problem in terms of number.

problem with the platelet itself.

The focus is on the specific number/range of the platelet count.

Abnormally increased or decreased Count determines associated conditions

The problem is the platelet itself, specifically, the function.

determines the platelets’ adhesion, aggregation, and release reaction.

Tests in Qualitative Platelet Count include:

Quantitative Test:

PLATELET COUNT

Platelets are hard to count when it is already extracted from the body, because:

Platelets have a high affinity for foreign substances and usually adhere to glass.

Platelets easily disintegrate _____________________

Platelets are hard to differentiate from debris, ______________________

Platelets are unevenly distributed in the blood ___________________________

If there are clots, the value will be ________________

Automated

Conventional Unit (cells/uL)

Manual

Conventional Unit (cells/uL)

Manual

SI Unit (x 10^9/L)

Automated

SI Unit (x 10^9/L)

General Normal Value of platelet count in manual method is:

General Normal Value of platelet count in automated method is:

Significant bleeding usually does not occur until the platelet count is less than_________

METHODS FOR PLATELET COUNT

Platelets are counted in relation to 1,000 RBCs in the blood smear.

𝑅𝐵𝐶 𝐶𝑜𝑢𝑛𝑡 (𝑚𝑚 3) / 1000 × 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝐶𝑜𝑢𝑛𝑡𝑒𝑑

The number of platelets counted was 85, and the patient’s RBC Count is 500,000 mm3. What is the platelet count?

This method directly counts using the platelet and does not consider other cells.

Principle: Whole blood is diluted with platelet diluting fluid in an RBC pipette or WBC pipette and

counted in a hemocytometer.

Two methods under the Direct Method:

Two methods under the Light Microscopy Methods (Brightfield):

Diluting Fluid: Reese and Ecker

Composed of: Brilliant Cresyl Blue, Sodium Citrate, Distilled Water, and Formalin

Count the platelets in the 4 corner large squares (as in WBC Count).

Get platelet count using the WBC Count formula.

𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝐶𝑜𝑢𝑛𝑡 = 𝑃𝑙𝑎𝑡𝑒𝑙𝑒𝑡𝑠 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝐷𝑒𝑝𝑡ℎ 𝐹𝑎𝑐𝑡𝑜𝑟

𝐴𝑟𝑒𝑎 𝐹𝑎𝑐𝑡𝑜𝑟

Composed of: Crystal Violet, Sodium Citrate, Distilled Water, and Formalin (40%)

Count the platelets in the 25 intermediate squares in the central large square for RBC Count.

Get platelet count using the RBC Count formula.

𝑃𝑙𝑡 𝐶𝑜𝑢𝑛𝑡 = 𝑃𝑙𝑡𝑠. 𝐶𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝑒𝑝𝑡ℎ 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 × 𝐴𝑟𝑒𝑎 𝐹𝑎𝑐𝑡𝑜𝑟

Supravital Stain

Brilliant Cresyl Blue

Supravital Stain

Crystal Violet

Methods under the Phase Contrast Microscopy Methods:

Diluting Fluid: 1% Ammonium Oxalate

AUTOMATED METHOD

Coulter S-Plus

AUTOMATED METHOD

Ortho ELT 81ds

AUTOMATED METHOD

Technicon Auto-Counter

AUTOMATED METHOD

Ultra-Flo 100

AUTOMATED METHOD

Baker Series 810

Estimation only and does not count directly.

Used to determine the approximate number of platelets per field, examine the thin area of the slide

(where only a few RBCs slightly overlap) using the oil immersion objective

Normal Value: 3-10 platelets/100 RBC or 5-20 platelets/200 RBC.

𝐸𝑠𝑡. 𝑃𝑙𝑡 𝐶𝑜𝑢𝑛𝑡/𝑢𝐿 = 𝐴𝑣𝑒 # 𝑜𝑓 𝑃𝑙𝑡𝑠/𝑂𝐼𝐹 × 20, 000

3-10 Platelets

5-20 Platelets

A normal wedge blood smear should demonstrate approximately

Platelet Count Estimation Interpretation:

0 to 49,000/uL

Platelet Count Estimation Interpretation:

50,000 to 99,000/uL

Platelet Count Estimation Interpretation:

100,000 to 149,000/uL

Platelet Count Estimation Interpretation:

150,000 to 199,000/uL

Platelet Count Estimation Interpretation:

200,000 to 400,000/uL

Platelet Count Estimation Interpretation:

401,000 to 599,000/uL

Platelet Count Estimation Interpretation:

600,000 to 800,000/uL

Platelet Count Estimation Interpretation:

Above 800,000/uL

THROMBOCYTOPENIA

Decreased platelet count due to:

Abnormally high platelet count, typically more than 450,000/mL.

Under this are Myeloproliferative Disorders, which include: Polycythemia Vera, Chronic Myelogenous Leukemia, Myelofibrosis with Myeloid Metaplasia (Primary Myelofibrosis) and Essential Thrombocytopenia

Occurs if the platelet counts are between 450,000/mL and 800,000 mL with no change in

platelet function.

There are no changes in platelet function, and it only indicates a high amount of platelets.

Causes of ___________________: Reactive Thrombocytosis Associated with Hemorrhage or Surgery

Causes of ___________________: Postsplenectomy Thrombocytosis

Causes of ___________________: Thrombocytosis Associated with Iron Deficiency Anemia

Causes of ___________________: Thrombocytosis Associated with Inflammation and Disease

Causes of ___________________: Exercise-Induced Thrombocytopenia

Causes of ___________________: Rebound Thrombocytosis

SECONDARY THROMBOCYTOSIS (ACTIVE)

Reactive Thrombocytosis after Hemorrhage or Surgery ______________________

SECONDARY THROMBOCYTOSIS (ACTIVE)

The spleen is the sequestration site of the platelets, so if the ___________ there would be no site for the platelets to be stored.

SECONDARY THROMBOCYTOSIS (ACTIVE)

Secondary Thrombocytopenia is mainly caused by __________________ that cause an

increase in the platelets.

Qualitative Tests

PLATELET FUNCTION TESTS

Most common signs and symptoms that suggest an acquired or a congenital disorder of platelet

function are:

Qualitative Tests

PLATELET FUNCTION TESTS

a vessel wound with exposed endothelial collagen or to a glass bead requires the presence of Plasma Von Willebrand Factor (VIII:vWF).

Qualitative Tests

PLATELET ADHESION

Endothelial Collagen

Qualitative Tests

PLATELET ADHESION

Glass Bead

Qualitative Tests

Tests to Measure Platelet Adhesion:

Qualitative Tests

Principle: Time that it takes for a standardized wound to stop bleeding.

Qualitative Tests

not affected by the coagulation mechanism

Qualitative Tests

Any problem with secondary hemostasis (clotting factors) does not affect the _________.

Qualitative Tests

Only affected by the primary hemostasis (platelets, vWF, collagen, and calcium).

Affects BT

Does not Affect BT

BLEEDING TIME

This is a comprehensive test of platelet action in vivo and is sensitive to abnormalities of:

Specific Medication that affects Bleeding Time

avoid 7 days before testing.

Specific Medication that affects Bleeding Time

should be discontinued 24 hours before testing.

Bleeding Time Method:

Bleeding Time

Finger is punctured (3mm deep) using a sterile lancet.

Bleeding Time

The earlobe is also a possible site of puncture

Bleeding Time

Blot a drop of blood with filter paper every 30 seconds; the filter paper must not touch the

wound/skin.

Bleeding Time

Do not milk the finger to force the bleeding.

Bleeding Time

Normal Value: 0-6 Minutes

Bleeding Time

Uses sphygmomanometer (inflated at 40 mmHg) at the patient’s upper forearm (above elbow).

Bleeding Time

Make two skin incisions (2 mm deep and 2 mm long), avoiding any subcutaneous veins.

Bleeding Time

Normal Value: 2-9 Minutes

Bleeding Time

A modification by C.H. Mielke Jr. and colleagues of the original Ivy’s bleeding time test.

Bleeding Time

Uses a template containing a standardized slit in the place of disposable lancets (has a standardized

depth).

Bleeding Time

Templates have been replaced by several commercially made devices, such as Simplate and

Surgicutt.

Bleeding Time

General Reference Range: 2-9 Minutes

Principle: As an anticoagulated blood is passed through a glass bead column, normal platelets with

normal vWF will adhere and aggregate to the beads

Then, after getting the measurement, transfer some amount of blood to the “U” shaped Glass Bead

Column, and then the platelets will adhere to the column.

Normal Value: 75-95% Platelet Retention

Clinical Significance of Decreased Platelet Retention:

Clinical Significance of Decreased Platelet Retention:

Clinical Significance of Decreased Platelet Retention:

Clinical Significance of Increased Platelet Retention:

Clinical Significance of Increased Platelet Retention:

Involves serial platelet counts on blood excluding from a forearm incision.

Normally, platelet counts decrease because of platelet adhesion to the wound.

These counts are compared with a venous blood platelet count as a control to calculate percent

platelet adhesion.

Normal Value: 15% to 45%

PLATELET FACTOR ASSAY METHOD:

Measures the amount of Platelet Factor 3.

Principle: Platelets facilitate the ability of the plasma to clot by providing a surface to which

certain clotting factors bind, thereby enhancing their reaction rates.

When platelets are incubated with activators (Kaolin and Epinephrine), platelets are stimulated to provide PF3 activity.

Thus, the clotting time of platelet-rich plasma (PRP) incubated with kaolin and epinephrine is

considerably shorter than that of PFP

This test simply compares the clotting time of the patient’s PRP with that obtained for a group of

normal individuals

PLATELET FACTOR 3 ASSAY

Shorter Clotting Time

PLATELET FACTOR 3 ASSAY

Longer Clotting Time

are heparin-binding proteins found in platelets’ alpha-granules.

As such, their levels in vivo are an indicator of the presence of ongoing platelet activation in a variety

of disease states,

Procedure: Radioimmunoassays (RIA KITS)

PLATELET FACTOR 4 AND BETA-THROMBOGLOBULIN ASSAY

As such, their levels in vivo are an indicator of the presence of ongoing platelet activation in a variety

of disease states, such as:

assist in the diagnosis of hereditary and acquired platelet disorders.

Principle: Platelet Aggregometry

Utilizes Spectrophotometry.

A platelet aggregometer measures and records a change in light transmission.

PLATELET AGGREGATION

Aggregating agents added to a stirred susension of PRP________________

PLATELET AGGREGATION

As a result, PRP __________________________________________________

Special Considerations/Patient Preparation in Platelet Aggregation

Special Considerations/Patient Preparation in Platelet Aggregation

Aggregating Agents: in Platelet Aggregation

Aggregating Agents: in Platelet Aggregation

Three Concentrations:

1 Low

2 High

3 Optimum

In _____________ the platelets aggregate and are followed by disintegration.

ADP

In _____________ the platelets aggregate and are followed by disintegration.

ADP

The transmittance is immediately lost, denoting a normal platelet

ADP

In _______________, only one wave of aggregation is observed.

ADP

In ___________, two waves are observed, collectively referred to as the

Biphasic/Double Wave Curve.

ADP

Optimum Concentration

In the _________, the platelet is changing its shape and preparing to aggregate.

ADP

Optimum Concentration

In the _________, the platelet releases its content and then will aggregate continuously.

In Low Concentration, the platelets aggregate and are followed by disintegration.

In High Concentration, only one wave of aggregation is observed.

In Optimum Concentration, two waves are observed, collectively referred to as the

Biphasic/Double Wave Curve.

Weak aggregating agent.

It will disintegrate once it reaches 30% transmittance.

Long lag phase before aggregating (low incubation time).

Also show a biphasic curve.

Clear viscous oil at room temperature, and is not readily soluble in concentrated aqueous solvents.

Stable for 2-3 weeks if kept at -20ºC in a sealed vial under nitrogen atmosphere.

Entered only with Hamilton micropipette

Stable at 4ºC for 2 weeks.

If the ristocetin cofactor is combined with vWF, platelets will aggregate.

Adhesion

Aggregation

RISTOCETIN

If there is no wave of transmittance, the platelets are either:

RISTOCETIN

If there is no transmittance, ________________________

RISTOCETIN

After adding the normal plasma, check the viscosity of the aggregation.

■ If waves appear, the patient is ______________

RISTOCETIN

After adding the normal plasma, check the viscosity of the aggregation.

■ If no waves appear, there is a___________________

RISTOCETIN

PRP + NP = Wave

RISTOCETIN

PRP + NP = No Wave

Principle: Agglutination of fixed platelets in response to ristocetin depends on the vWF present

in the plasma.

Normal Value: 45% - 140%

Principle: Within one hour after the whole blood is collected, allow the blood to clot in a clean glass

tube at 37ºC.

measured based on the amount of expressed serum.

Measures the entire function of platelets from adhesion to retraction.

Normal clot retraction requires:

Clot retraction methods used:

Dimpling Phenomenon occurs when serum appears on the side of the blood drop on top of the castor oil.

Normal Value: 15 - 45 Minutes

HIRSBOECK/CASTOR OIL

Abnormal characteristics of extruded serum:

seen in leukemia, after meals, and diabetes.

HIRSBOECK/CASTOR OIL

Abnormal characteristics of extruded serum:

seen in jaundice because of liver conditions.

HIRSBOECK/CASTOR OIL

Abnormal characteristics of extruded serum:

seen in multiple myeloma.

The blood has retracted if the serum is already seen on the side of the tubes and the blood

cells are attached to the glass rod.

After removing the rod, a certain amount of cells will be left out (lower than 5 mL).

%𝐶𝑙𝑜𝑡 𝑅𝑒𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 = 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝑒𝑟𝑢𝑚 𝐿𝑒𝑓𝑡 𝑖𝑛 𝑇𝑢𝑏𝑒/ 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐵𝑙𝑜𝑜𝑑 𝑈𝑠𝑒𝑑 × 100

The amount of blood used is constantly 5 mL.

Normal Value: 44 - 67%

MACFARLANE METHOD

What is the % clot retraction if the blood left after removing the retracted blood is 2 mL?

Interaction of platelets with fibrinogen and fibrin must also be normal for clot retraction to occur.

MACFARLANE METHOD

Abnormal clot retraction is seen in:

serves as a supravital stain to visualize the platelets.

serves as a supravital stain to visualize the platelets.

serves as an anticoagulant to prevent clumping due to adherence to glass.

is added to suspend the other reagents.

used to preserve the diluting fluid.