1-PLATELET DETERMINATION Flashcards by Dave U.

Normal Platelets shape

nonnucleated blood cells | biconvex (resting) | pseudopod (active)

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average diameter of platelets

2.5µm(2-4µm

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MPV

8-10 fL (5-7 fL in citrated blood)

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Normal platelet count

150-450 x 10
9/L

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How much is stored in spleen and how much is circulating

2/3 in circulation | 1/3 sequestered in spleen

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platelets tend to “round up” in this anti coagulant

EDTA

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Peripheral blood film platelet appearance

circular to irregular, lavender, anuclear, granular

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Appear in compensation for
thrombocytopenia

Reticulated/ Stress Platelets

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Reticulated/ Stress Platelets dimensions

larger: 6 µm | MPV: 12-14 f

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 largest cells in the BM

Megakaryocytes

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polypoid cell

Megakaryocytes

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Number of megakaryocytes in BM in regards to total BM cells

0.5%

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Normal BM aspirate smear

: 2-4
megakaryocyte/LPF

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maturation time of megakaryocytes in the BM

4-5 days

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Life span of platelets in the peripheral circulation

9-12 days/ 8-10 days

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number of platelets in 1 liter (L) or 1 microliter (µL) of whole blood

PLATELET COUNT

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REASONS WHY PLATELETS ARE HARD TO COUNT

 Platelets adhere to foreign surfaces.
 Platelets easily disintegrate.
 They are hard to differentiate from debris.
 Platelets are unevenly distributed in the blood because they tend to clump.

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SPECIMEN for manual platelet count

Venous blood anticoagulated with EDTA

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METHODS

Indirect and Direct method

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platelets are counted in relation to 1,000 RBCs in the bloodsmear

Indirect Method

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whole blood is diluted with platelet diluting fluid in an RBC pipette and counted in a hemocytometer

. Direct Method

Direct Methods for manual platelet count

Light Microscopy Method
Phase Microscopy Method
AUTOMATED PLATELET COUNT

Light Microscopy Methods

Rees and Ecker’s
Guy and Leake’s

Phase Microscopy Methods

Brecher-Cronkite Method

Unopette, Tocantin’s Method, Nygard’s Method, Walker andSweeney’sMethod

Rees and Ecker’s diluting fluid composition

 Sodium citrate  prevents platelet aggregation  Formaldehyde  preservative  Brilliant cresyl blue  stain/dye  Distilled water

Appearance of platelets in R and E

small | round/oval | highly refractile | bluish

Guy and Leake’s  Diluting Fluid (isotonic) composition:

 Sodium oxalate  prevents platelet aggregation  Formaldehyde  preservative  Crystal violet  stain/dye  Distilled water

Appearance of platelets: in G and L

highly refractile | lilac

 Brecher-Cronkite Method Diluting Fluid used:

1% ammoniumoxalate (hypotonic)

lyses RBCs andallowsplatelets toformpseudopods

1%ammoniumoxalate

Appearance of platelets in B and C method

small (2-4 μm) | round/oval | displays a light purple sheen

Dilutions

 1:100 (most common)  1:20  less than 50 platelets counted on each side  1:200  greater than 500 platelets counted on each side

 Inadequate mixing or poor collection consequence

platelet clumping  false ↓ platelet count

Short draw: excess anticoagulant consequence

 swelling of platelets  fragmentation false ↑ platelet count

 occurs in EDTA anticoagulated samples  adherence of platelets around neutrophils, producing a ring or satellite effect  causes pseudothrombocytopenia

Platelet satellitosis

Platelet satellitosis solution

: use sodium citrate as anticoagulant; multiply the obtained platelet count by 1.1 for accuracy

AUTOMATED PLATELET COUNT methods

Beckman Coulter Unicel DxH 800 Sysmex XN Series Abbott CELL-DYN Sapphire Siemens ADVIA2120i

Impedence volume and conductivity and five-angle light scatter movement

Beckman Coulter Unicel DxH 800

Impedance; light scatter; fluorescent staining, forward light scatter, and side fluorescent light detection

Sysmex XN Series

Dual-angle light scatter analysis; impedance count for verification; optional CD61 monoclonal antibody count

Abbott CELL-DYN Sapphire

Low-angleandhigh-anglelight scatter; refractive index

Siemens ADVIA2120i

What increases as EDTA blood is standing

MPV every 1 and 3 hours increases further with time

Presence of microcytes or schistocytes consequence on automated

↑ platelet count

Platelet clumps consequence on automated

↓ platelet count

Leukemia consequence on automated

↑ platelet count

Old specimen consequence on automated

↓ platelet count

DISPOSABLE BLOOD CELL COUNT DILUTION SYSTEMS

LEUKOCHEK

It is an example of capillary pipette and diluent reservoir system which is commercially availableforWBC and platelet counts

LEUKOCHEK

LEUKOCHEK specimen

 EDTA-anticoagulated whole blood or blood obtained from capillary puncture

REAGENT AND MATERIALS in leukochek

 Plastic reservoir (containing 1.98 mL of 1% buffered ammonium oxalate)  Capillary pipette (calibrated to accept 20 μL of blood)  Hemacytometer and coverslip

In instances of significant anemia or erythrocytosis, use the following formula for the platelet estimate:

Average no. of platelets × total RBC count/ 200 RBCs

If platelet count is normal, there must be:

 About 1 platelet per 10-30 red blood cells  7-21 platelets per oil immersion field