Hematology 2 Laboratory Must Knows

Vincent Lawrence D. Marcos

S.Y. 2022-2023 Cagayan State University

Procedures Prior Platelet Function Test

Two procedures performed prior to platelet function test:
● Platelet count (Neubauer Counting Chamber)
● Platelet estimate (Peripheral Blood Film)

Platelet Count
● Number of platelets in 1 Liter (L) or 1 microliter (μL) of whole blood.
● Delta Checking
○ Checking previous results to have a basis in reporting current results..
● Automated method results of low pt count are counterchecked using manual method.

Reasons why Platelets are hard to count:

● Platelets adhere to foreign substances. (Satellitosis for example: solution: use
bluetop).
● Platelets easily disintegrate.
● Platelets are hard to disintegrate with debris.
● Platelets are unevenly distributed because they tend to clump.

Normal Platelet Count: 150-400 x 109/L

Principle of Platelet count:

● Whole blood is diluted with platelet diluting fluid in an RBC pipette and counted using
a hemocytometer.
○ Neubauer
■ Platelets are counted in the 25 smallest squares.
■ 10 squares are counted if there is an existing idea of platelet count.
■ 25 squares if there is no existing idea.
● Specimen: Whole Blood (EDTA)
○ Sodium Citrate anticoagulant in cases of satellitosis.
● Methods
○ Indirect
■ Platelets are counted in relation with 1000 RBCs.
■ Not reliable.
○ Direct method
■ Platelet is counted using diluting fluid and hemocytometer.
■ RBC thoma pipette is used.

Light Microscopy Method

● Rees and Eckers
○ Diluting fluid composition: Brilliant Cresyl blue, Sodium Citrate, Formalin, and
Distilled water.
○ Platelets appear light blue.
● Guy and Leake’s
○ Diluting fluid composition: Crystal Violet, Sodium Citrate, Formalin, and
Distilled water.

1
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

Phase Contrast Microscopy Method

● Brecher-Cronkite
○ Dilution fluid: 1% ammonium oxalate
■ Capable of lysing RBCs
● Unopette
○ Dilution fluid: 1% Ammonium oxalate
○ Replaced by LeukoChek
● Tocantin’s method
● Nygard’s method
● Walker and Sweeney’s method

Dilution Factor
● 1:100 is most common
● <50 platelet count on both sides
○ Change dilution into 1:20
● >500 platelet count on both sides
○ Change dilution into 1:200
● Diluted blood samples are stable for 8 hours.
*Neubauer counting chambers should be read within 30 minutes because it dries easily.

Automated Method: Use of machines to count platelets.

Procedure:

1. Collect Sample
2. Using a thoma pipette, aspirate 5 microliter of whole blood.
3. Aspirate diluting fluid up to the 101 mark of the thoma pipette.
4. Mix the diluting fluid and whole blood by shaking the pipette until it becomes a
homogeneous mixture.
5. Blot the first 2 to 3 drops of the mixture so that the mixture from the bulb will be
charged. Charge the mixture into the Neubauer Counting Chamber.
6. Incubate the Counting Chamber inside a moist chamber for 3-10 minutes to allow the
settling of the platelets, to avoid the movement of the platelets due to the flow of the
liquid.
7. Count the platelet under a Light microscope.

Sources of Error:
● Inadequate mixing or poor collection leads to platelet clumping resulting in
pseudothrombocytopenia.
● Skin puncture specimen is less desirable because blood in skin puncture clots easily.
● Platelet Satellitosis
○ Adhesion of Platelets on neutrophils causing pseudothrombocytopenia.
○ The solution is to recollect samples in a blue top tube or sodium citrate tube.
○ The final platelet count is multiplied by 1.1 for accuracy by correcting the
anticoagulant whole blood ratio.

2
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

Platelet Estimate (Peripheral Blood Film Examination)

● Count the number of platelets in 10 consecutive fields under the microscope and
calculate the average number of platelets per field.
● Average number of platelets per field is multiplied by 20,000.
● Example: 278 platelets in 10 fields
○ (278 ÷ 10) × 20,000 = 556,000 cells/mm3 or cells/μL (in conventional units)
○ Or 556 × 10*9/L (in SI units)
● Platelet estimate is compared with the result yielded by the machine.
● In instances of significant anemia or erythrocytosis, average no. of platelets × total
RBC count/200 RBCs
○ If platelet is normal, there must be:
■ About 1 platelet per 10-30 red blood cells.
■ 7-20 platelets per oil immersion field.

Platelet Function Tests

Bleeding Time (BT)
● Screening test for primary hemostasis.
○ For detecting disorders for platelet function and von Willebrand’s disease.
○ Evaluates the vascular system.
○ It is the measure of the interaction of platelets and the vascular system,
particularly adhesion; specifically in the capillaries.
○ In vivo measurement platelet adhesion and aggregation on locally injured
vascular subendothelium.
○ Directly affected by the platelet count and ability to form a plug.

Bleeding Time test methods:

1. Duke Method
○ Earlobe or fingertip: puncture (3mm deep) using a sterile lancet.
○ A pricker is usually used to control the depth of puncture/incision.
○ Blood is blotted every 30 seconds using a filter paper and the filter paper
should not touch the surface.
○ Normal Values: Ranges from 2-4 minutes
2. Ivy Method
○ It uses a device that gives a standardised length and depth of incision.
○ Principle: A blood pressure cuff is placed on the patient’s arm above the
elbow, inflated, and maintained at a constant pressure (mmHg) throughout
the procedure. One (or two) standardized incisions (are) made on the volar
surface of the forearm. The length of time required for bleeding to stop is
recorded as the bleeding time.

3. Mielke Method (modified ivy method)

○ It is similar to Ivy method but it uses a template instead of a device.

3
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

○ Standardized method
○ It is not currently used in hospitals.
○ Bard-parker or a similar blade placed in a special handle containing a gauge
is used along with polystyrene or plastic template.

Procedure
1. Locate the area for the bleeding time.
○ Volar surface: 5cm below the fold of the elbow.
2. Clean the site of incision.
3. Place BP cuff above the elbow and apply pressure of 40mmHg or lower for
newborns.
○ <2 lbs = 20mmHg
○ 2-4 lbs = 25mmHg
○ >4 lbs = 30mmHg
4. Prepare Bleeding time device
○ Caution: putting too much pressure in applying BT device will increase
incision depth that will result in falsely prolonged BT result.
5. Activate the trigger and remove it 1 second after.
○ Incision must be made and bleeding time started 30-60 secons after inflating
the BP cuff.
○ Start timer once blood comes out.
6. Blot blood on the puncture site every 30 seconds by clean circular filter paper.
7. When bleeding ceases, stop the time and remove BP cuff.
8. Place bandage on the puncture site.

Clinical Application
● Increased bleeding time

4
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

○ Platelet count: <30,000 to 50,000/μL

■ Bleeding time increases up to more than 30 minutes.
○ Platelet count: >100,000/μL
■ Impaired platelet function or defect or subendothelial factor.
○ Dysfunctional platelets
■ Von Willbrand Disease
■ Following ingestion of aspirin which impairs the prostaglandin pathway
that inhibits platelet activation.
■ Aspirin-containing compounds.
■ Anti-inflammatory drugs (aspirin, ibuprofen)
■ Anticoagulants
■ Some antibiotics and certain other drugs
■ Drug therapy: most common cause of prolonged bleeding time.

Aspirin Tolerance test

● Useful in distinguishing normal and abnormal platelets.
● BT is measured before and after 2 hours of aspirin (650mg) ingestion.
○ Difference in BT is evaluated.
● Aspirin effect lasts for the duration of the platelet life cycle (7-10 days).

Copley-Lalitch Method (Copley-Lalitch immersion test)

● Procedure:
○ Clean the finger.
○ Puncture 6mm deep
○ Immerse the wounded finger in a sterile NSS warmed at 37°C until bleeding
stops.
○ Same as Adelson-Crosby method but has different proponents.

MacFarlane’s Method
● It uses earlobe as a puncture site.
● The same as Copley-Lalitch immersion test and Adelson-Crosby method.

Tourniquet test (Capillary Fragility test)

● Crude method of capillary fragility and integrity of blood vessels.
● Degree of thrombocytopenia correlates with tourniquet test.
● This is performed on patients with low Platelet count
● Normal result: none to few petechiae produced.
● Principle:
○ Inflated BP cuff is used to apply pressure to the capillaries for 5 minutes. The
arm is then examined for petechiae.
● Procedure:
○ Examine the forearm, hand and fingers to make certain no current petechiae
is present.
○ Apply the blood pressure cuff on the upper arm above the elbow, and take
blood pressure reading.

5
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

○ Inflate the blood pressure cuff to a point halfway between the systolic and
diastolic pressures and maintain for 5 minutes.
○ Remove blood pressure cuff and proceed after 5 to 10 minutes.
○ Examine the forearm, hands, and fingers for petechiae.
■ Disregard petechiae within ⅕ inch of the BP cuff because the
petechiae produced in that area is due to the pressure and not
because of fragile capillary.
○ The test results may be graded roughly as follows:
■ 1+ (0-10) - a few petechiae on the anterior part of the forearm.
■ 2+ (10-20) - many petechiae on the anterior part of the forearm.
■ 3+ (20-50) - multiple petechiae over the whole arm and the back of the
hand.
■ 4+ (>50) - Confluent petechiae on the arm and back of the hand.
● Confluent: too much petechiae tend to pile up and will look like
ecchymosis, when in fact they are just clumps of petechiae
forming.
○ Clinical application:
■ Thrombocytopenia
■ Decreased fibrinogen
■ Vascular purpura
■ Senile purpura
■ Scurvy/Vitamin D deficiency

Clot Retraction Time (CRT)

 When blood coagulation is complete, the clot normally undergoes retraction. During
clot retraction, serum is expressed from the clot, and the clot becomes denser.
 When clot contracts, it becomes smaller in size.
 Clot retraction process is dependent on:
o Functionally normal platelets.
o Calcium
o ATP
 Clot retraction happens when there is an outside-in signaling through the platelet receptor
GPIIbIIIa. This will make the platelets capable of exerting contractile force within the formed
clot, the clot becomes denser and smaller making it separate with the vessel wall.
 Thrombosthenin - contractile protein located within the plateles, enables platelets to exert
contractile force.

CRT methods: Qualitative CRT

o Hirschboeck/Castor oil:
o Place castor oil on Red top tube (3/4)
o Add one drop of fresh blood.
o Observe for “dimpling” phenomenon
 Start timer from the time blood is dropped.
 Stop timer when dimpling phenomenon is observed.

6
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

o Normal Values: 15-45 minutes.

o <15 minutes: Thrombotic tendency
o >45 minutes: hemorrhagic tendency

Quantitative CRT
o MacFarlane Method
o Specimen: 5ml fresh whole blood.
o Uses tube containing glass rod.
o Temperature: 37°C
o Incubation time: 1 hour
o Computation: %CR = amount of serum left in the tube/amount of blood used x
100.
o Normal values: 44 - 67%
o Procedure
o A glass stirring rod is placed in a test tube, then the test tube is covered.
o The whole set-up is incubated at 37°C for 1 hour.
o After incubation, check the volume for the amount of expressed serum in the
tube divided by the amount of blood used multiplied by 100.
o During extraction of samples in red top tube, the tube is filled with blood and
let stand for a period of time. The red cells clot and separate from the slides
of the test tube from which serum is distinguishable - this is an example of
clot retraction.
o As the clot retracts, it expresses serum.
 This is the serum seen in red top tubes.
o Example of computation:
 Expressed serum or amount of serum left in tube: 3mL
 Amount of blood used: 5mL
 CR% = (3mL/5mL) x 100 = 60%
 Normal clot retraction time
o Coagulation onto the surface of the rod is caused by Factor XII since it is
linked to in vitro coagulation.
 Factor XII is activated through exposure to negatively charged
surfaces.
 After activation, coagulation cascade will initiate until clot formation
followed by clot retraction.
o Stefanini Method
o Fresh whole clotted blood is placed in a 37°C water bath and inspected at
1,2,4 and 24 hours for the presence of a retracted clot.
o Specimen: 3mL of whole blood placed in a glass test tube (without
anticoagulant).
o Procedure:
o Obtain 3 mL of blood and dispense carefully into a glass test tube.
o Place the test tube of blood in the 37C water bath and allow the blood to clot.

7
Hematology 2 Laboratory Must Knows
Vincent Lawrence D. Marcos
S.Y. 2022-2023 Cagayan State University

o As soon as the blood has clotted, inspect the clot at 1, 2, 4, and 24 hours for
the formation of a retracted clot.
o Results are reported as the length of time it took for the clotted blood to
retract.

o Normal: clot retraction occurs at 2-4 hours.

o Poor: clot retraction occurs after 4 hours and within 24 hours.
o None: clot retraction occurs only after 24 hours.

Clinical significance:
o Glanzmann’s thrombasthenia
o Thrombocytopenie (platelet count: <100,000/uL)
o Dysfibrinogenemia or hypofibrinogenemia - formed clot is small; increased RBCs
expressed from the clot (RBC fallout)
o Paraproteinemias
o Disseminated Intravascular Coagulation - formed clot is small and ragged; increased
RBC fallout (too much red cells expressed)
o Increased RBC count: limited degree of clot retraction
o Decreased RBC count: increased degree retraction

Relationship between the Degree of Clot Retraction and RBC, Hematocrit, Platelet and
Fibrinogen
o The degree of clot retraction is directly proportional to the amount/number and
function of platelet
o The degree of clot retraction is inversely proportional to the RBC count, MCV,
hematocrit, and fibrinogen levels.

Normal Result:
o Clot retraction begins within 30 seconds after the blood has clotted
o At the end of 1 hour, clot retraction is observed with most retraction occurring within
the first 4 hours.
o Clot retraction should be complete within 4 hours.