Manual

Overview of Manual Cell Counts

Definition: Manual cell counts are performed by trained laboratory personnel using a microscope and a specialized counting chamber (hemocytometer) to enumerate cells in a fluid sample
Clinical Significance: Although automated cell counters are now commonly used, manual cell counts remain important in certain situations:
- When automated cell counts are inaccurate or unavailable
- For counting cells in body fluids where automated methods may not be validated
- To confirm abnormal results obtained by automated methods
Fluids Analyzed:
- Whole Blood: Red blood cells (RBCs), white blood cells (WBCs), and platelets
- Body Fluids: Cerebrospinal fluid (CSF), synovial fluid, serous fluids (pleural, peritoneal, pericardial), and others

Principles of Manual Cell Counting

Dilution: The sample is diluted with a specific diluent to reduce the cell concentration to a manageable level
Counting Chamber (Hemocytometer): A specialized glass slide with a precisely ruled grid of known dimensions The Neubauer counting chamber is the most common type
Microscopy: A microscope is used to visualize the cells within the counting chamber
Cell Identification: Cells are identified based on their morphology and staining characteristics
Calculation: The cell count per unit volume is calculated using a formula that takes into account the dilution factor, the area of the counting chamber, and the depth of the chamber

Equipment and Materials

Microscope: With a calibrated eyepiece and 10x and 40x objectives
Counting Chamber (Hemocytometer): Neubauer chamber is most common
Coverslip: A special coverslip designed for use with the counting chamber to create a known volume above the counting grid
Pipettes: Accurate pipettes for measuring the sample and diluent
Diluent: An appropriate diluent for the specific cell type being counted:
- WBC Count: Acetic acid (3%) or Turk’s solution (lyses red blood cells)
- RBC Count: Isotonic saline (0.85% NaCl)
- Platelet Count: Ammonium oxalate (1%) or sodium citrate (3.8%)
- Body Fluid Cell Counts: Isotonic saline (0.85% NaCl) or crystal violet stain
Tally Counter: A manual or electronic tally counter to keep track of the cells being counted

Procedure for Manual Cell Counts

Sample Preparation:
1. Mix the Sample: Ensure that the blood or body fluid sample is well-mixed
2. Prepare the Dilution:
  - Use a calibrated pipette to accurately measure the sample and diluent
  - Prepare the appropriate dilution according to established laboratory procedures
    - WBC Count: Typically a 1:20 or 1:100 dilution
    - RBC Count: Typically a 1:200 dilution
    - Platelet Count: Typically a 1:100 dilution
    - Body Fluid Cell Counts: May require no dilution or up to a 1:20 dilution, depending on the cell concentration
  - Mix the sample and diluent thoroughly
3. Allow to Stand: For WBC counts using a lytic diluent, allow the mixture to stand for at least 10 minutes to allow the red blood cells to lyse
Charging the Counting Chamber:
1. Clean the Hemocytometer and Coverslip: Use lens paper to thoroughly clean the hemocytometer and coverslip
2. Position the Coverslip: Carefully place the coverslip on the hemocytometer, ensuring that it is properly seated and covers the counting area
3. Charge the Counting Chamber: Gently introduce a small amount of the diluted sample into the counting chamber by capillary action. Avoid overfilling or introducing air bubbles
4. Allow Cells to Settle: Allow the cells to settle for a few minutes before counting
Microscopic Examination and Counting:
1. Position the Counting Chamber: Place the hemocytometer on the microscope stage and secure it with the clips
2. Focus: Use the 10x objective to focus on the counting grid
3. Counting Rules: Follow consistent counting rules to avoid over- or undercounting cells:
  - Count cells that touch the upper and left-hand boundaries of the counting area
  - Do not count cells that touch the lower and right-hand boundaries
  - Count cells within the defined area of the counting chamber
4. Counting Patterns:
  - Systematically count the cells within the designated squares of the counting chamber
  - Use a consistent counting pattern (e.g., serpentine pattern) to ensure that all cells are counted
5. WBC Count: Count the cells in the four large corner squares of the Neubauer chamber
6. RBC Count: Count the cells in the five small squares within the central large square of the Neubauer chamber
7. Platelet Count: Count the platelets in the twenty-five small squares within the central large square of the Neubauer chamber
8. Body Fluid Cell Counts: Count the cells in all nine large squares of the Neubauer chamber
9. Record the Counts: Use a tally counter to keep track of the number of cells counted
10. Perform Duplicate Counts: Perform duplicate counts on a second chamber to assess precision
Calculation of Results:
1. Apply the Appropriate Formula: The formula for calculating the cell count depends on the dilution factor and the area and depth of the counting chamber
  - General Formula: Cell Count (cells/μL) = (Number of Cells Counted x Dilution Factor) / (Area Counted (mm2) x Depth of Chamber (mm))
  - Specific Formulas (Neubauer Chamber):
    - WBC Count: Cell Count (cells/μL) = (Number of Cells Counted x Dilution Factor) / (4 mm2 x 0.1 mm)
    - RBC Count: Cell Count (cells/μL) = (Number of Cells Counted x Dilution Factor) / (0.2 mm2 x 0.1 mm)
    - Platelet Count: Cell Count (cells/μL) = (Number of Cells Counted x Dilution Factor) / (1.0 mm2 x 0.1 mm)
    - Body Fluid Cell Counts: Cell Count (cells/μL) = (Number of Cells Counted x Dilution Factor) / (9 mm2 x 0.1 mm)
2. Average Duplicate Counts: If duplicate counts were performed, calculate the average of the two counts
3. Report the Result: Report the cell count in the appropriate units (e.g., cells/μL)

Specific Procedures for Blood Cell Counts

White Blood Cell (WBC) Count:
- Diluent: Acetic acid (3%) or Turk’s solution (lyses red blood cells)
- Dilution: Typically 1:20 or 1:100
- Counting Area: Four large corner squares of the Neubauer chamber
- Counting Cells touching the upper and left-hand boundaries, but not those touching the lower and right-hand boundaries
Red Blood Cell (RBC) Count:
- Diluent: Isotonic saline (0.85% NaCl)
- Dilution: Typically 1:200
- Counting Area: Five small squares within the central large square of the Neubauer chamber
- Avoid air bubbles
Platelet Count:
- Diluent: Ammonium oxalate (1%) or sodium citrate (3.8%)
- Dilution: Typically 1:100
- Counting Area: Twenty-five small squares within the central large square of the Neubauer chamber
- Allow platelets to settle for 10-15 minutes before counting
- Use phase-contrast microscopy if available to enhance platelet visualization

Specific Procedures for Body Fluid Cell Counts

Cerebrospinal Fluid (CSF):
- Diluent: Isotonic saline (0.85% NaCl) or crystal violet stain
- Dilution: May require no dilution or up to a 1:20 dilution, depending on the cell concentration
- Counting Area: All nine large squares of the Neubauer chamber
- Report RBC count and total nucleated cell (TNC) count
- Perform a cytospin preparation to differentiate the types of nucleated cells (e.g., neutrophils, lymphocytes, monocytes)
Serous Fluids (Pleural, Peritoneal, Pericardial):
- Diluent: Isotonic saline (0.85% NaCl) or crystal violet stain
- Dilution: May require no dilution or up to a 1:20 dilution, depending on the cell concentration
- Counting Area: All nine large squares of the Neubauer chamber
- Report RBC count and total nucleated cell (TNC) count
- Perform a cytospin preparation to differentiate the types of nucleated cells (e.g., neutrophils, lymphocytes, mesothelial cells)
Synovial Fluid:
- Diluent: Isotonic saline (0.85% NaCl) or crystal violet stain
- Dilution: May require no dilution or up to a 1:20 dilution, depending on the cell concentration
- Hyaluronidase Pre-Treatment: If the synovial fluid is viscous, pretreat with hyaluronidase to reduce viscosity and improve cell dispersal
- Counting Area: All nine large squares of the Neubauer chamber
- Report RBC count and total nucleated cell (TNC) count
- Perform a cytospin preparation to differentiate the types of nucleated cells (e.g., neutrophils, lymphocytes, monocytes, synovial lining cells)

Quality Control and Troubleshooting

Use Calibrated Equipment:
- Ensure that pipettes and counting chambers are properly calibrated
Maintain Clean Equipment:
- Thoroughly clean the hemocytometer and coverslip before each use
- Use fresh, clean diluent
Follow Established Procedures:
- Adhere to standardized procedures for dilution, charging the counting chamber, counting cells, and calculating results
Count an Adequate Number of Cells:
- Count at least 100 cells (or more) to improve accuracy, especially for body fluid cell counts with low cell concentrations
Perform Duplicate Counts:
- Perform duplicate counts on a second chamber to assess precision
- The two counts should agree within an acceptable range (e.g., +/- 10%)
Identify and Correct Errors:
- Uneven Cell Distribution: Ensure that the cells are evenly distributed in the counting chamber
- Air Bubbles: Avoid introducing air bubbles when charging the chamber
- Contamination: Prevent contamination of the sample or diluent
- Inaccurate Pipetting: Use calibrated pipettes and ensure accurate measurement of the sample and diluent
- Improper Cell Identification: Develop expertise in identifying different cell types to minimize counting errors

Reporting Results

Report the cell counts in the appropriate units (e.g., cells/μL, x 10^9/L)
Include the reference range for each parameter
Note any abnormal results or flags
Document all quality control procedures and corrective actions taken

Key Terms

Hemocytometer: A specialized counting chamber used for manual cell counts
Neubauer Chamber: The most common type of hemocytometer
Diluent: A fluid used to dilute a sample
Total Nucleated Cell (TNC) Count: The number of all nucleated cells in a body fluid sample
Cytospin Preparation: A method for concentrating cells from a fluid sample onto a slide for microscopic examination
Hyaluronidase: An enzyme used to reduce the viscosity of synovial fluid
Controls: Testing known samples to ensure proper testing technique

Cell Counts

Automated