7-4 COMPLETE BLOOD COUNT

LEARNING OBJECTIVE: Identify the five parts of a complete blood count, and recognize the testing procedures for the following: Unopette® Red Blood Cell Count, Microhematocrit, Unopette White Blood Cell Count, and Differential White Blood Cell Count.

A complete blood count consists of the following five tests:

The complete blood count, commonly referred to as a CBC, is used in the diagnosis of many diseases. Blood collected for these tests are capillary or peripheral blood and venous blood. CBCs may be performed either manually or by using automated hematology analyzers. The manual method is used in isolated locations and on board some naval vessels where a hematology analyzer installation is not practical. For this reason, and because machines break down on occasion, the manual method will be covered in the following sections.

COUNTING BLOOD CELLS

To manually count red blood cells (erythrocytes) and white blood cells (leukocytes), you will need a microscope and an instrument called a hemacytometer. See figure 7-6. The hemacytometer is a thick glass slide with three raised parallel platforms on the middle third of the device. The central platform is subdivided by a transverse groove to form two halves, each wider than the two lateral platforms and separated from them and from each other by moats. The central platforms each contain a counting chamber and are exactly 0.1 mm lower than the lateral platforms.

Each counting chamber has precisely ruled lines etched into the glass, forming a grid. This grid or ruled area is so small that it can only be seen with the aid of a microscope. The grid used by most laboratories is the Improved Neubauer Ruling. See figure 7-7 for an example of the Improved Neubauer Ruling. The Improved Neubauer Ruling is 3 by 3 mm (9 mm2) and subdivided into nine secondary squares, each 1 by 1 mm (1 mm2).

A thick cover glass, ground to a perfect plane, accompanies the counting chamber (fig. 7-6). Ordinary cover glasses have uneven surfaces and should not be used. When the cover glass is in place on the platform of the counting chamber, there is a space exactly 0.1 mm thick between it and the ruled platform.

 

Figure 7-6.—Top and side views of a hemacytometer.

Counts of red blood cells and white blood cells are each expressed as concentration: cells per unit volume of blood. The unit of volume for cell counts is expressed as cubic millimeters (mm3) because of the linear dimensions of the hemacytometer chamber.

TOTAL RED BLOOD CELL COUNT

The total red blood cell (erythrocyte) count is the number of red cells in one cubic millimeter of blood. The normal red blood cell count is as follows:

Adult male............. . .4.2 to 6.0 million per mm
Adult female ............ .3.6 to 5.6 million per mm
Newborn. ............... .5.0 to 6.5 million per mm
 

Figure 7-7.—Improved Neubauer Ruling.

As we said earlier, the red cell count is used in the diagnosis of many diseases. For example, a red cell count that drops below normal values may indicate anemia and leukemia. On the other hand, a red cell count that rises above the normal values may indicate dehydration.

The Unopette® Method is used to manually count red blood cells. Material requirements and the step-by-step procedures for performing this procedure are provided in the following sections.

Materials Required for Unopette Procedure

The Unopette procedure consists of a disposable diluting pipette system that provides a convenient, precise, and accurate method for obtaining a red blood cell count. To perform a red blood cell count using the Unopette method, you will need to obtain the following materials:

Figure 7-8.—Unopette® for RBC count.

The Unopette procedure for counting red blood cells is as follows:

  1. Puncture the diaphragm in the neck of the diluent reservoir with the tip of the capillary shield on the capillary pipette. See figure 7-9.

Figure 7-9.—Puncturing the diaphragm of diluent with the capillary pipette shield.

  1. After obtaining free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary pipette. Holding the capillary pipette slightly above the horizontal, touch the tip to the blood source (see fig. 7-10, view A). The pipette will fill by capillary action. When blood reaches the end of the capillary bore in the neck of the pipette, filling is complete and will stop automatically. The amount of blood collected by the capillary tube is 10 ml. Wipe any blood off the outside of the capillary tube, making sure no blood is removed from inside the capillary pipette. (An alternative source of blood is a thoroughly mixed fresh venous blood sample obtained by venipuncture. See figure 7-10, view B.)

 

 Figure 7-10.—Drawing blood into the Unopette capillary tube:
A. From a finger puncture;
B. From a venous blood sample.

  1. With one hand, gently squeeze the reservoir to force some air out, but do not expel any diluent (fig. 7-11). Maintain pressure on the reservoir. With the other hand, cover the upper opening of the capillary overflow chamber with your index finger and seat the capillary pipette holder in the reservoir neck (see fig. 7-11).

Figure 7-11.—Preparing reservoir to receive blood from the capillary tube.

  1. Release pressure on the reservoir and remove your finger from the overflow chamber opening. Suction will draw the blood into the diluent in the reservoir.
  2. Squeeze the reservoir gently two or three times to rinse the capillary tube, forcing diluent into but not out of the overflow chamber, releasing pressure each time to return diluent to the reservoir. Close the upper opening with your index finger and invert the unit several times to mix the blood sample and the diluent. See figure 7-12.

Figure 7-12.—Mixing blood sample and diluent.

  1. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield.
  2. Immediately prior to cell counting, mix again by gentle inversion, taking care to cover the upper opening of the overflow chamber with your index finger.
  3. Place the cover glass on the hemacytometer counting chamber, making sure cover glass is clean and free of grease. (Fingerprints must be completely removed.)
  4. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position, releasing pressure to draw any fluid in the capillary tube into the reservoir. Invert and fill the capillary pipette by gentle pressure on the reservoir. After discarding the first 3 drops, load (charge) the counting chamber of the hemacytometer by gently squeezing the reservoir while touching the tip of the pipette against the edge of the cover glass and the surface of the counting chamber (fig. 7-13). A properly loaded counting chamber should have a thin, even film of fluid under the cover glass (fig. 7-14, view A). Allow 3 minutes for cells to settle. If fluid flows into the grooves (moats) at the edges of the chamber or if air bubbles are seen in the field, the chamber is flooded and must be cleaned with distilled water, dried with lens tissue, and reloaded (fig. 7-14, view B). If the chamber is underloaded, carefully add additional fluid until properly loaded.

Figure 7-13.—Loading the counting chamber.

Figure 7-14.—Loading hemacytometer:
A. Hemacytometer properly loaded;
B. Hemacytometer improperly loaded.

10. Place the loaded hemacytometer into apetri dish with a piece of dampened tissue to keep the hemacytometer from drying out (fig. 7-15). Allow 5 to 10 minutes for the cells to settle.

Figure 7-15—Loaded hemacytometer placed inside petri dish.

  1. Once the cells have settled, place the hemocytometer on the microscope. Use the low-power lens to locate the five small fields (1, 2,3,4, and 5) in the large center square bounded by the double or triple lines. See figure 7-16. Each field measures 1/25 mm2, 1/10 mm in depth, and is divided into 16 smaller squares. These smaller squares form a grid that makes accurate counting possible.
  2. Switch to the high-power lens and count the number of cells in field 1. Move the hemacytometer until field 2 is in focus and repeat the counting procedure. Continue until the cells in all five fields have been counted. Note the fields are numbered clockwise around the chamber, with field 5 being in the center. Count the fields in this order. To count the cells in each field, start in the upper left small square and follow the pattern indicated by the arrow in field 1 of figure 7-16. Count all of the cells within each square, including cells touching the lines at the top and on the left. Do not count any cells that touch the lines on the right or at the bottom.

Figure 7-16.—Hemacytometer counting chamber.

  1. Total the number of cells counted in all five fields and multiply by 10,000 to arrive at the number of red cells per cubic millimeter of blood.

Example

Total number of cells counted = 423.

Multiply: 423 x 10,000 = 4,230,000

Total red cell count = 4,2300,000 cells/mm3

 

NOTE

The number of cells counted in each field should not vary by more than 20. A greater variation may indicate poor distribution of the cells in the fluid, resulting in

HEMOGLOBIN DETERMINATION

A routine test performed on practically every patient is the hemoglobin determination. Hemoglobin determination, or hemoglobinometry, is the measurement of the concentration of hemoglobin in the blood. Hemoglobin’s main function in the body is to carry oxygen from the lungs to the tissues and to assist in transporting carbon dioxide from the tissues to the lungs. The formation of hemoglobin takes place in the developing red cells located in bone marrow.

Hemoglobin values are affected by age, sex, pregnancy, disease, and altitude. During pregnancy, gains in body fluids cause the red cells to become less concentrated, causing the red cell count to fall. Since hemoglobin is contained in red cells, the hemoglobin concentration also falls. Disease may also affect the values of hemoglobin. For example, iron deficiency anemia may drop hemoglobin values from a normal value of 14 grams per 100 milliliters to 7 grams per 100 milliliters. Above-normal hemoglobin values may occur when dehydration develops. Changes in altitude affect the oxygen content of the air and, therefore, also affect hemoglobin values. At higher altitudes there is less oxygen in the air, resulting in an increase in red cell counts and hemoglobin values. At lower altitudes there is more oxygen, resulting in a decrease in red cell counts and hemoglobin values.

The normal values for hemoglobin determinations are as follows:

  Grams per 100 ml blood Percent
Women. 12.5 to 15. 83 to 110
Men 14 to 17 97 to 124
Newborns 17 to 23  97 to 138

Methods for hemoglobin determination are many and varied. The most widely used automated method is the cyanmethemoglobin method. To perform this method, blood is mixed with Drabkin’s solution, a solution that contains ferricyanide and cyanide. The ferricyanide oxidizes the iron in the hemoglobin, thereby changing hemoglobin to methemoglobin. Methemoglobin then unites with the cyanide to form cyanmethemoglobin. Cyanmethemoglobin produces a color which is measured in a colorimeter, spectrophotometer, or automated instrument. The color relates to the concentration of hemoglobin in the blood.

Manual methods for determining blood hemoglobin include the Haden-Hausse and Sahli-Hellige methods. In both methods, blood is mixed with dilute hydrochloric acid. This process hemolyzes the red cells, disrupting the integrity of the red cells’ membrane and causing the release of hemoglobin, which, in turn, is converted to a brownish-colored solution of acid hematin. The acid hematin solution is then compared with a color standard.

HEMATOCRIT (PACKED CELL VOLUME) DETERMINATION

The hematocrit or packed cell volume (PCV) determines the percentage of red blood cells (RBCs) in whole blood.

The normal hematocrit value for men is 42% to 52%; for women, 37% to 47%; and for newborns, 53% to 65%. When hematocrit determinations are below normal, medical conditions such as anemia and leukemia may be present. Above-normal hematocrit determinations indicate medical conditions like dehydration, such as occur in severe burn cases.

Currently, automated hematology analyzers supply most hematocrits. However, when hematology analyzers are not available, hematocrit determinations can be manually performed by the microhematocrit method or macrohematocrit method. Both methods call for the blood to be centrifuged, and the percentage of packed red cells is found by calculation.

The microhematocrit method is the most accurate manual method of determining blood volume and should be used whenever feasible. Material requirements and the step-by-step procedures for performing the microhematocrit method will be covered in the following sections.

Materials Required for Microhematocrit Procedure

To perform a hematocrit using the micro­hematocrit method, the following materials are required.

Microhematocrit Procedure

To perform the microhematocrit method, you should follow the steps listed below:

  1. Fill the capillary tube two-thirds to three-quarters full with well-mixed, oxalated venous blood or fingertip blood. (For fingertip blood use heparinized tubes, and invert several times to mix.)
  2. Seal one end of the tube with clay.
  3. Place the filled tube in the microhematocrit centrifuge, with the plugged end away from the center of the centrifuge.
  4. Centrifuge at a preset speed of 10,000 to 12,000 rpm for 5 minutes. If the hematocrit exceeds 50 percent, centrifuge for an additional 3 minutes.
  5. Place the tube in the microhematocrit reader. Read the hematocrit by following the manufacturer’s instructions on the microhematocrit reading device.

TOTAL WHITE BLOOD CELL COUNT

The total white cell (leukocyte) count determines the number of white cells per cubic millimeter of blood. A great deal of information can be derived from white cell studies. The white blood cell count (WBC) and the differential count are common laboratory tests, and they are almost a necessity in determining the nature and severity of systemic infections. Normal WBC values in adults range from 4,500 to 11,000 cells per cubic millimeter; in children the range is from 5,000 to 15,000 cells per cubic millimeter; and in newborns the range is from 10,000 to 30,000 cells per cubic millimeter.

White blood cell counts are performed either manually or with automated hematology analyzers. Only the manual method will be covered in this chapter. After a brief discussion on abnormal white blood cell counts, we will cover the Unopette method for manually counting white blood cells.

Abnormal White Cell Counts

When white cell counts rise above normal values, the condition is referred to as leukocytosis. Leukocytosis frequently occurs when systemic or local infections (usually due to bacteria) are present. Counts for infections are highly variable. Examples of some infections and their representative white cell counts are as follows:

Dyscrasia (the diseased condition) of blood-forming tissues, such as occurs in leukemia (due to a malfunctioning of lymph and marrow tissues) also results in leukocytosis, with extremely high white cell counts. These white cell counts sometimes exceed 1,000,000/mm3.

Other physiological conditions that can cause leukocytosis and a white cell count as high as 15,000/mm3 may occur as follows:

An abnormally low count, known as leukopenia, may be caused by the following conditions:

Materials Required for Unopette Procedure

The Unopette method uses a disposable diluting pipette system that provides a convenient, precise, and accurate method for obtaining a white blood cell count. When the Unopette method is used, whole blood is added to a diluent. The diluent lyses (destroys) the red blood cells, but preserves the white blood cells. Once the red cells are completely lysed, the solution will be clear. The diluted blood is then added to a hemacytometer. Once the hemacytometer is loaded, the cells should be allowed to settle for 10 minutes before counting proceeds.

The following materials are required to perform a white blood cell count using the Unopette method:

Unopette Procedure

The Unopette disposable diluting pipette system used to count WBCs is almost identical in shape and application to the Unopette system for RBC counts. The only major difference is that the reservoir contains a different diluent and the capillary pipette capacity differs (RBC 10 l and WBC 20 l). To assist you in performing the Unopette procedure for WBCs, we will refer to illustrations for the Unopette procedure for RBCs in this section.

The Unopette procedure for counting white blood cells is as follows:

  1. Puncture the diaphragm in the neck of the reservoir with the tip of the capillary pipette shield. See figure 7-9.
  2. After you obtain free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary pipette. Hold the capillary pipette slightly above the horizontal and touch the tip to the blood source (fig. 7-10, view A). The pipette will fill by capillary action. When blood reaches the end of the capillary bore in the neck of the pipette, filling is complete and will stop automatically. The amount of blood collected by the capillary tube is 20 l. Wipe any blood off the outside of the capillary tube, making sure no blood is removed from inside the capillary pipette. (An alternative source of blood is a thoroughly mixed fresh venous blood sample obtained by venipuncture. See figure 7-10, view B.)
  3. With one hand, gently squeeze the reservoir to force some air out, but do not expel any diluent (fig. 7-11). Maintain pressure on the reservoir. With the other hand, cover the upper opening of the capillary overflow chamber with your index finger and seat the capillary pipette holder in the reservoir neck (fig. 7-11).
  4. Release pressure on the reservoir and remove your finger from the overflow chamber opening. Suction will draw the blood into the diluent in the reservoir.
  5. Squeeze the reservoir gently two or three times to rinse the capillary tube, forcing diluent into but not out of the overflow chamber, releasing pressure each time to return diluent to the reservoir. Close the upper opening with your index finger and invert the unit several times to mix the blood sample and diluent. See figure 7-12.
  6. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield.
  7. Immediately prior to cell counting, mix again by gentle inversion, taking care to cover the hole with your index finger.
  8. Place the coverglass on the hemacytometer counting chamber, making sure the coverglass is clean and grease-free. (Fingerprints must be completely removed.)
  9. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position. Release pressure to draw any fluid in the capillary tube into the reservoir. Invert and fill the capillary pipette by gentle pressure on the reservoir. After discarding the first 3 drops, load (charge) the counting chamber of the hemacytometer by gently squeezing the reservoir while touching the tip of the pipette against the edge of the coverglass and the surface of the counting chamber (fig. 7-13). A properly loaded counting chamber should have a thin, even film of fluid under the coverglass (fig. 7-14, view A). Allow 3 minutes for the cells to settle. If fluid flows into the grooves (moats) at the edges ofthe chamber or if you see air bubbles in the field, the chamber is flooded and must be cleaned with distilled water, dried with lens tissue, and reloaded (fig. 7-14, view B). If the chamber is underloaded, carefully add additional fluid until properly loaded.
  10. Place the loaded hemacytometer into apetri dish with a piece of dampened tissue to keep the hemacytometer from drying out (fig. 7-15). Allow 5 to 10 minutes for the cells to settle.
  11. Once the cells have settled, place the hemacytometer on the microscope. Using the high-power objective, count the WBCs in the four corner fields of the hemacytometer chamber (fields A, B, C, and D of figure 7-16). Each field is composed of 16 small squares. To count the cells in each field, start in the upper left small square and follow the pattern indicated by the arrow in field B of figure 7-16. Count all of the cells within each square, including cells touching the lines at the top and on the left. Do not count any cells that touch the lines on the right or at the bottom.
  12. When all the cells in the 4 fields have been counted, multiply the count by 50. This will give you the total number of white cells per cubic millimeter of blood.

Example

25 cells in field #1
23 cells in field #2 26 cells in field #3 26 cells in field #4
Total red cell count = 4,2300,000 cells/mm3
100 total cells in all fields

Multiply:

100x50 =5,000

Total white cell count = 5,000 cells/mm3

DIFFERENTIAL WHITE BLOOD CELL COUNT

A total white blood cell count is not necessarily indicative of the severity of a disease, since some serious ailments may show a low white cell count. For this reason, a differential white cell count is performed. A differential white cell count consists of an examination of blood to determine the presence and the number of different types of white blood cells. This study often provides helpful information in determining the severity and extent of an infection, more than any other single procedure used in the examination of the blood.

The role of white blood cells, or leukocytes, is to control various disease conditions. Although these cells do most of their work outside the circulatory system, they use the blood for transportation to sites of infection.

Five types of white cells are normally found in the circulating blood. They are

Cell Identification

To perform a differential white cell count, you must be able to identify the different types of white cells. The ability to properly identify the different types of white cells is not difficult to develop, but it does require a thorough knowledge of staining characteristics and morphology (the study of the form and structure of organisms). This knowledge can be gained only by extensive, supervised practice.

To acquaint you with the developmental stages of each type of leukocyte, a colorized illustration (fig. 7-17) has been provided. This illustration also displays the developmental stages of the red blood cell (erythrocyte) and the blood platelet cell (thrombocyte). To further assist you, identifying characteristics of each type of leukocyte as they appear on a stained blood smear will be covered in the following sections.

Figure 7-17.—Development of blood cells.

Laboratories use a blood smear to obtain a differential white cell count. To prepare a blood smear, a blood specimen is spread across a glass slide, stained to enhance leukocyte identification, and examined microscopically. Material requirements and the step-by-step procedure for performing a blood smear will be covered later in this chapter.

NEUTROPHILS.—Neutrophils account for the largest percentage of leukocytes found in a normal blood sample, and function by ingesting invading bacteria. On a stained blood smear, the cytoplasm of a neutrophil has numerous fine, barely visible lilac-colored granules and a dark purple or reddish purple nucleus (see figure 7-17). The nucleus may be oval, horseshoe, or “S”-shaped, or segmented (lobulated). Neutrophils are subclassified according to their age or maturity, which is indicated by changes in the nucleus. The subclassifications for neutrophilic cells are metamyelocyte, band, segmented, and hypersegmented.

Neutrophilic Metamyelocyte.—A neutrophilic metamyelocyte, also called a “juvenile” cell, is the youngest neutrophil generally reported. The nucleus is fat, indented, and is usually “bean”-shaped or “cashew nut”-shaped (fig. 7-17).

Neutrophilic Band.—A neutrophilic band, sometimes called a “stab” cell, is an older or intermediate neutrophil. The nucleus has started to elongate and has curved itself into a horseshoe or S-shape. As the band ages, it matures into a segmented neutrophil (fig. 7-17).

Segmented Neutrophil.—A segmented neutrophil is a mature neutrophil. The nucleus of a segmented neutrophil is separated into two, three, four, or five segments or lobes (fig. 7-17).

Hypersegmented Neutrophil.—A hyperseg­mented neutrophil is a mature neutrophil. The nucleus of a hypersegmented neutrophil is divided into six or more segments or lobes (fig. 7-17).

EOSINOPHIL.—Eosinophils aid in detoxifi­cation. They also break down and remove protein material. The cytoplasm of an eosinophil contains numerous coarse, reddish-orange granules, which are lighter colored than the nucleus (fig. 7-17).

BASOPHIL.—The function of basophilic cells is unknown. It is believed, however, that basophilic cells keep the blood from clotting in inflamed tissue. Scattered large, dark-blue granules that are darker than the nucleus, characterize the cell as a basophil (fig. 7-17). Granules may overlay the nucleus as well as the cytoplasm.

LYMPHOCYTE.—The function oflymphocytes is also unknown, but it is believed that they produce antibodies and destroy the toxic products of protein metabolism. The cytoplasm of a lymphocyte is clear sky blue, scanty, with few unevenly distributed, azurophilic granules with a halo around them (fig. 7-17). The nucleus is generally round, oval, or slightly indented, and the chromatin (a network of fibers within the nucleus) is lumpy and condensed at the periphery.

MONOCYTE.—The monocyte, the largest of the normal white blood cells, destroys bacteria, foreign particles, and protozoa. Its color resembles that of a lymphocyte, but its cytoplasm is a muddy gray-blue (fig. 7-17). The nucleus is lobulated, deeply indented or horseshoe-shaped, and has a relatively fine chromatin structure. Occasionally, the cytoplasm is more abundant than in the lymphocyte.

Materials Required for the Differential Count Procedure

To perform a differential count, the following materials are required:

The procedure for the differential white cell count is done in 4 steps:

  1. Make the blood smear
  2. Stain the cells
  3. Count the cells
  4. Report the count

Each step of this procedure will be discussed in the following sections.

MAKING THE BLOOD SMEAR.—The simplest way to count the different types of white cells is to spread them out on a glass slide. The preparation is called a blood smear. There are two methods of making a blood smear: the slide method (covered in this chapter) and the cover glass method.

It is very important to make a good blood smear. If it is made poorly, the cells may be so distorted that it will be impossible to recognize them. You should make at least two smears for each patient, as the additional smear should be examined to verify any abnormal findings.

To prepare a blood smear for a differential count:

  1. Using a capillary tube, collect anticoagulated blood from a venous blood sample.
  2. Deposit a drop of blood from capillary tube onto a clean, grease-free slide. Then place the slide on a flat surface, blood side up.
  3. Hold a second slide between your thumb and forefinger and place the edge at a 23-degree angle against the top of the slide that holds the drop of blood (see figure 7-18, view A). Back the second slide down until it touches the drop of blood. The blood will distribute itself along the edge of the slide in a formed angle (see figure 7-18, view B).
  4. Push the second slide along the surface of the other slide, drawing the blood across the surface in a thin, even smear (see figure 7-18, view C). If this is done in a smooth, uniform manner, a gradual tapering effect (or “feathering”) of the blood will occur on the slide. This “feathering” of the blood is essential to the counting process and is the principal characteristic of a good blood smear (see figure 7-18, view D). When you are making the smear, prevent blood from reaching the extreme edges of the slides. Allowing the smear to reach the edges of the slide will aggravate the tendency of large cells to stack up on the perimeter of the smear. A smear with wavy lines or blanks spots should be discarded, and a new smear made.
  5. Once the blood smear is made, let it dry (it will take a few minutes). Then write the patient’s name in pencil on the bottom edge of the slide, as illustrated in figure 7-18, view D). Proceed to step 2, staining the cells.

Figure 7-18.—Making a blood smear:
A. Placing second slide at a 23 °angle; B. Blood distributing itself along second slide’s edge;
C. Drawing blood across surface of slide; D. Example of a properly prepared blood smear.

STAINING THE CELLS.—Once a blood smear is made, it should be stained. Staining the blood smear highlights the differences among the different types of leukocytes for easier recognition during the counting process. The most popular stain used for this purpose is Wright’s stain. Wright’s stain is a methyl alcohol (methanol) solution of an acid dye and a basic dye. The acid dye in Wright’s stain is known as eosin and is red in color. The basic dye in Wright’s stain is known as methylene blue and is blue in color. Generally, white cells are identified by their affinity to the dye they prefer. For example, cells that prefer the acid dye (eosin) are called eosinophils. Other cells that prefer the basic dye are called basophils.

WARNING

Wright’s staining solution contains methanol, which is considered a hazardous material. It is classified as flammable, a poison, and an irritant. Methanol must be kept away from heat, sparks, and open flames. Good ventilation in usage areas is paramount since exposure to vapors can irritate eyes, nose, throat, and mucous membranes of the upper respiratory tract. When not in use, methanol containers should be closed tightly and stored upright to prevent leakage. Gloves and protective clothing (e.g., lab coat or apron) and eyewear should be worn to avoid contact with the solution. Absorption through skin can cause permanent blindness. Death may result from ingestion or exposure to high vapor concentrations of methanol.

There are a variety of staining products on the market today. Some of these staining products have combined Wright’s solution with other staining solutions, such as Giemsa stain. When using a new product, you should always review the manufacturer’s usage and safety recommendations.

The staining process that we will cover in this chapter is known as a quick stain. A quick stain has very few equipment requirements and only a few procedural steps. An example of a quick stain is One Step II Wright-Giemsa Stain Solution® by Criterion Sciences. To stain a blood smear with this product, follow the steps below.

  1. Prepare two staining containers by filling one with One Step II stain solution and the other with deionized or distilled water. The use of tap water instead of deionized or distilled water is not recommended since the pH of tap water varies. If tap water is used, its pH should between 5.8 and 7.03.
  2. Immerse the slide (blood smear) in the stain for 15 to 30 seconds. (To prevent debris or precipitate from contaminating the slide, do not add new stain to old.)
  3. Remove the slide and allow excess stain to drain from the edge of the slide.
  4. Immerse the slide in the deionized or distilled water for 5 to 15 seconds. (Change the water when it becomes dark blue or when film forms on the surface.)

NOTE

Rinse time is critical and must be shorter than the stain time.

  1. Drain excess water and wipe the back of the slide to reduce background color.
  2. Place slide in horizontal position on table and allow to air dry.

NOTE

Do not attempt to accelerate drying time by placing slide on a warmer or in front of a fan. The film of water on the slide is important for the color development.

  1. Once the slide is dry, proceed to step 3, counting the cells.

COUNTING THE CELLS.—Once the blood smear has been stained, it is placed under a microscope, and the differential count is conducted.

To perform a differential white cell count:

  1. Place the slide under the microscope. Switch the oil immersion objective (red) (100X) into position above the stage. Turn the coarse adjustment to raise the oil immersion objective about 1 inch above the opening in the stage. Open the condenser and switch on the microscope light.
  2. Place a large drop of immersion oil on the thin area of the blood smear. See figure 7-19.

Figure 7-19.—Placement of immersion oil on blood smear.

  1. Hold the slide so the thin area is on your left. Then fix the slide firmly in the jaws of the mechanical (movable) stage. Move the mechanical stage so the drop of oil on the slide is directly over the bright light coming up from the condenser.
  2. Using the coarse control knob, you should now slowly lower oil immersion objective into the drop of oil (on the slide). When the objective is in the drop of oil, continue turning the coarse adjustment until the objective is touching the glass slide.
  3. Now, while continually looking through the eyepiece, VERY SLOWLY rotate the coarse adjustment toward you until you see some cells. After you have brought the cells into view with the coarse adjustment, bring the cells into perfect focus by rotating the fine adjustment.

NOTE

Always rotate the fine adjustment back and forth when identifying cells. This step will help you see the various layers of the cell and thereby help you to identify the different types of white cells.

  1. Count 100 consecutive white cells, pressing the correct key on the cell counter for each type of white cell identified. (If the cell counter is not available, record cell type and number of cells encountered on a piece of paper.) Follow path similar to one illustrated in figure 7-20 to count cells.
  2. Total each type of white cell. If you count 20 lymphocytes among the 100 cells, the differential count for lymphocytes is 20%. Continue this process until your count totals 100%. This differential count is referred to as a relative count. Another differential count that may be requested is an absolute count. To perform an absolute count, multiply the total white cell count by the individual cell percentages. See the example below.

Example

Patient has a total white cell count of 8,000.
Differential count shows 20% leukocytes.

Multiply:

8,000 x 0.20 (20%) = 1,600

Patient has 1,600 lymphocytes/mm3

 

Figure 7-20.—Counting path for differential count.

NOTE

When performing the white cell count, you may observe abnormal white cells such as distorted lymphocytes, smudge cells, and disintegrated cells. Distorted lymphocytes, which appear squashed or distorted, are caused by excessive pressure on the cell during the process of making the smear. Distorted cells should be recorded as normal lymphocytes. Smudge cells are white cells that have ruptured and only the nucleus remains. A few smudge cells may be found in a normal blood smear. Smudge cells should not be added to the count or recorded. Disintegrated cells are ruptured cells, but the nucleus and cytoplasm still remain. Disintegrated cells should not be counted as one of the 100 cells, but should be recorded on the report as “disintegrated cells.”

  1. Once the differential count is completed, proceed to step 4, reporting the count.

NOTE

If it is desirable to save a smear for reexamination, remove the immersion oil by placing a piece of lens tissue over the slide and moistening the tissue with xylene. Draw the damp tissue across the slide, and dry the smear with another piece of lens paper.

REPORTING THE COUNT.—When you have calculated the differential count, the report is given according to either the Schilling classification or filament and nonfilament classification methods. We will be covering the Schilling classification, since it is the simplest and most popular method.

The Schilling Classification.—The Schilling classification was established when Victor Schilling, a German hematologist, noticed that in many diseases there is an increase in the percentage of immature neutrophils. The blood chart he developed reported the percentages of the different neutrophilic cell types and (in part) was arranged in the following manner:

Normal
%
Myelocytes Metamyelocytes Band Cells Segmented
Cells
0 0 2 to 6 55 to 75

Note that the immature cells are on the left side of the chart. If percentages of immature cell increased, Schilling referred it as a “shift to the left.” When theshift to the left was accompanied by a low white cell count, Schilling called it a “degenerative shift to the left.” A degenerative shift to the left is seen in such diseases as typhoid fever. This shift is caused by a depression of the cell factories in the bone marrow.

When the shift to the left is accompanied by a high white cell count, it is called a “regenerative shift to the left.” A regenerative shift to the left is seen in such diseases as pneumonia. This shift is caused by a stimulus of the cell factories in the bone marrow.

A “shift to the right” implies an increase in hypersegmented neutrophils. It may be seen in pernicious anemia, an anemia caused by the malabsorption of vitamin B12.

The Schilling classification for an adult differential white cell count is provided in table 7-2.

Table 7-2.—Schilling Classification of the Differential White Cell Count

Cell Normal %
Neutrophilic myelocytes 0
Neutrophilic metamyelocytes 0
Neutrophilic band cells 2 to 6
Neutrophilic segmented cells 55 to 75
Lymphocytes 20 to 35
Monocytes 2 to 6
Eosinophilic segmented cells 1 to 3
Basophilic segmented cells 0 to 1
 

NOTE

Normal values for differential counts vary with the age of the patient. For example, children’s blood normally contains 0% to 2% basophils, 0% to 5% eosinophils, 25% to 75% neutrophils, 30% to 70% lymphocytes, and 0% to 8% monocytes. Normal values may also be adjusted by hospitals that have evaluated the normal differential value for their local population.

General Interpretations of Leukocyte Changes.—Together, the total white cell count and differential count aid physicians in interpreting the severity of infections. Some general interpretations of leukocyte changes are as follows: