The heart of the manual cell count is the hemacytometer, or counting chamber. The most common one is the Levy chamber with improved Neubauer ruling. It is composed of two raised surfaces, each in the shape of a 3-mm × 3-mm square (total area 9 mm²) separated by an H-shaped moat. As shown in Figure 14-1, this large square is made up of nine 1-mm × 1-mm squares, and each of the white blood cell (WBC) squares is divided further into 16 squares with the center square subdivided into 25 smaller squares. Each of these smallest squares is or 0.04 mm². A coverslip is placed on top of the counting surfaces. The distance between each counting surface and the coverslip is 0.1 mm; the total volume is 0.9 mm³. Hemacytometers and coverslips must meet the specifications of the National Bureau of Standards, as indicated by the initials “NBS” on the chamber. When the dimensions of the hemacytometer are thoroughly understood, the area counted can be changed to facilitate the counting of specimens with extremely low or high counts.

Figure 14-1 Hemacytometer and a close-up view of the counting areas as seen under the microscope. The areas for the standard white blood cell count are labeled W, and the areas for the standard red blood cell count are labeled R. The entire center square, outlined in blue, is used for counting platelets.

Calculations

The general formula for manual cell counts is as follows and can be used to calculate any type of cell count:

Total count=cells counted×dilution factorarea(mm2)×depth(0.1)

Total count=cells counted×dilution factor×10area(mm2)

White Blood Cell Count

The leukocyte or WBC count represents the number of WBCs in 1 L of whole blood. Whole blood from a capillary puncture or anticoagulated with ethylenediaminetetraacetic acid (EDTA) is mixed with a diluting fluid that contains a weak acid (acetic or hydrochloric) or a buffered ammonium oxalate solution to lyse the nonnucleated erythrocytes. The typical dilution for the WBC count is 1 : 20. A hemacytometer is charged (filled) with the well-mixed dilution and placed under a microscope to count the number of cells.

Procedure

1. Clean the hemacytometer and coverslip with alcohol and dry thoroughly with a lint-free tissue.

2. Make a 1 : 20 dilution, placing 25 mcL of well-mixed blood into 475 mcL of weak acetic acid solution in a small test tube.

3. Cover the tube and mix by inversion.

4. Allow the dilution to sit for 10 minutes to ensure that the red blood cells (RBCs) have lysed. The solution will be clear once lysis has occurred. Leukocyte counts should be performed within 3 hours of dilution.

5. Mix again by inversion and fill a plain microhematocrit tube.

6. Charge both sides of the hemacytometer by holding the microhematocrit tube at a 45-degree angle and touching the tip to the coverslip edge where it meets the chamber floor.

7. After charging the counting chamber, place it in a moist chamber (Box 14-1) for 10 minutes before counting the cells to give them time to settle. Care should be taken not to disturb the coverslip.

BOX 14-1 How to Make a Moist Chamber

A moist chamber may be made by placing a piece of damp filter paper in the bottom of a Petri dish. An applicator stick broken in half can serve as a support for the chamber.

8. While keeping the hemacytometer in a horizontal position, place it on the microscope stage.

9. Lower the condenser on the microscope and focus by using the low-power (10×) objective lens. The cells should be distributed evenly in all of the squares.

10. For a WBC 1 : 20 dilution, count all of the cells in the four corner squares, starting with the square in the upper left-hand corner (see Figure 14-1). Cells that touch the top and left lines should be counted; cells that touch the bottom and right lines should be ignored (Figure 14-2). See Figure 14-3 for the appearance of WBCs in the hemacytometer under the microscope.

Figure 14-2 One square of a hemacytometer indicating which cells to count. Cells touching the left and top lines (*solid circles*) are counted. Cells touching bottom and right (*open circles*) are not counted.

11. Repeat the count on the other side of the counting chamber. The difference between totals for the two sides should be less than 10%. A greater variation could indicate an uneven distribution, which requires that the procedure be repeated.

12. Average the two sides. Using the average, calculate the WBC count using the first of the two equations given earlier.

Example

When a 1 : 20 dilution is used, the four large squares on one side of the chamber yield counts of 23, 26, 22, and 21. The total count is 92. The four large squares on the other side of the chamber yield counts of 28, 24, 22, and 26. The total count is 100. The difference between sides is less than 10%.

The average of the two sides of the chamber is 96. Using the average in the formula:

WBC count=cells counted×dilution factorarea counted(mm2)×depth=96×204×0.1=4800/mm3or4.8×109/L

General reference ranges for males and females in different age groups can be found on the inside front cover of this text. Laboratory values may vary slightly according to the population tested and should be established for each institution.

Sources of Error and Comments

1. The hemacytometer and coverslip should be cleaned properly before they are used. Dust and fingerprints may cause difficulty in distinguishing the cells.

2. The diluting fluid should be free of contaminants.

3. If the count is low, a greater area may be counted (e.g., 9 mm²) to improve accuracy.

4. The chamber must be charged properly to ensure an accurate count. Uneven flow of the diluted blood into the chamber results in an irregular distribution of cells. If the chamber is overfilled or underfilled, the chamber must be cleaned and recharged.

5. After the chamber is filled, allow the cells to settle for 10 minutes before counting.

6. Any nucleated erythrocytes (NRBCs) present in the sample are not lysed by the diluting fluid. The NRBCs are counted as WBCs because they are indistinguishable when seen on the hemacytometer. If five or more NRBCs per 100 WBCs are discovered on the differential, the WBC count must be corrected for these cells. This is accomplished by using the following formula:

Uncorrected WBC count×100Number of NRBCs per 100 WBCs+100

Report the result as the “corrected” WBC count.

7. The accuracy of the manual WBC count can be assessed by performing a WBC estimate on the peripheral blood smear (see Chapter 15). Boxes 14-2 and 14-3 show another method of WBC estimation.

BOX 14-2

How to Determine the WBC Estimating Factor

1. Perform automated WBC counts on 30 consecutive fresh patient blood samples. Make sure the WBC count is in control.

2. Prepare and stain one peripheral blood film for each sample.

3. For each film, under oil-immersion microscopy, find an area where 50% of the red cells are overlapping in doublets or triplets. Then count the number of WBCs in 10 consecutive fields.

4. Divide by 10 the total number of WBCs found to obtain the average number per single oil-immersion field.

5. Divide the automated WBC count by the average number of WBCs per oil field.

6. Add the numbers obtained in step 5 and divide by 30 (the number of observations in this analysis) to obtain the average ratio of the WBC count-to-WBCs per oil-immersion field.

7. Round the number calculated in step 6 to the nearest whole number to obtain an estimation factor.

Modified from Stiene-Martin EA: Clinical hematology: principles, procedures, correlations, ed 2, Philadelphia, 1998, Lippincott.

BOX 14-3

How to Perform a White Blood Cell (WBC) Estimate

1. Scan a thin area, using the 50× oil immersion lens.

2. Observe 10 fields, counting all WBCs in each field.

3. Average the number of WBCs seen per oil immersion field.

4. Multiply the number of WBCs per oil immersion field by 3000 and compare with the WBC count.

From U.S. Army Medical Department Center and School Standard Operating Procedure, Fort Sam Houston, Texas, November 1, 2001. Other methods of WBC estimation are discussed in Chapter 15.

Platelet Count

A phase-contrast microscope is used in the reference method for performing a manual platelet count. Platelets are adhesive to foreign objects and to each other, which makes it difficult to count them. They also are small and can be confused easily with dirt. In this procedure, whole blood, with EDTA as the anticoagulant, is diluted with 1% ammonium oxalate, which lyses the nonnucleated erythrocytes. The platelets can be counted with the use of a phase-contrast microscope as described by Brecher and Cronkite.¹

Procedure

1. Make a 1 : 100 dilution, placing 20 mcL of well-mixed blood into 1980 mcL of ammonium oxalate in a small test tube.

2. Mix the dilution thoroughly and charge the chamber. (Note: A special thin flat-bottomed counting chamber is used for phase platelet counts.)

3. Place the charged hemacytometer in a moist chamber (see Box 14-1) for 15 minutes to allow the platelets to settle.

4. Platelets are counted using the 40× objective lens. The platelets have a diameter of 2 to 4 µm and appear round or oval, displaying a light purple sheen when phase-contrast microscopy is used. The shape and color help distinguish the platelets from highly refractile dirt and debris. “Ghost” RBCs often are seen in the background.

5. Count the 25 small squares in the center square of the grid (see Figure 14-1). The area of this center square is 1 mm². Each side of the hemacytometer should be counted, and the difference between the totals should be less than 10%.

6. Calculate the number of platelets per liter by using the first equation given earlier. For example, if 200 platelets were counted in the entire center square,

200×1001×0.1=200,000/mm3or200×109/L

7. Platelet counts should be verified by performing an estimate on the Wright-stained peripheral blood smear (see Chapter 15).

General reference ranges for males and females according to age groups can be found on the inside front cover of this text.

Sources of Error and Comments

1. Inadequate mixing and poor collection of the sample can cause the platelets to clump on the hemacytometer. If the problem persists after redilution, a new sample is needed. A finger-stick sample is less desirable because of the adhesive quality of the platelets.

2. Dirt in the pipette, hemacytometer, or diluting fluid may cause the counts to be inaccurate.

3. If fewer than 50 platelets are counted on each side, the procedure should be repeated by diluting the blood to 1 : 20 using calibrated pipettes. If more than 500 platelets are counted on each side, a 1 : 200 dilution should be made. The appropriate dilution factor should be used in calculating the results.

4. If the patient has a normal platelet count, the red cell area (see Figure 14-1) may be counted. Then, the area is 0.2 mm² on each side.

5. The phenomenon of “platelet satellitosis” may occur when EDTA anticoagulant is used. This refers to the adherence of platelets around neutrophils, producing a ring or satellite effect (see Figure 15-1). Using sodium citrate as the anticoagulant should correct this problem. Because of the dilution in the citrate tubes, it is necessary to multiply the obtained platelet count by 1.1 for accuracy (see Chapter 15).

Erythrocyte Counts

Manual erythrocyte counts are rarely performed because of the inaccuracy of the count and questionable necessity. Use of other, more accurate manual RBC parameters, such as the microhematocrit and hemoglobin concentration, is desirable. Table 14-1 contains information on performing manual erythrocyte counts.

TABLE 14-1

Manual Cell Counts with Most Common Dilutions, Counting Areas

Cell Counted	Diluting Fluid	Dilution	Objective	Area Counted
Leukocyte	Weak acid	1 : 20	10× or 40×	4 mm²
Leukocyte	Weak acid	1 : 100	10×	9 mm²
Erythrocyte	Isotonic saline	1 : 100	40×	0.2 mm² (5 small squares of center mm²)
Platelet	Ammonium oxalate	1 : 100	40× phase	1 mm²