What's the next step after getting variable cell counts of 12, 28, 18, and 15 in a 1:20 dilution of a body fluid on a hemocytometer?

Hemocytometer Cell Count Analysis

When there is significant variability between cell counts in the four large corner squares of a hemocytometer (12,28,18, and 15), you should clean the hemocytometer and recharge it with a well-mixed dilution.

Analysis of the Current Cell Count Results

The cell counts from the four large corner squares (12,28,18, and 15) show considerable variability, particularly between the lowest count (12) and the highest count (28). This level of variation indicates:

• Potential uneven distribution of cells in the hemocytometer chamber
• Possible inadequate mixing of the sample
• Potential clumping of cells in the dilution

Statistical Assessment

The coefficient of variation (CV) for these counts can be calculated:

• Mean count = (12 + 28 + 18 + 15) ÷ 4 = 18.25
• Standard deviation is high relative to the mean
• The count of 28 is more than 50% higher than the mean, indicating an outlier or significant distribution problem

Proper Hemocytometer Procedure

According to quality control guidelines for cell counting, reliable cell counts should demonstrate consistency across the counting area 1. When significant variability is observed:

1. The hemocytometer should be thoroughly cleaned to remove any residual cells or debris
2. The cell suspension should be thoroughly mixed to ensure homogeneous distribution
3. A fresh sample should be loaded into the hemocytometer

Mixing Technique

• Gently but thoroughly mix the diluted sample immediately before loading
• Avoid creating bubbles which can interfere with even cell distribution
• Use proper pipetting technique when loading the chamber

Why Other Options Are Not Appropriate

• Option A (Compare this cell count to the one on the other side): While comparing counts between chambers can be useful, the primary issue here is the significant variability within a single chamber, which must be addressed first 1.

• Option B (Increase the number of mm² counted): Counting more squares would not resolve the fundamental issue of uneven cell distribution in the sample 1.

• Option D (Report the white count as 3.7 x 10⁹/L): Calculating and reporting results from such variable data would produce unreliable results. The calculation would be:

  • Mean count = 18.25 cells
  • Dilution factor = 20
  • Area counted = 4 mm²
  • Depth = 0.1 mm
  • Formula: (18.25 × 20)/(4 × 0.1) = 365 × 10⁴/mL = 3.65 × 10⁹/L

  However, reporting this value would be inappropriate given the high variability in the raw counts 1.

Quality Control Standards

Quality control guidelines for cell counting emphasize the importance of consistency in cell distribution 1. When performing cell counts:

• Cell distribution across the counting chamber should be uniform
• Counts from different areas of the chamber should be within an acceptable range of variation
• Significant deviations suggest technical issues with sample preparation or loading

Manual cell counting with a hemocytometer remains the gold standard for many applications 2, 3, but proper technique is essential for accurate results.

Conclusion

The correct approach when faced with highly variable cell counts in a hemocytometer is to clean the hemocytometer and prepare a fresh, well-mixed sample for counting. This ensures the reliability and accuracy of the cell count results, which is critical for subsequent calculations and experimental procedures.