7.4 Manual Cell Counts, Manual Hemoglobin Methods, and Hematology Quality Control
Key Takeaways
Each large square of the improved Neubauer chamber is 1 mm² with a 0.1 mm depth, so it holds 0.1 µL; cells per µL equal count × dilution ÷ (area × 0.1).
Turk's solution lyses red cells with acetic acid and stains white cell nuclei with gentian violet, while 1% ammonium oxalate is used for manual platelet counts.
The WBC count is corrected for nucleated red cells by multiplying it by 100 and dividing by 100 plus the NRBCs per 100 WBCs.
In the copper sulfate donor screen, a drop of blood that sinks in solution of specific gravity 1.053 indicates hemoglobin of at least about 12.5 g/dL.
Bull's moving average monitors MCV, MCH, and MCHC in batches of patient samples, and a batch mean outside about ±3% of target signals an instrument problem.
Manual Cell Counts, Manual Hemoglobin Methods, and Hematology Quality Control
Cell counts carry 30% and hemoglobin and hematocrit 15% of the Hematology blueprint, so manual methods are tested even though analyzers now do most routine counting. Manual counts remain the backup when an analyzer fails, the check when an analyzer flags a result, and the method for body fluids and some special counts. This section covers the hemocytometer, diluting fluids and counting formulas, the eosinophil and platelet counts, corrections and sources of error, older hemoglobin methods, and quality control for the hematology laboratory.
1. The Improved Neubauer Hemocytometer
- The ruled area is a square of 3 mm × 3 mm (9 mm²) divided into nine 1-mm² squares. The coverslip sits 0.1 mm above the ruled surface, so each 1-mm² square holds 0.1 µL (0.1 mm³).
- The four corner squares (each divided into 16 smaller squares) are used for white cell counts.
- The central square is divided into 25 smaller squares (each 0.2 mm × 0.2 mm = 0.04 mm²), each with 16 tiny squares. Red cell counts use five of these (the four corners and the center), a total area of 0.2 mm². Platelet counts commonly use the whole central square (1 mm²).
- Charging: Clean the chamber and coverslip, mix the diluted sample well, and fill both sides by capillary action without overfilling or trapping air bubbles. Let the cells settle for a few minutes (about 10 to 15 minutes in a moist chamber for platelets).
- Counting rule: Count cells touching the top and left boundary lines and ignore those touching the bottom and right lines, so no cell is counted twice. The two sides should agree within about 10%.
The General Formula
2. Diluting Fluids and Worked Counts
| Count | Typical Dilution | Diluting Fluid and Principle | Area Counted |
|---|---|---|---|
| WBC | 1:20 | Turk's solution (glacial acetic acid lyses red cells, gentian violet stains nuclei) or 1% to 3% acetic acid | Four corner squares (4 mm²) |
| RBC | 1:200 | Hayem's fluid, Gower's solution, or isotonic saline (keeps red cells intact) | Five small central squares (0.2 mm²) |
| Platelets | 1:100 | 1% ammonium oxalate lyses red cells; count under phase-contrast microscopy (Brecher-Cronkite method) | Central square (1 mm²) |
| Eosinophils | 1:10 to 1:32 | Pilot's, Hinkelman's, or Dunger's fluid with eosin or phloxine stains eosinophil granules while lysing red cells | Entire ruled area (9 mm²), often both sides |
Worked Examples
- WBC: 1:20 dilution; 180 cells counted in 4 mm². WBC = 180 × 20 ÷ (4 × 0.1) = 3,600 ÷ 0.4 = 9,000/µL (9.0 × 10^9/L).
- RBC: 1:200 dilution; 450 cells counted in 0.2 mm². RBC = 450 × 200 ÷ (0.2 × 0.1) = 90,000 ÷ 0.02 = 4,500,000/µL (4.5 × 10^12/L).
- Platelets: 1:100 dilution; 250 platelets counted in 1 mm² (one side). Platelets = 250 × 100 ÷ (1 × 0.1) = 250,000/µL (250 × 10^9/L).
Correcting the WBC for Nucleated Red Cells
Nucleated red cells (NRBCs) survive the lysing fluid and are counted as white cells. When the differential shows 5 or more NRBCs per 100 WBCs:
Example: uncorrected WBC 12,000/µL with 20 NRBCs per 100 WBCs → 12,000 × 100 ÷ 120 = 10,000/µL.
Estimating Counts from the Blood Smear
- Platelet estimate: Average the platelets in 10 oil-immersion fields of the monolayer and multiply by about 20,000 (normal is roughly 8 to 20 platelets per field). Look at the feathered edge for platelet clumps before accepting a low count.
- WBC estimate: Average WBCs per high-dry (40×) field and multiply by about 2,000. A smear estimate that disagrees with the analyzer count is investigated before the result is released.
Sources of Error in Manual Counts
- Inherent counting (Poisson) error: the fewer cells counted, the larger the error, which is why more squares are counted when counts are low.
- Poor mixing, clotted samples, wrong dilution, unclean or overfilled chambers, and counting debris or yeast as cells.
- Platelet counts are falsely low with clumping or satellitism and falsely high when red cell fragments or debris are counted.
3. Manual Hemoglobin Methods
The ICSH reference method (cyanmethemoglobin) and automated methods are covered in the automated CBC section. Older methods still appear in board questions:
| Method | Principle | Notes |
|---|---|---|
| Sahli (acid hematin) | 0.1 N HCl converts hemoglobin to brown acid hematin, which is diluted with water until it matches a glass standard | Uses a 20 µL TC Sahli pipette; visual comparison makes it imprecise; does not measure all forms of hemoglobin equally |
| Oxyhemoglobin | Dilute ammonia; oxyhemoglobin read photometrically | Fast but does not measure carboxyhemoglobin, methemoglobin, or sulfhemoglobin accurately |
| Alkali hematin | Sodium hydroxide converts hemoglobin to alkali hematin | HbF resists alkali and is underestimated |
| Copper sulfate specific gravity | A drop of blood sinks in a copper sulfate solution of specific gravity 1.053 if hemoglobin is at least about 12.5 g/dL | A rapid donor screening method; a drop that floats fails the screen |
| Azide methemoglobin (HemoCue) | Point-of-care photometer converts hemoglobin to azidemethemoglobin | Used for donor screening and point-of-care testing |
- Microhematocrit: Capillary tubes filled about three-quarters with blood are sealed and spun at about 10,000 to 15,000 × g for about 5 minutes. Trapped plasma raises the spun hematocrit slightly, more so in spherocytosis, sickle cell anemia, and macrocytosis. Including the buffy coat in the reading falsely raises the result.
4. Hematology Quality Control
- Commercial control materials at two or three levels are run each shift or 24 hours and plotted on Levey-Jennings charts, with Westgard rules applied as in clinical chemistry. Hematology controls are stabilized cells with a short shelf life.
- Calibration uses commercial calibrators or fresh whole blood assigned values by reference methods. It is verified after major maintenance, after a component is replaced, and whenever controls show a shift.
- Bull's moving average (X̄B): The analyzer averages the MCV, MCH, and MCHC of consecutive patient samples in batches (commonly 20). Red cell indices are very stable in a hospital population, so a batch mean outside about ±3% of the target signals an instrument problem even when controls pass.
- Delta check: A patient's result is compared with their previous result. A change too large to be explained biologically (for example, a large MCV change within 24 hours without transfusion) suggests a misidentified or contaminated specimen.
- Rule of three: Hemoglobin × 3 should approximate the hematocrit, and RBC × 3 should approximate the hemoglobin, in normocytic normochromic samples (see the automated CBC section).
- Retained patient samples and duplicate testing detect drift. In manual counts, duplicate counts or the two sides of a chamber should agree within the laboratory's limit.
- External quality assessment: In the Philippines, clinical laboratories take part in DOH-designated external quality assessment programs run by national reference laboratories.
Blood is diluted 1:20 in Turk's solution, and 92 cells are counted in the four large corner squares of an improved Neubauer hemocytometer. The differential shows 15 nucleated red cells per 100 WBCs. What is the corrected WBC count?
4,000/µL
4,600/µL
5,290/µL
6,900/µL
A hematology analyzer's Bull's moving average for MCHC rises 5% above target over the last three batches, although the morning commercial controls were acceptable. What does this indicate?
Normal biological variation in the patient population, which needs no action
An analytical drift in hemoglobin or red cell measurement that needs investigation
A delta-check failure caused by a single misidentified patient specimen in the batch
Lipemia in one patient specimen that falsely raised the MCHC of the whole batch
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