Hema 2 Lec Module 3: Laboratory Evaluation of Platelets

Quantitative Determination

of Platelets

Qualitative Determination

of Platelets

2 Types of Laboratory Evaluation of Platelets

150 - 450 x 10^9 per liter

Reference ranges vary from

Platelet count

is a fundamental component in the evaluation of a patient, especially one with signs and symptoms of bleeding or thrombosis.

Thrombocytopenia

Decreased platelet count

Thrombocytosis

Increased platelet count

Sysmex

(Automated Method)

For flow cytometry

Coulter machine

(Automated Method)

Measure impedance

Automated Methods

Most common, most rapid

Flow cytometry and electrical impedance

are the most commonly employed ways of determining platelet quantity: _____________ and _____________ are the most frequently used

Coulter principle

What principle does electrical impedance utilize

Coulter principle

A stream of cells in a suspension (blood) passes through a small aperture across which an electrical current is applied

Size

Each cell that passes through alters the electrical impedance and can thus be differentiated based on the ____. (Amplitude generated)

RBCs or WBCs

Falsely elevate

(Electrical Impedance)

When giant platelets are present in the sample, because of their large size, they can be mistaken for _______ that can falsely _____ the platelet count

Platelets

False Elevate

A severely anemic patient with many microcytic RBCs (small RBCs) in the sample can be mistaken for _____ and that can falsely _______ the platelet count

Flow Cytometry

The suspension of cells or particles is aspirated into a channel surrounded by a narrow fluid system

Focused laser beam

The cells pass through one at a time through a ______ for flow cytometry

Flow Cytometry

The light is either scattered or absorbed at certain angles when it strikes a certain cell.

Forward Angle

Assesses the size of the passing cell

Side Scatter

Assesses the internal complexity (e.g. presence in granules or certain organelles, etc.)

8-20

There are ______ platelets per oil immersion field (OIF) in a properly prepared smear, where erythrocytes barely touch or just overlap

10

at least ___ different OIFs should be carefully examined for platelet estimation

20,000

The average number in 10 fields can be multiplied by a factor of _______ to arrive at an approximation of the quantitative platelet concentration (it will not give an accurate platelet count)

F

The estimation of platelets from a blood smear DOES NOT replace actual quantitative measurement, it SHOULD be done as a cross-check of automated counts

T/F: The estimation of platelets from a blood smear does replace actual quantitative measurement, it should not be done as a cross-check of automated counts

Platelet Satellitism

Platelet Clumping

Giant platelets

Instances when an examination of the blood film is necessary even after automated counting:

Platelet Satellitism

Platelet appear to encircle the neutrophils almost entirely.

Platelet Satellitism

Can result in inaccurate automated platelet counts (especially when they start to clump up)

Platelet Satellitism

Occurs rarely when EDTA is used as anticoagulant in blood collection

Re-smear using sodium citrate-anticoagulated blood

What is the remedy for Platelet Satellitism?

Platelet Clumping

Commonly seen from blood taken from difficult extractions (e.g. the blood was in the hub of the syringe too long and they started to aggregate)

Giant Platelets

Leads to falsely low platelet counts and falsely high RBC counts in automated counters

Re-draw / re-extraction

Remedy for Platelet Clumping

Giant Platelets

Seen in certain disorders such as immune thrombocytopenic purpura and Bernard-Soulier syndrome

Brecker-Cronkite Manual Method of Platelet Counting

Principle: Whole blood is diluted with 1% ammonium oxalate, which completely hemolyzes erythrocytes. Platelets can then be counted using a phase contrast microscope.

EDTA-anticoagulated whole blood

Preferred Specimen for Brecker-Cronkite Manual Method of Platelet Counting

T

T/F: For Brecker-Cronkite Manual Method of Platelet Counting, capillary blood may be used if venous blood is not available

5 hours

24 hours

The assay must be performed within______ of the time that the blood specimen is collected or within __________, if the specimen is refrigerated.

Reagents, Supplies and Equipment for Brecker-Cronkite Manual Method of Platelet Counting

1% ammonium oxalate

RBC pipettes

Phase hemocytometer

Phase contrast microscope

1% ammonium oxalate

Must be stored refrigerated and filtered prior to use; discarded if turbid

0.5

101

Blood sample is drawn exactly up to the ____ calibration mark, thereafter oxalate solution is drawn up to the ____ calibration mark to produce a 1:200 dilution

at least 8 hours

The 1:200 dilution of the Brecker-Cronkite Manual Method of Platelet Counting is stable for _____

thrombocytopenic

WBC pipette is used to make a 1:20 dilution if the patient is severely _____

Phase hemocytometer

Has a flat bottom rather than the concave type used for other cellular elements

Phase contrast microscope

Background should appear dark while platelets, leukocytes appear illuminated

Phase contrast microscope

With HPO, platelets should appear round to oval in shape and have a faintly purple-orchid appearance.

T

T/F: Both phase hemocytometer and phase contrast microscope are not available in most laboratories

5 squares

(Calculation in Brecker-Cronkite Manual Method of Platelet Counting)

How many squares are counted on each side of the hemocytometer?

10

Total number of platelets counted on each side should be within ___ of each other (internal control).

T

(Calculation in Brecker-Cronkite Manual Method of Platelet Counting)

T/F: The total number of platelets counted on each side is added and divided by 2, this number represents the average number of platelets counted in 5 squares

Thrombocytopenia

In cases of ___________, the dilution may have to be adjusted to 1:100 in an RBC pipette or 1:20 in a WBC pipette

Average # of Platelets in 5 squares x Dilution Correction Factor x Volume Correction Factor

(the dilution and volume correction factor are derived in the same manner as for the erythrocyte count)

Platelets / uL =

Sources of error:

Specimen age

Platelet clumping: clumps will prevent / preclude any accurate counting

Debris in the diluting fluid: diluting fluid must be filtered prior to every use and any gross turbidity is already enough reason to dispose of it

Incorrect dilution: hard to use the RBC and WBC pipettes

Platelet adherence to glass

platelet function

If a platelet count is within reference ranges, but a patient has a suggestive bleeding history, an assessment of __________ should be conducted.

FOUR QUALITATIVE ASSESSMENT OF PLATELETS

Bleeding Time, Clot Retraction Time, Platelet Aggregation/Aggregometry, Antiplatelet Antibody Assays

Bleeding Time

An in vivo measurement of platelet adhesion on locally injured vascular sub-endothelium.

IVY METHOD & 40

What method reflects platelet function by timing the length of time two standardized punctures of the ventral forearm takes to stop bleeding while a blood pressure cuff inflated to about _________mmHg is in place on the upper arm.

3 to 8

thrombocytopenia can also prolong the test; as the platelet count drops below 100 x 109 /L (100,000/uL), the bleeding time increases progressively from a normal time of ________minutes to more than 30 minutes.

impaired platelet function & defect of subendothelial factors

A prolonged bleeding time in a patient with a platelet count greater than 100 x 109 /L indicates either an __________ or a _____________ that prevent adequate adhesion of platelets to the vessel wall.

-Von Willebrand Disease

-Bernard-Soulier syndrome

-presence of vascular problems

-after aspirin ingestion

Examples when the bleeding time is prolonged in qualitative platelet disorders

SIMPLATE or SURGICUTT METHOD

the procedure is similar to the Ivy method with the difference being the use of a Simplate bleeding device (shown) instead of a lancet.

Duke's method

earlobe or fingertip as the site of incision

Mielke's method

uses a disposable blade and a plastic or similar template with a preformed slit; deemed outdated and difficult to standardize or reproduce

clot retraction

When platelets are activated, the process can be visibly observed as ______________

contractile

The ________ abilities of platelets also result in the contraction formed clots

(1) the number and quality of platelets, (2) fibrinogen concentration, (3) fibrinolytic activity, and (4) packed red cell volume (Hct/PCV)

Clot retraction reflects:

hematocrit level

the degree of clot retraction is limited to the extent that fibrin contracts by _____________

Erythrocytes

The fibrin clot enmeshes the cellular elements of the blood, primarily what?

lesser

the greater the hematocrit, the _____________ the degree of clot retraction

smaller

the __________ the hematocrit, the greater the degree of clot retraction

directly

The degree of clot retraction is _________ proportional to the number of platelets

hematocrit, fibrinogen

The degree of clot retraction is inversely proportional to the ________ and the level of ________.

platelet quantity and function

Clot reaction time is useful as a gross screening test of _________ (especially platelet activation), as well as fibrinolysis

impaired

When clot dissolution/fibrinolysis is very active, the fibrin clot may be dissolved almost as quickly as it is formed, and thus clot retraction is _________.

clot retraction

The procedure involves placing a test tube of fresh, nonanticoagulated whole blood in a 37°C water bath, and examining it at 1, 2, 4, and 24 hours for ________________

1 hour & 24 hours

At 37°C, normal clot retraction should begin within______ and be complete by _________.

ADP, collagen, epinephrine, snake venom, arachidonic acid, thrombin, and ristocetin

What agents are used to aggregate platelets in vitro?

platelet-rich

The principle of the test is that ________ plasma is treated with a known aggregating agent (agonists)

cloudiness or turbidity

If aggregated, the degree of light transmittance in the form of varying levels of ____________ can be measured using a spectrophotometer

curve

Depending on the type of aggregating agent used, what help assess the effectiveness of platelet function that is generated on a graph.

Agonists that cause primary and secondary aggregations

What makes agonist have a distinct shape in their curve?

antiplatelet drug therapy

In vitro, platelet aggregation assays, aside from identifying abnormal platelet function, also monitor _____________.

turbidimetric aggregometer

In platelet aggregation the procedure is performed on a ___________ which serves as a standardized spectrophotometer

light

As aggregation proceeds, more ______ passes through the sample.

increased

The occurrence of platelet aggregation will result in _________ light transmittance

Antiplatelet antibody assaty

Not routinely done test

Immune thrombocytopenic purpura.

What antibodies against platelets may appear in the plasma of patients in certain clinical conditions?

Complement fixation methods, lysis of chromium 51-labelled platelets, assays of platelet-bound immunoglobulins, and competitive inhibition assays.

What techniques in antiplatelet antibody assays are available?

platelet refractoriness

Antiplatelet antibodies have also been implicated in ______________ or the lack of response of thrombocytopenic patients to multiple transfusions with platelet concentrates