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Specimens

INTRODUCTION

Blood cells are subject to quantitative variations as well as the qualitative variations described in Lesson 4. Some diseases stimulate the production of blood cells while others prevent or diminish the production of blood cells. For this reason a cell count gives valuable information to the physician concerning his patient's condition. Furthermore, in the case of the leukocyte count, the total count is necessary to calculate absolute counts for each type of leukocyte. This is done by multiplying the total count by the percentage of the particular cell type.

Cell counts can be performed by a variety of methods. Erythrocytes and leukocytes are counted by manual methods or automated methods. Other cell counts are performed only by manual methods. It is important when performing a cell count to maintain good quality control. Great care should be taken when performing any cell count.

The following paragraphs outline procedures for white blood cell (WBC) count, total eosinophil count, and reticulocyte count. The WBC counts are routinely done; they are performed either by the hemacytometer method (manually) or by automated methods. Total eosinophil counts are performed by a hemacytometer method (manually) using special diluting fluids to accentuate these cells. Reticulocytes are demonstrated by using a supravital stain. Semen analysis and cerebrospinal fluid (CSF) counts use a hemacytometer to perform the procedure as well. They are included in the next Lesson.

WHITE BLOOD CELL COUNT

Unopette 1: 20 or 1:100 dilution.

Principle

 Whole blood is mixed with a weak acid solution to dilute the blood and hemolyze the red blood cells. Then loaded into a Hemacytometer and counted.

Specimen

Whole blood may be obtained from a venous EDTA sample or a free flowing capillary puncture and diluted 1:100 with a Unopette.

Reagents

A prepared kit is available that uses the Unopette system for white blood cell counts. The reservoir contains 3% glacial acetic acid. A 25 ul capillary pipette is used to aspirate the blood sample and make a 1:20 dilution in the reservoir. (Alternatively, the Unopette containing ammonium oxalate and result in a 1:100 dilution may be used). Functions of reagents are:

  • Diluent.
  • Lyse RBCs.
  • Preserve WBCs and platelets.

Procedure

  1. Dilute the specimen - let stand for 10 minutes to allow red cells to hemolyzed.
  2. Expel first 3 to 4 drops of diluted specimen to clean capillary bore.
  3. Charge the Hemacytometer (both sides) with the diluted specimen.
  4. Cells must settle for a minimum 3 to 5 minutes after placing Hemacytometer in moist chamber.
  5. Count white blood cells. See figure 5-1.

Use low power objective and low light.

  • Viewed under low power, leukocyte nuclei appear slightly iridescent but not retractile; cells should have a visible cell wall and nucleus; use fine focus to differentiate them from artifacts.
  • Count all WBCs within the 9 large squares (1:100) and those WBCs touching upper and right-hand perimeter lines.
  • Count second side of Hemacytometer in the same manner.
  • Validation that each side of the chamber was charged equally - Total number of cells counted on each side of the counting chamber should agree within 10 percent of each other - calculate acceptable range using lower count.
NOTES:
  • Count both sides of the Hemacytometer to calculate the average and to provide a more accurate WBC count.
  • Count cells that fall on top and left line of the squares but not the bottom and the right.

Figure 5-1. Hemacytometer counting chamber (WBCs).
Areas marked A, B, C, and D are used to count white blood cells.

Calculation

The Unopette system delivers a dilution of 1:20.

The depth of the counting chamber is 0.1 mm and the area counted is 4 sq mm (4 squares are counted, each with an area of 1.0 sq mm therefore, 4 X 1.0 sq mm = a total of 4 sq mm). The volume counted is: area X depth = volume. 4 mm2 X 0.1 mm = 0.4 mm3 (cubic millimeters).

Here is the formula:

 For example:

Average of the 2 chamber counts. Total the amount and divide by 2:

Sources of Error.

  • Improper collection of blood specimens causes variable results.
  • Wet or dirty pipets.
  • Not allowing cells to settle for an adequate amount of time.
  • Poor pipetting technique causes high or low counts. Poor pipetting technique includes:
  • Undershooting Unopette with blood.
  • Overfilling Unopette with blood.
  • Air bubbles in the shaft.
  • Not mixing the blood specimen thoroughly.
  • Failure to expel 3 or 4 drops in the pipet tips before charging the Hemacytometer.
  • Overfilling the chamber of the hemacytometer, which causes erroneously high counts.
  • Not mixing the diluted specimen prior to filling the Hemacytometer.
  • Uneven distribution of cells in the counting chamber causes erroneous results.
  • Counting artifacts.
  • Dirty or scratched Hemacytometer.
  • Failure to mix anticoagulated blood thoroughly before use.

Discussion.

(1) The counting chamber must be scrupulously clean and free of debris that might be mistaken for cells.

(2) The minimum blood sample recommended for performing routine white blood cell counts is that obtained using one pipet and counting two chambers as previously outlined.

(3) If nucleated erythrocytes are present, the count is corrected by the following formula:

(4) The percent nucleated erythrocyte is obtained from the differential count.

Normal Values

  • Adults (both sexes): 4,500 to 11,500 WBCs per cu mm or 4.5-11.5 X 109 WBCs/L.
  • Childhood: 6,000 to 14,000 WBCs per cu mm or 6.0-14.0 X 109 WBCs/L.
  • Birth: 9,000 to 30,000 WBCs per cu mm or 9.0-34.0 X 109 WBCs / L.

TOTAL EOSINOPHIL COUNT

Principle

A sample of blood is diluted with a solution that selectively stains the eosinophils and eliminates all other leukocytes and erythrocytes from view. Following mixing, the specimen is introduced into the counting chamber and the number of eosinophils in a known volume of blood is counted.

Reagent

A prepared kit is available that uses the Unopette system for absolute eosinophil counts. The reservoir contains phloxine B solution in propylene glycol and distilled water. A 25 ul capillary pipette is used to aspirate the blood sample and make a 1:32 dilution in the reservoir. Alternatively, stains including Pilot’s solution or Randolph’s stain, may be prepared by the laboratory as described elsewhere.

Procedure.

  1. Add the sample for the Unopette pipette to the reservoir.
  2. Mix by gently shaking the pipets for 30 seconds. Prolonged and harsh shaking will tend to cause rupturing of the eosinophils.
  3. Let stand for 10 minutes to allow red cells to hemolyze.
  4. Expel first 3 to 4 drops of diluted specimen to clean capillary bore.
  5. Using one pipet, charge both chambers of a hemacytometer and with the other pipet charge both chambers of the second Hemacytometer.
  6. Allow both hemacytometers to stand for 15 minutes to permit staining of the eosinophils. To prevent evaporation, the hemacytometers are placed on a damp towel and covered with Petri dish covers.
  7. Under low-power magnification, count the red-stained eosinophils in the entire ruled area (9 sq mm) each of the four chambers (a total area of 36 sq mm). The chamber has a depth of 0.1 mm so the total vo1ume is 3.6 cu mm.

Calculations.

Sources of Error.

  • Improper collection of blood specimens causes variable results.
  • Wet or dirty pipets.
  • Not allowing cells to settle for an adequate amount of time.
  • Poor pipetting technique causes high or low counts. Poor pipetting technique includes:
  • Undershooting Unopette with blood.
  • Overfilling Unopette with blood.
  • Air bubbles in the shaft.
  • Not mixing the blood specimen thoroughly.
  • Failure to expel 3 or 4 drops in the pipet tips before charging the Hemacytometer.
  • Overfilling the chamber of the hemacytometer, which causes erroneously high counts.
  • Not mixing the diluted specimen prior to filling the Hemacytometer.
  • Uneven distribution of cells in the counting chamber causes erroneous results.
  • Counting artifacts.
  • Dirty or scratched Hemacytometer.
  • Failure to mix anticoagulated blood thoroughly before use.

Discussion.

(1) Fuchs-Rosenthal or Levy Hemacytometer is preferable to a standard counting chamber since its greater volume (4.0 X 4.0 X 0.2 mm) allows for counting of more cells, thereby reducing the statistical error. Counting two of these chambers is equivalent in accuracy to seven standard chamber counts.

(2) In eosinopenia, it is necessary to set up more chambers to provide an optional number of cells to be counted.

(3) The eosinacetone diluting fluids are unsatisfactory and should not be used.

(4) Estimation of eosinophils on a stained blood smear is too inaccurate for use because of poor cellular distribution.

(5) The propylene glycol in Pilot's solution renders the erythrocytes invisible, and the sodium carbonate causes lysis of all the leukocytes except the eosinophils.

The phloxine stains the eosinophils.

(6) In the Thorn test an eosinophil count must be made prior to the initiation of the test proper. This establishes the patient's total eosinophil count, to which the response of the adrenal cortex to adrenocorticotropic hormone (ACTH) can be judged. The ACTH is then injected and, at an interval of 4 hours, another eosinophil count is made. The interpretation of this test is as follows:

(a) Normal--approximately a 50 percent drop in eosinophils.

(b) Cushing's disease (hyperadrenalism)--0-30 eosinophils per cu mm.

(c) Addison's disease (hypoadrenalism) no change in eosinophil count.

(7) Nasal smears are also submitted for eosinophil evaluation. These smears are stained with Wright's stain and examined for the presence of eosinophils.

Normal Value. Normal value: 150-300 eosinophils per cu mm.

RETICULOCYTE COUNT

Principle

Nonnucleated immature erythrocytes contain nuclear remnants of RNA and the cell is known as a reticulocyte. To detect the presence of this RNA, the red cells must be stained while they are still living. This process is called supravital staining. With supravital staining, the RNA appears as a reticulum within the red cell.

Reagent

(1) New Methylene Blue Solution. Dissolve 0.5 grams of new methylene blue, 1.4 grams of potassium oxalate, and 0.8 grams of sodium chloride in distilled water. Dilute to 100 ml. Filter before use.

(2) Brilliant Cresyl Blue Solution. Dissolve 1.0 grams of brilliant cresyl blue in 99 ml of .85 per cent sodium chloride. Filter before use.

Procedure.

  1. Mix equal amounts of blood and new methylene blue stain (2-3 drops or 50 uL each) and allow to incubate at room temperature for 3-6 min.
  2. This allows the reticulocytes adequate time to take up the stain.
  3. At the end of 15 minutes, mix the contents of the tube well.
  4. Place a small drop of the mixture on two clean glass slides and prepare a thin smear (prepare two smears).
  5. Counter stain with Wright's stain, if desired.
  6. Allow smear to air-dry.
  7. Place the slide on the microscope stage and, using the low power objective, locate the thin portion of the smear in which the red cells are evenly distributed and are not touching each other.
  8. Switch to oil immersion magnification and count the number of reticulocytes in five fields of 200 RBCs.

Calculation

Sources of Error

  • Equal volumes of blood and stain give optimum staining conditions. An excess of blood causes the reticulum to understain. An excess of stain usually obscures the reticulum.
  • Crenated erythrocytes and rouleaux formation make an accurate count difficult to perform.
  • Stain precipitated on erythrocytes causes them to appear as reticulocytes.
  • Dirty slides cause uneven spreading.
  • The dye solution should have adequate time to penetrate the cell and stain the reticulum.

Discussion

Reticulocytes are nonnucleated erythrocytes that exhibit blue reticulum strands within their cytoplasm when stained supravitally. When stained only with Wright's stain, they are buff-pink in color and larger and darker than erythrocytes.

Reticulocytes serve as an index of the activity of the bone marrow in blood regeneration. As such, these counts are of value in following anti-anemia therapy. Satisfactory response to therapy is evidenced by an increase of reticulocytes in the peripheral blood. Increased reticulocyte counts also occur whenever there is rapid bone marrow activity as in leukemia or blood regeneration associated with hemorrhage or hemolysis. Decreased reticulocyte counts occur in conditions in which the bone marrow is not producing adequate red blood cells, such as aplastic anemia.

Several methods for staining and counting reticulocytes are in common use. Compared to the use of alcoholic solutions of dye, methods employing saline solutions of new methylene blue can give slightly higher values for reticulocytes. For comparative studies, the same method should be used throughout the work.

Precipitated stain is often confused with reticulum but can be recognized by its presence throughout the smear and apart from the red cells. Precipitation can be eliminated as a source of error by frequently filtering the stain.

An alternate method of counting reticulocytes utilizes the Miller disk that is placed inside the microscope eyepiece. This disc consists of 2 squares as shown below in figure 5-2. The area of the smaller square (B) is a tenth that of square A. Therefore, if there are 40 red cells in square A, there should be four red cells present in square B. When employing this method to count reticulocytes, the red cells in square B are counted in successive fields on the slide, until a total of 500 red cells have been counted. At the same time, the reticulocytes in square A are enumerated. At the completion of the count, theoretically, the reticulocytes obtained in this way are divided by 50, in order to obtain the percent reticulocytes present in the blood.

Figure 5-2. Miller disc.

Normal Values

  • Birth to 1 day. Two and one-half to 6.0 percent.
  • 1 day to 2 weeks. 0.30 to 1.5 percent.
  • 2 weeks to adult. 0.50 to 2.20 percent.

 

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