HealthSeba

Laboratories use two main techniques depending on infrastructure and workload.

Available Methods

• Manual method (Hemocytometer method)

• Electronic cell counter (Automated hematology analyzer)

Manual Platelet Count Method

Small laboratories and teaching institutions frequently perform manual estimation due to its low cost and reliability when performed carefully. Moreover, this method remains valuable when automation is unavailable.

Equipment Used

• Platelet pipette

• Platelet diluting fluid

• Improved Neubauer counting chamber

Platelet Pipette

Identification

The platelet pipette closely resembles the RBC pipette. Inside the bulb, a mixing bead ensures uniform dilution. Clear graduations appear at:

• 0.5

• 1

• 101

Dilution Principle

When blood reaches the 0.5 mark and diluting fluid fills up to the 101 mark, the final dilution becomes 1:200.

Platelet Diluting Fluid

Commonly Used Fluid

• 1% Ammonium oxalate

Functions

• Lyses red blood cells

• Preserves platelets

• Prevents platelet clumping

As a result, platelets remain clearly visible under microscopy.

Procedure for Manual Platelet Count

1: Dilution

First, draw blood up to the 0.5 mark of the platelet pipette. Next, add diluting fluid until the 101 mark. Then mix thoroughly and allow the mixture to stand for 10–15 minutes so that red cells lyse completely. This step achieves a dilution of 1:200.

2: Charging the Chamber

After mixing, place the coverslip correctly on the Neubauer chamber. Carefully charge the chamber while avoiding air bubbles. Subsequently, allow platelets to settle evenly before counting.

3: Counting

Count platelets in:

• Central large square (area = 1 mm²)

• Subdivided into 25 medium squares

Chamber depth remains 0.1 mm.

Total volume counted:
1 × 1 × 0.1 = 0.1 mm³

Counting Rules

To maintain accuracy, follow standardized counting rules:

• Count platelets touching the top and left boundary lines

• Exclude platelets touching the bottom and right boundary lines

Under high-power objective, platelets appear as:

• Small refractile bodies

• Purple-colored dots

Applying these rules consistently improves reproducibility in **platelet counting **.

Calculation of Platelet Count

Let N represent the number of platelets counted.

Dilution: 1:200
Volume counted: 0.1 mm³

Formula

Platelet count = (N × Dilution) / Volume

Platelet count = (N × 200) / 0.1

Platelet count = N × 2000 /mm³

Example

If counted platelets = 150

Platelet count = 150 × 2000
Platelet count = 300,000/mm³

Therefore, the platelet count equals 3.0 lakh/mm³.

Advantages of Manual Method

Manual **platelet counting ** offers several benefits:

• Useful in small laboratories

• Low operational cost

• No expensive equipment required

• Reliable backup when analyzers fail

Normal Range of Platelet Count

SI Unit:
150 – 400 × 10⁹/L

Abnormal Platelet Levels

1. Low Platelet Count (Thrombocytopenia)

Platelet count below 150,000/mm³ indicates thrombocytopenia.

Common causes include:

• Dengue

• Viral infections

• Aplastic anemia

• Leukemia

• Chemotherapy

• Hypersplenism

Severe risk levels:

• <50,000/mm³ → High bleeding risk

• <20,000/mm³ → Spontaneous bleeding risk

2. High Platelet Count (Thrombocytosis)

Platelet count above 400,000/mm³ indicates thrombocytosis.

Causes include:

• Infection

• Inflammation

• Iron deficiency anemia

• Polycythemia vera

• Post-surgical state

Excessively high values may increase thrombotic risk.

Errors in Platelet Count

1. Technical Errors

• Improper dilution

• Inadequate mixing

• Dirty chamber

• Air bubbles

• Delayed counting

• Improper charging

2. Platelet Clumping

• EDTA-induced clumping

• False low platelet count

3. Misidentification

• Dust particles mistaken as platelets

• RBC fragments counted incorrectly

4. Statistical Error

• Uneven distribution of platelets

Counting additional squares reduces this error and improves platelet counting accuracy.