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:
Figure 7-9.—Puncturing the diaphragm of diluent with the capillary pipette shield.
Figure 7-10.—Drawing blood into the Unopette capillary
A. From a finger puncture;
B. From a venous blood sample.
Figure 7-11.—Preparing reservoir to receive blood from the capillary tube.
Figure 7-12.—Mixing blood sample and diluent.
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.
Figure 7-16.—Hemacytometer counting chamber.
Total number of cells counted = 423.
Total red cell count = 4,2300,000 cells/mm3
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.
To perform the microhematocrit method, you should follow the steps listed below:
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:
- a shielded capillary pipette (20 microliter (1ul) capacity), and
- a plastic reservoir containing a premeasured volume of diluent (1:100 dilution).
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:
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
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:
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:
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.
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.
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:
Figure 7-19.—Placement of immersion oil on blood smear.
Patient has 1,600 lymphocytes/mm3
Figure 7-20.—Counting path for differential count.
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:
|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
|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|
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: