Tests for

Prepared by: Robina C. Enguana,

RMT
Contents
▪ Algorithm for Diagnosing Platelet Disorders
▪ Platelet Count
▪ Platelet Function Tests
a. Bleeding Time
b. Clot Retraction
c. Platelet Adhesiveness Test
d. Platelet Aggregometry
e. Platelet Function Analyzer: PFA-100
▪ Capillary Resistance Test
 Algorithm for Diagnosing Platelet
Disorders
Bleeding Time Platelet Count

Quantitative or
Qualitative Quantitative
Qualitative
platelet platelet
platelet
abnormality abnormality
abnormality

Platelet aggregation
studies, closure time,
lumi-aggregation, etc.
 Review of Platelet Function
• BV wall damage exposes highly adhesive substrates (e.g., P selectin,
vWF, collagen, and many other extracellular matrix component)

• stable adhesion through receptor-ligand interaction

• vWF + GP Ib

•shape change, degranulation and spreading upon areas of exposed subendothelium

•release of ADP, generation of TxA 2, expression of negatively charged surface phospholipid

• GP IIb/IIIa + fibrinogen

• Aggregated platelets contract resulting to a denser and smaller clot

PLATELET COUNT
• Reference method: phase-contrast microscopy
using 1% ammonium oxalate (Brecher and
Cronkite)

• a 1:100 dilution is made and loaded in the

counting chamber

• platelets are counted in 25 small squares at the

central square
 Morphologic Evaluation of
Platelets
Normal EDTA blood (prepared up to 1 hour after collection)
❑ 2 – 4 μm in diameter, round to oval and separated from one another
❑ mean platelet volume (MPV) = 6.5 - 12fL
❑ fine purple granules fill the cytoplasm
❑ distribution: 7 – 20 platelets/OIF (of 100-200 RBCs)

Fingerstick blood
❑ platelets with irregular shapes and increased clumping
❑ if characteristic clumping is not observed, suspect Glanzmann
thrombasthenia
Normal Platelets, Wright stain

EDTA blood Capillary blood

Giant platelet, EDTA blood, Wright
stain
BLEEDING TIME

• Duke’s method
• Ivy’s method
• Template method
• Copley-Lalitch method
BLEEDING TIME
–Duke’s method – fingertip and earlobe
puncture
–Ivy’s method – uses BP cuff
–Template method
• Puncture on forearm
CLOT RETRACTION
TIME
• Clot retraction is proportional to platelet
count
– Castor oil / Hirschboeck
– Stefanini
– MacFarlane
 Hirschboeck/Castor Oil Method

Stefanini Method

MacFarlane Method
PLATELET
ADHESIVENESS TEST
• measures adhesion and aggregation through the use
of glass bead column
PLATELET
ADHESIVENESS TEST
Decreased in:
• Glanzmann’s Increased in:
thrombasthenia • venous
• vWD thrombosis
• Chediak-Higashi • pulmonary
syndrome embolism
• some myeloproliferative • carcinoma
disorders • pregnancy
• uremia • following
• ingestion of splenectomy
aspirin • use of oral
contraceptives
PLATELET
AGGREGATION TEST
• Detects in vitro platelet aggregation
– Citrated PRP + aggregating agent
❑Epinephrine
Bernard Soulier (lacks gp1b)
❑ADP -abnormal ristocetin
❑Collagen VWF (deficiency or defect of VWF)
❑Ristocetin -abnormal ristocetin
LIGHT TRANSMISSION PLATELET
AGGREGOMETRY
• Involves a series of tests performed on WB or PRP,
using several agonists (platelet activators).
• The agonist is added to the suspension and a
dynamic measure of platelet clumping is recorded.
• Simultaneously to platelet aggregation,
luminometry test can be performed. In that case,
ATP release is assayed using a luminescent marker.
LIGHT TRANSMISSION PLATELET
AGGREGOMETRY

❑ Primary Aggregation → temporary aggregation

❑ Secondary Aggregation → formation of full aggregates after
release of delta-granule ADP
 Agonists
1. Thrombin
2. ADP
3. Epinephrine
4. Collagen
5. Arachidonic acid
6. Ristocetin
 PFA-100
PFA-100 • Closure Time (CT)
→ the time taken for blood,
drawn through a fine capillary,
to block a membrane coated
with collagen and epinephrine
(CEPI) or collagen and ADP
(CADP)

→ measured in seconds
 PFA-100
PFA-100
 CAPILLARY RESISTANCE
/FRAGILITY TEST
TOURNIQUET / RUMPEL-LEEDE /
➢Uses BP
HESS TESTcuff
➢Detects the ability of the
capillaries to resist pressure
➢Correlates with
thrombocytopenia
 #rciconnects

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