Common Tests for Primary Hemostasis

Why are platelets difficult to count accurately?

adhere to foreign surfaces, disintegrate easily, resemble debris, and tend to clump

What is the general normal platelet count range?

150 to 450 × 10⁹/L

How does platelet count change after age 65 in men?

decreases to 122–350 × 10⁹/L

How does platelet count change after age 65 in women?

decreases to 140–379 × 10⁹/L

What platelet count is associated with a high risk of clinically important bleeding?

Less than 100,000/μL (100 × 10⁹/L)

What is the reference method for manual platelet counting?

Phase-Contrast Microscope Method by Brecher and Cronkite

What diluent is used in the Phase-Contrast Method, and why?

1% ammonium oxalate

What is the dilution ratio of EDTA whole blood used in the Phase-Contrast Method?

1:100

In which part of the hemocytometer are platelets counted in this method?

25 small squares of the central large square (1 mm²)

How is the accuracy of the manual thrombocyte count verified?

platelet count estimate on a Wright-stained peripheral blood smear from the same specimen

Can a light microscope be used as an alternative?

Yes, but visualizing platelets may be more difficult.

In performing the count, how much blood should be added in ammonium oxalate?

20 uL of blood into 1,980 uL of 1% ammonium oxalate

What type of hemocytometer is used for phase-contrast platelet counts?

special thin, flat-bottomed hemocytometer

How long should the hemocytometer sit in a moist chamber before counting?

15 mins

At what magnification are platelets counted in this method?

400x total magnification (using a 40x objective lens).

What is the diameter of platelets under phase-contrast microscopy?

2 to 4 um

What is the shape of platelets under phase-contrast microscopy?

round or oval

What is the color of platelets under phase-contrast microscopy?

light purple sheen

What may be seen in the background while viewing platelets?

ghost erythrocytes

What is the acceptable difference between counts on both sides of the chamber?

< 10%

What should you do if the difference between counts on each side is greater than 10%?

repeat

What is the formula for percentage difference?

(V1-V2)/(V1 + V2/2) x 100

What magnification should be used to examine platelets for estimation?

OIO (100x)

How many platelets per oil immersion field (OIF) are expected in a normal blood smear?

approx 7 to 21 platelets per 100x field

How many oil immersion fields should be counted to estimate platelet count?

10

What is the formula to estimate platelet count per microliter (μL)?

estimated platelet count/μL = average number of platelets per OIF × 20,000

What type of blood smear is preferred for platelet estimation?

normal (wedge) blood smear

What formula should you use in cases of significant anemia or erythrocytosis?

average no. of platelets/field x total RBC count / 200 RBCs per field

What is the average number of RBCs per oil immersion field in the optimal assessment area?

200

What is the platelet estimate range for a marked decrease?

0 to 49,000/uL

What is the platelet estimate range for a moderate decrease?

50,000 to 99,000/uL

What is the platelet estimate range for a slight decrease?

100,000 to 149,000/uL

What is the platelet estimate range for a low normal?

150,000 to 199,000/μL

What is the platelet estimate range for normal?

200,000 to 400,000/μL

What is the platelet estimate range for slight increase?

401,000 to 599,000/μL

What is the platelet estimate range for a moderate increase?

600,000 to 800,000/uL

What is the platelet estimate range for a marked increase?

above 800,000/uL

What is the required platelet count range for platelet-rich plasma (PRP) used in light-transmittance aggregometry?

200,000 to 300,000/uL

What type of anticoagulant is used to prepare PRP for platelet aggregometry?

Sodium citrate

What is the centrifugation setting used to prepare PRP from whole blood?

50 × g for 30 minutes

Why must the stopper remain in place during centrifugation of the blood sample for PRP preparation?

To maintain the pH

At what temperature should PRP be stored until the test begins?

18 to 24C (ambient temperature)

When should PRP-based aggregometry be initiated after centrifugation?

No less than 30 minutes

Within how many hours of blood collection must be the PRP-based LTA test be completed?

4 hrs

How much whole blood is needed to produce adequate PRP for platelet aggregometry?

9 to 12 mL

At what platelet count is light-transmittance aggregometry considered UNRELIABLE?

< 100,000/μL

What are some examples of aggregating reagents?

ADP, collagen, epinephrine (adrenaline), ristocetin

What are some disorders with normal response with A.C.E.?

Bernard Soulier Syndrome
Von Willebrand Disease

What are some disorders with abnormal response with A.C.E.?

Glanzmann’s thrombasthenia

What are some disorders with normal response with ristocetin?

Glanzmann’s thrombasthenia

What are some disorders with abnormal response with ristocetin?

Bernard Soulier’s Syndrome
Von Willebrand Disease

What two platelet functions does lumiaggregometry measure?

platelet aggregation and ATP release (from dense granules)

What type of sample is used in platelet lumiaggregometry?

whole blood diluted with saline

In lumiaggregometry, what compound is released from platelet dense granules?

ATP

What reagent is used to detect ATP in lumiaggregometry?

firefly-derived luciferin-luciferase reagent

What type of signal is produced during ATP detection in lumiaggregometry?

cold chemiluminescence (proportional to ATP concentration)