o Manual CBC count: Hgb, Hct, WBC differential count II. DIRECT METHOD
 Capillary blood: irregular borders (due to filopodia/hairlike  employs dilution of blood using RBC/WBC pipet w/ the use of a
projections) I. INDIRECT METHOD hemacytometer
  of irreg is assoc w/ relative time bet. pricking & smearing  Platelets & RBCs are counted simultaneously in a blood smear
 PC/µL or PC/L is calc based on RBC ct obtained using hemacytometer A. Brecker & Cronkite Method
Distinctive Areas of a Wright-Stained Blood Smear:  results are less reliable: RBC factor is 50  reference method
1. Granulomere/Chromomere – central area filled w/ purplish granules  Phase-contrast microscopy (w/ green or gray filter)
2. Hyalomere – pale blue cytoplasm A. Dameshek Method (Wet Method)  Appearance of platelets:
o Diluent: Rees-Ecker diluent o Green filter: Dark
Lifespan: 8-11 days in circulating blood  3.8 g Na Citrate (prevents clumping of platelets) o Gray filter: Pink/purple
24 hrs outside the body (extracted blood)  0.2 ml 40% formaldehyde (preservative)  Flat-bottomed hemacytometer (focus can be easily adjusted)
Stored blood in blood banks is deficient of viable platelets  0.1 g brilliant cresyl blue (dye)  no. 1 or ½ thin coverslip (thick coverslip retards refraction of light)
*Platelet concentrate is prepared fr fresh blood  100 ml dH2O  2 RBC pipets (classic method); 1 RBC pipet (modern method)
Functions:  Filtered before use to remove any debris present  Diluent: 1% Ammonium Oxalate
1. Maintaining integrity of BVs – “leak-free”; fill gaps  Doesn’t lyse RBC & WBC (platelet is 1/5 – 1/10 the size of RBC)  stable for 8 hrs
2. Hemostasis  Dilution is stable only for 30mins  stock bottle (brown) & refrigerated
a. adhere to injured BVs
 Amt needed for the day is filtered before use
b. aggregate @ site of injury forming 1 platelet plug Special steps in procedure:  Adv: lyse RBCs, but not WBCs & platelets
c. release biochemicals important in hemostasis 1. After finger puncture, wipe 1st drop of blood then place a large drop  Dilution = 1:100
 release serotonin & thromboxane A2 (for vasoconstriction) & of diluent over the punctured site - to avoid exposure to air & Procedure:
ADP (for clumping) disintegration of platelets a. moisten the inner wall of each RBC pipet w/ diluent
d. Source of Platelet factor 3 – for prodxn of throboplastin Ratio = 1:5 (blood to diluent)  aspirate diluents into the bulb & expel excess fluid (because
3. Initiate Clot Retraction – mediated by Thrombosthenin (contractile 2. Transfer a portion on a cover glass & invert on a slide platelets can adhere to glass surfaces)
CHON produced by platelets); process by w/c serum is expressed fr the 3. Allow to stand for 15mins b. prepare fingertip for puncture
clot 4. examine under OIO (diaphragm partly closed) & count platelets & RBCs c. wipe off the 1st drop of blood
until 1000 RBCs are recorded; platelets are lilac colored, tiny, d. fill end pipet w/ blood up to 1 mark, then diluent up to 101 mark to
PLATELET COUNT glistening make 1:100 dilution
*More difficult to do compared to RBC & WBC cuz: e. mix blood & diluting fluid using the pipet shaker
1. very small 𝑅𝐵𝐶/µ𝐿
Calculation: Platelet count/µL = # of platelets x f. discard 1st few drops & charge the hemacytometer using a different
2. disintegrate easily when expose to air 1000
NV: 500, 000 – 900, 000/µL or 500 – 900x109/L pipet for each chamber
3. tendency to stick/clump together g. place the hemacytometer in a Petri Dish w/ wet cotton ball or wet
4. adhere on to glass surfaces/any foreign body filter paper (to prevent evaporation) & allow to stand for 15mins
Wet mount - RBCs tend to concentrate @ the edges of coverslip, so
5. difficult to differentiate fr dust, dirt & bacteria h. examine under HPO of phase-contrast microscope
counting is done in central area (falsely ↑ ratio of platelets
6. not well distributed in circulation
to RBC)  platelets seen as sm., glistening, oval/round w/ irregular borders
 Modified Dameshek i. count platelets in 10 R squares on each counting chamber
Thrombocytosis – ↑ in no. of platelets above normal
 uses a siliconized medicine dropper in diluting blood  total of 20 R squares
Thrombocytopenia – ↓ in no. of platelets below normal
 allowable difference bet each chamber is ±10 platelets
N condition: represents only 2/3 of total platelet mass; 1/3 found in spleen
B. Fonio Method (Dry Method) o if >10, repeat mixing and charging to countercheck the test
o diluent: 14% Magnesium Sulfate (MgSO4) o if no. of platelets in 20 R squares is <100, add more squares
PHYSIOLOGIC VARIATION OF PLATELET COUNT
 Doesn’t lyse RBCs until 100 platelets are recorded
Spleen
Special steps in procedure: o if no. of platelets in 50 R squares is50, repeat procedure
 platelet reservoir in man
Ratio = 1:3 (blood to diluent) w/ 1:10 or 1:20 dilution using the WBC pipet
 release of splenic pool can be caused by:
1. transfer mixture on 1 end of a clean slide
o admin of epinephrine no. of platelets
2. make a wedge smear w/ a spreader slide Calculation: Platelet count = × reciprocal of dilution x 250
o variety of stresses: excitement, hypoxia, strenuous exercise, ↑ no.of R squares
3. air dry the smear 1 1
altitude
4. stain w/ Wright’s stain Factor 250 = ×
 Neonates (1st 4 days): slightly ↓ than adults 10 25
5. examine under OIO & count platelets & RBCs until 1000 RBCs are
 Menstruating:  PC shortly before & during 1st day
recorded (do the counting @ 1/5 – 1/3 part from end of smear) NV: 150, 000 – 400, 000/µL or 150 – 400x109/L
 Arterial blood: slightly ↑ platelet count than venous blood
NV: 250, 000 – 500, 000/µL or 250 – 500x109/L
 Venous blood: slightly ↑ platelet count than peripheral blood
 N absent in lymph/other body fluids
B. Rees-Ecker Method  Drug-induced (Quinine, Quinidine, Penicillin & Sulfa drugs)
 progenitor of Brecker & Cronkite  Bacterial infections (Diphtheria, Typhoid fever)
 ordinary L/M 2. Due to Platelet Sequestration
 Diluent: Rees-Ecker diluent  Massive splenomegaly
 same as Dameshek method  Liver diseases
 Only 1 RBC pipet is needed  Portal hypertension
 Dilution = 1:200  Lymphomas
Procedure: (only the differences from B&C) 3. Due to Immune Destruction of platelet
a. draw blood up to 0.5 mark, then diluent up to 101 mark  Autoimmune thrombocytopenia
b. place the hemacytometer in a Petri Dish w/ wet cotton ball (to - presence of anti-platelet IgG
prevent evaporation) & allow to stand for 10mins - formerly ITP (idiopathic thrombocytopenic purpura)
c. examine under HPO of L/M 4. After Massive Blood Transfusion
d. count platelets in 25 R squares on each counting chamber (a total - dilution of circulating platelets w/ banked blood
of 50 R squares) - takes 3-4 days of platelet count to return to normal

no. of platelets
Calculation: Platelet count = × reciprocal of dilution x 250
no.of R squares
or
Platelet count = no. of platelets × 1000
NV: 150, 000 – 400, 000/µL or 150 – 400x109/L

C. 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, Blood to 0.5 mark, 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

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
 Polycythemia vera
 Thrombocythemia
- platelet count as ↑ as 1,000,000/µl
2. Post Splenectomy
3. After admin of epinephrine
4. After blood loss (including surgery)
5. Accompanying BM recovery
 After cytotoxic chemotherapy
 After treatment of Vit B12
B. THROMBOCYTOPENIA
1. due to ↓ platelet production
 Aplastic anemia
 Paroxysmal nocturnal hemoglobinuria
 Leukemia (acute, chronic)
 Metastatic lymphoma or carcinoma
 Folate & Vit B12 deficiency
 Cytotoxic & immunosuppressive chemotherapy
 Viral infections (dengue)