MANUAL PLATELET COUNT

Platelets or thrombocytes function primarily in the hemostatic mechanism of the

body. They function in the coagulation of blood and therefore associated with bleeding
and clotting. Platelets count important in helping to diagnose bleeding disorders. They
are difficult to count because of several reasons; platelets are small and difficult to
discern, and they disintegrate easily and are difficult to distinguish from debris. They
have an adhesive character and become attached to surfaces or to particles of debris in
the diluting fluid or to each other causing uneven distribution of cells. The clumping
tendency of platelets is decrease if EDTA is used as an anticoagulant.
The two most commonly employed methods of manual platelet counts utilizes a
1:100 or 1:200 dilution of blood applied to an Improved Neubauerhemocytometer
chamber. The two methods differ in the way the platelets are made visible. The
Tocantins method using Rees-Ecker diluent employs a citrate-formaldehyde buffer with
brilliant cresyl blue as a platelet stains for light microscopy. This diluents fixes and
preserves red blood cells as well as platelets to prevent their disintegration.

The more popular phase-contrast microscopy method uses method uses phase
microscope and 1% ammonium oxalate diluting fluid which lyses red cells and allow
platelets to form pseudopods.The Unopette system measures 20 μl of blood and diluted
in 1.98 ml of 1-g/dL ammonium oxalate. This hemolyzes the RBCs, leaving WBCs and
platelets intact.

PRINCIPLE

Blood is mixed with a diluents in which red cells are either left intact or lyzed. A
hemocytometer is filled with the diluted fluid, and the platelets are counted under
the microscope.

SPECIMEN
 If the blood sample is from a finger prick, the puncture must be clean and the
blood free flowing. Wipe away the first drop of blood.
If blood sample is from venous blood, it must be collected in dry plastic or
siliconized glass syringe with a short needle not smaller than 21 gauge. The
needle must be removed before the blood is delivered into plastic container with
EDTA.
The blood and anticoagulant must be mixed gently, to avoid frothing, without any
delay.

TOCANTINS METHOD

MATERIALS
RBC pipet
Rubber tubings
3 ml syringe
Venous blood with EDTA
Mechanical pipet shaker
Rees-Ecker diluents
Rubber aspirator
Concentrated bleach
Distilled water
Acetone
Glass containers
Cotton

PROCEDURE
1. Rinse the bore of the RBC pipet by drawing the diluting fluid up and down in the
stem of the pipet. Expel the fluid from the pipet.

2. Fill the RBC pipet to 0.5mark, and wipe off the outside of the pipet.

3. Immediately draw the diluting fluid up to 101 mark.

4. Mix the content of the pipet by shaking for at least 10 minutes by hand or by
mechanical mixer.

5. Discard 3 – 5 drops of mixture from the bulb into the waste cloth.
6. Hold the pipet at a 45° angle. Firmly place a dry finger over dry end of pipet to
stop sample flow and touch tip to the point where the coverglass and
hemocytometer meet (Fig. 3-1).
Do not disturb coversglass.
 Mount in duplicate

7. Slowly release finger to allow mixture from bulb to flow evenly and completely
under coverglass and over surface of ruled area. Be sure to fill area completely,
but do not overfill or underfill.

Figure 3-1 Filling the counting chamber

8. Fill the opposite side of hemocytometer in the same manner

9. Place the charge hemacytometer in a moist chamber for 15 minutes to allow the
platelets to settle.
 How To Make A Moist Chamber
Place a piece of damp filter paper/cotton in the bottom of a petri dish. An
applicator stick broken in half can serve as a support for the chamber.

10.Count platelets with the use of the 40x objective lens.

 Platelets may appear round or oval displaying dark blue sheen color.

11.Count the 25 small squares in the center square of the grid (1mm 2).

12.Repeat the count on the other side of the counting chamber. Average the two
sides.

13.Show platelet location on the ruled area.

IDENTIFICATION

Instructor’s Signature: ___________________________Date:

TRIAL 1 CHAMBER 1

Instructor’s Signature: ___________________________Date:

TRIAL 2 CHAMBER 1

Instructor’s Signature: ___________________________Date:

TRIAL 1 CHAMBER 2

Instructor’s Signature: ___________________________ Date:

TRIAL 2 CHAMBER 2
 Instructor’s Signature: ___________________________Date:

RESULTS:
Actual Platelet Count
 CHAMBER 1
Side 1 Side 2

CHAMBER 2

Side 1 Side 2

Compute the following:

CHAMBER 1

A. Dilution = Volume of the bulb (100)

Volume of blood

Resulting dilution=

B. Average = Platelets counted in side 1 + Platelets counted in side 2

2

C. Platelets/ mm3 = Average number of Platelets x Depth factor (mm) (10) x Dilution factor
Area counted (mm2) (1)
 Platelets/ mm3 =

PLATELETS / mm3 =

Instructor’s Signature:___________________________Date:

PRECAUTIONS AND TECHNICAL ERRORS

1. In platelets counts, it is imperative that peripheral blood be freely flowing when
obtained from a finger puncture.

2. If a platelet count is requested in combination with other counts for the same
patient and same finger puncture will be used, it is necessary to take the blood
for platelet count first, before performing the remaining counts. The finger must
not be squeezed excessively when the blood is drawn into the pipette.

3. Pipettes and counting chamber must be clean and free from lint because
platelets may be confused with dirt and debris. Ethyl alcohol and a lint free cloth
are recommended for cleaning the counting chamber.

4. Rapid dilution of the blood is essential or platelets may form clumps and the
blood may clot.

5. The platelet count must be performed within three hours of dilution of the
sample.

6. The microscope light must be decreased to provide good contrast and to enable
platelets to be seen in the hemocytometer chamber. Constant focusing of the
microscope is necessary to identify the platelets.
 7. If platelets are clumped or are distributed unevenly in the counting chamber, the
chamber should be cleaned and refilled (after remixing blood dilution). If clump
are still present, new sample should be obtained.

8. To minimize the error for manual platelet counts, duplicates are always prepared
and duplicate counts done on both sides of two counting chambers. Duplicate
should agree within 10% to be acceptable. Discrepancies between the two
chambers of 20% or more necessitate repeat counts. A fresh specimen may be
necessary.

9. When platelet count is low, a larger volume on each side may need to be
counted to improve accuracy and precision. For very low count (Less than 50 x
109/L), a 1:20 dilution should be made in a white cell diluting pipet and a new
dilution factor used in the calculation.

Instructor’s Signature: __________________________ Date:

B. FONIO’S METHOD
MATERIALS
Venous blood with EDTA
Clean glass slides
Spreader slide
Slide rack
Microscope
Wright’s stain
Immersion oil

PROCEDURE
1. From the specimen for manual platelet count, transfer a drop of blood on a glass
slide.

2. Make a wedge smears and allow to dry.

3. Stain with Wright’s and examine under the oil immersion objective (100x).

4. In an area of the smear where the RBCs barely touch, count the 1000 RBC and
all the platelets seen within the count.
Platelets may appear round or oval displaying lilac to purple color .

IDENTIFICATION:

Instructor’s Signature: __________________________Date:

COMPUTATION:
A. Platelets/ mm3= Platelet count X RBC count 1000

Platelets/ mm3 =

Instructor’s Signature: ____________________________ Date:

PLATELET COUNT
PLATELETS
 Aka “THROMBOCYTES” or “THROMBOPLASTIDS”
 Fragments of megakaryocyte, non-nucleated, irregular in size
 2-4 m in diameter
 Life span: 8-11 days in circulation (turnover of 35,000/L/day)
 Average count: 150-450 x 109/L
 Platelet distribution:
o 2/3 in the circulation
o 1/3 in the spleen
 Function:
o Hemostasis
 Aggregation and formation of primary platelet plug
 Thromboplastic activity – to initiate clotting as the source of thrmboplastin
 Clot retraction (thrombothenin)
o Maintain capillary integrity
 Increase platelet:
o Polcythemia vera
o Idiopathic thrombocythemia
o Chronic myelogenous leukemia
o Splenectomy
 Decrease platelet:
o Thrombocytopenia purpura
o Aplastic anemia
o Acute leukemia
o Gaucher’s disease
o Pernicious anemia
o Chemotherapy
o Radiation therapy

Platelets are difficult to count because:

o They easily disintegrate
o Small, colorless, refractile bodies
o Difficult to distinguish from debris
o Unevenly distributed in the blood
o Have a tendency to clump with each other

***EDTA helps to decrease platelet clumping, but the mean platelet volume (MPV) will increase during the
first hour in the tube
***BEST to measure MPV – 1-3 hours after obtaining the specimen

METHODS
1. INDIRECT METHOD – platelets are counted in relation to 1000 RBCs in the smear
A. Dameshek method
B. Fonio’s method
C. Olef’s
D. Cramer and Bannerman
E. Modified Indirect Platelet Count
- count platelet in 10 consecutive OIO field and multiply by 200
2. DIRECT METHOD – most accurate way of platelet count uses RBC pipet, diluting fluid or Unopette for
platelet and WBC count
A. Rees – Ecker
B. Guy’s and Leake method
C. Phase Microscopy method (Brecher-Cronkite)
- recommended method
- 1% ammonium oxalate
D. Walker and Sweeney method
E. Unopette method – 1:100
F. Van Allen’s method
G. Nygard’s method
H. Tocantin’s method
I. Kristerson as modified by Leiupert
J. Feissly and Ludin method

AUTOMATION:
A. Impedance counting
B. Laser Light scattering
a. Coulter Counter
b. Technicon Hemolab
c. Fisher Autocytometer
d. MK-4 Platelet Counter

Common Artifacts in Automated Platelet Counting

 Non-technical factors may produce falsely low platelet count in instruments but normal in smear
o Platelet cold agglutinins
o Abnormal amount of plasma protein in various paraproteinemias
o Previous contacts of platelet with foreign surface such as dialysis membrane
o Giant platelets
o Platelet satellitosis
o Lipemia
o EDTA-induced platelet clumping
 Platelet count is low on smear but normal in instrument (analyzer)
o Patient receiving chemotherapy for acute leukemia/lymphoma
o White cell cytoplasmic fragments are counted as platelets
o RBC fragments are counted as platelets
 High platelet count may be due to:
o Microspherocytes
o Fragments of leukemic cells
o Pappenheimer bodies

Rees-Ecker
Sodium citrate – 3.8g
Brilliant Cresyl Blue – 0.1g
Neutral HCHO – 0.2mL
Distilled water – 100mL

NOTE:
CLUMPS – use 0.109 M Sodium Citrate as anticoagulant and multiply by 1.1
 50 platelet – 1:20 dilution
 platelet – 1:200 dilution

REES-ECKER Method:
- count platelet in 25 intermediate squares
= platelet ct x 10 x 200 x 25 (1)
25 OR
- count platelet in 4 large corner squares
= platelet ct x 10 x 200
4 OR
- count platelet in 5 central squares
= platelet ct x 10 x 200 25 (5)
5 OR
- count platelet in each ruled area
= platelet ct x 10 x 200
2