DIAGNOSTIC PROCEDURES FOR PRIMARY

HEMOSTASIS

f.
g.

1. Capillary Fragility Test (CFT)

Torniquet test
To evaluate fragility of capillary walls
o Weak = venous pressure = rupture
o Scurvy (vitamin C deficiency)
To identify platelet deficiency, CFT correlates with
the degree of thrombocytopenia
Procedure
a.

Check blood pressure

Ex. 120/90 =

b.
c.

120+ 90
2

= 105 mmHg

Apply sphygmomanometer (5 mins)

Count petechiae fter 15-30 mins

Interpretation of Result
1
+
2
+
3
+
4
+

0-10
1120
2150
>50

Depress trigger and simultaneously start the

timer
Blot with filter paper every 30 seconds

Reference Range: 2-9 minutes

**Above platelet count of 100x109/L should fall within
the reference range
C. In vitro Bleeding Time Device

Dode Behring PFA 100 (Platelet function

Analyzer)
High Sheer Flow System
Platelets occlude an aperture within membranes
coated with:
o Collagen/ epinephrine (for primary screening)
o Collagen/ ADP (for differentiation of dysfunction
due to aspirin)
3. Platelet Estimation in PBS
Ideal requires a fresh drop of capillary blood
without anticoagulant.
Sample collected in EDTA may also be used
Films made after 5hrs from blood collection may
exhibit plenty of artifacts
Platelets from skin puncture: irregular in shape,
activated
Platelets from EDTA: round
Stain with Romanowsky stain
10-30 RBCs= 1-3 platelets

2. Bleeding Time
A. Conventional Method/ Modified Ivy-Duke
Original test
1912: Duke
1941: modified by Ivy
Used among 0-3 years old patients (finger) and
adult patients with no site for simplate method
Procedure
a.
b.
c.
d.

e.
f.

Cleanse site (70% alcohol)

Skin puncture (2mm depth)
Start timer
Without touching the wound, blot drop of blood
with filter paper every 30 seconds
**Blotting the wound directly would yield false
increase result
Record at the nearest 30 seconds
Report as more than 20 minutes if bleeding does
not stop after 20 minutes

Reference Range 2-4 mins

B. Simplate Method
Procedure
a.
b.
c.
d.
e.

Select site at the volar area of the arm

(muscular)
Place sphygmomanometer
Simplate/ surgicutt (5 mm wide, 1 mm depth)
Inflate to 40 mmHg
2 punctures for quality control

Platelet Satellitism
-

Occurs in EDTA blood sample

Platelet adheres on cell membrane of neutrophil
Yield false decrease platelet count

Verification of Low Platelet Count (<50,000/uL)

a.
b.
c.

Vortex for 2 mins, run specimen on machine for

10,000 rpm
Recollect specimen on blue top (citrated blood)
PBS

Quality Assurance
1.
2.

Even cellular distribution

No overlapping
Space should be approximately diameter of RBC
Platelet clumps or other abnormal cells in the
feathery edge (platelet satellitism)

Examine entire smear for validation of

abnormally low platelet count

Decreased Platelet Count

a.

Platelet clumps

b.
c.

Platelet satellitism
Giant platelets

False Elevation of Platelet Count

a.

b.

Cytoplasmic tags/ fragments

o Small particles w/c lack platelet organelles
o Result from the tendency of cytoplasmic
fragments to separate from leukemic blast
cells
o Same size and shape as platelets
o Resembles platelets in Wrights stain
Cryoglobulins
o Proteins that tend to precipitate on low
temperature
o Appears as small round globules
o Exhibits in patients with cryoglobulinemia
(mycoplasma pneumonia infection)

Methods for Platelet Count

a.

Microscopic Examination
-

To evaluate quality of smear

To ascertain approximate number of platelets in
OIO (100X)
For the detection of rouleaux formation
For the detection of fibrin clots

b.

Platelet Estimate:

platelets20 fields x 100 x 14,000

100

**answer must be compared with the result obtained

from the instrument. Any discrepancy must be
investigated

Platelets are counted in relation to 1,000 RBCs in

the blood smear

platelet count=

Interpretation of Platelet Estimate

Marked decrease
Moderate decrease
Slight decrease
Low normal
Normal
Slight increase
Moderate increase
Marked increase

Light Microscopy

Reese-Ecker
o
Sodium Citrate, BCB, d.H2O
o Prevents coagulation and hemolysis
o Preserves RBC
o Provides necessary low specific gravity to
facilitate settling of platelet
o Provides fixation to reduce the
adhesiveness of the platelets

Guy & Leake

o Sodium oxalate, formalin, crystal violet,
sodium citrate
Phase Contrast Microscopy (recommended)

Becker-Cronkite
o Ammonium oxalate (hemolyzes RBC)
o Utilizes phase contrast microscopy

Unopette
o Developed in 1950
o Commercially prepared diluents

Indirect Method
-

0-49,000 uL
50, 000- 99,000 uL
100,000- 149,000 uL
150,000- 199,000 uL
200,000- 400,000 uL
401,000- 599,000 uL
600, 000- 800,000 uL
>800,000 uL

Thrombocytopenia purpura
Aplastic anemia
Acute leukemia
Gauchers disease
Chemotherapy and radiation

Dameckek: BCB, Na Citrate, formalin, sucrose,

d.H2O
Fonios: 14% Magnesium sulphate
Olefs

Significant Platelet Levels

<150,000 uL
30,000- 50,000 uL
<30,000 uL

Manual Method for Platelet Count

1.

<5,000 uL

Platelet Count

Reference Range
150,000- 400,000 uL
150-400x109/L

Polycythemia vera
Idiopathic thrombocytopenia
CML
Splenectomy

Thrombocytopenia

Abnormally low
Possible bleeding with
trauma
Possible spontaneous
bleeding
Severe spontaneous
bleeding

Correlation between platelet count and bleeding time

1.
-

Thrombocytosis
-

RBC count
1, 000 RBC x platelet counted

2.
3.
-

Normal PC, Prolonged BT

Quality platelet abnormality
o Aspirin
o gpIIb/IIIa deficiency
o von Willebrand syndrome
o Primary vascular abnormality
Decreace PC, Normal BT
Autoimmune thrombocytopenia
Decrease PC, Very Prolonged BT
Quantitative + qualitative platelet dysfunction