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Lesson 7-1
 Differential Leukocyte Count and Other Procedures

DIFFERENTIAL LEUKOCYTE COUNT

The critical examination of a blood smear includes the following: quantitative and qualitative study of platelets, differential count quantitating the three types of leukocytes (granulocytes, lymphocytes, monocytes), and morphological characteristics of erythrocytes and leukocytes. Staining the blood smears is a critical part of the examination.  To accurately perform the differential count it is necessary for a technician to recognize all the characteristics of normal blood cells. This includes normal biological variation. For instance, not every lymphocyte is exactly the same size, nor do all lymphocytes have exactly the same number of azurophilic granules.

Certain morphological and histochemical characteristics are utilized to differentiate blood cells. A review of the significant features promotes a better understanding of blood differentials. Cellular characteristics such as relative size, shape, cytoplasmic granulation, nuclear- cytoplasmic ratio, nuclear configuration, chromatin or nucleoli are very important.

Experience is the foremost teacher in hematology. It is readily acquired in a busy hematology section where the opportunity for differential analysis occurs frequently. Experience can be diversified and interesting if proficiency slides and material from cases of confirmed diagnoses are maintained as study sets. This study material should be available to all technicians in the laboratory.

All routine blood smears should be kept until the physicians have reviewed the differential reports. A 1-week period is usually adequate. Occasionally, a review of a specific problem slide results in findings that were not originally apparent and reinforces confidence in the laboratory by the medical staff. This practice also adds to the experience and proficiency of the technician.


EXAMINATION OF PERIPHERAL BLOOD SMEARS

Principle

The stained blood smear permits the study of the appearance and the identification of the different kinds of leukocytes, and the appearance of erythrocytes and thrombocytes (blood platelets).

Differential Leukocyte Count.

  • Inspect the smear under low power magnification. Locate the thin end of the smear where there is no overlapping of erythrocytes.
  • Switch to oil immersion. Identify and count 100 consecutive leukocytes and record each cell type separately on the differential counter. Begin at the thin end of the smear and count the white cells observed as the slide is moved in a vertical direction. When near the edges of the smear, move the slide horizontally for a distance of about two fields, then proceed vertically back across the smear. See Figure 7-1. Continue this "snake-like" movement until 100 leukocytes have been counted and classified.

Figure 7-1. Examination of and peripheral blood smears.

  • If the WBC count is between 20,000 and 50,000 per cu mm of blood, count and classify 300 leukocytes. When the count is greater than 50,000 per cu mm of blood, count and classify 500 leukocytes.
  • The number of each type of leukocyte is expressed as a percent of the total number of white cells counted. Absolute values may be calculated by multiplying the percent value by the total leukocytic count.

Erythrocyte Morphology.

  • Study the erythrocytes and report any evidence of rouleaux formation or signs of immaturity.
  • Report the erythrocyte morphology with reference to size, shape, staining characteristics, and inclusions. Report the degree of the specific morphological characteristic (for example, moderate hypochromia).
  • If nucleated erythrocytes are found (usually these are metarubricytes), report the number per 100 leukocytes counted.

Qualitative Platelet Evaluation.

  • Observe the thrombocytes in several oil immersion fields to obtain a rough estimation of their numbers (normal, increased, or decreased). Normal is an average of 8 to 10 per oil immersion field.
  • Note any abnormality in morphology (giant platelets, etc.) If the thrombocytes appear to be significantly decreased, a thrombocyte count and/or a clot retraction test may be indicated.

Discussion

 All abnormal white cells (for example, immature, hypersegmented, toxic, atypical lymphocytes, etc.) should be classified or described and reported in percent, separately. Cells that are ruptured, fragmented, or degenerated are not included in the differential count, but should be noted separately and reported as the number seen per 100 leukocytes.

In view of the gradual transition from the metamyelocyte to the banded neutrophil and then to the segmented neutrophil, exact classification is sometimes difficult. In such cases, classify the cell according to the more mature form.

Size considerations in differentiating blood cells require a defined linear standard. The micron (.001 mm) is usually used in reference to microscopic dimensions. Ocular micrometers are available through Federal medical supply channels and are easily calibrated, using a hemacytometer that has standardized dimensions. In routine screening of blood smears, an experienced technician relates the size of a normocytic erythrocyte (seven to eight microns) to the size of the white cell to be differentiated, since erythrocytes are usually present throughout the microscopic field. Finally, it should be understood that personal visual discrimination is an inaccurate gauge of linear measure. Some reference measure should be employed.

The shape of blood cells often depends upon the smear and staining technique. Variations that have no clinical significance occur from physical and chemical distortions that result from technical error. These variations are avoided with careful technique. Each routine smear should be scanned initially to evaluate the smear and stain quality before differential analysis.

Cytoplasmic granulation--neutrophilic, basophilic, or eosinophilic--is an important morphological observation. Differences in granule color in Wright-stained preparations are caused by the variable dye affinity of specific granules. The intensity of colors and the relative blueness or redness of the erythrocytes is used to evaluate the quality of the stain. The familiar basophilic (blue), eosinophilic (red), and neutrophilic (pink) granules are quite obvious in routine blood smears. The presence, absence, type, and quantity of granules are characteristic attributes used to differentiate leukocytes.

The size ratio of nucleus to cytoplasm (N:C) is a differentiating characteristic. For instance, a cell with a nuclear mass equal to the cytoplasmic mass would have an N:C ratio of 1:1. The total cell mass is usually greater in the more immature cells and decreases as the cell matures. The nuclear mass usually decreases also as the cell matures. Of course, lymphocytes are the exception to this generality.

The nuclear configurations of leukocytes help distinguish these cells. Round, oval indented, band, or segmented are terms used to describe variations in shape. These normal configurations can be distorted by physical and chemical factors. Some of the leukocytes are so fragile that in thick blood smears their normal configuration may be distorted by the pressure of erythrocytes forced against them. These artifacts should be recognized as such in an intelligent evaluation of blood differentials.

In addition to nuclear shape and size, the internal nuclear morphology shows differential inclusions. The chromatin appears finely reticulated in some cells, or as a coarse network, or even clumped, in others. The parachromatin, a lighter staining material beside the chromatin, is scant or abundant. The appearance of the chromatin and the quality of parachromatin are utilized to differentiate blood cells. The presence, absence, and number of nucleoli in the nucleus are the most distinctive characteristics of immature nuclei in blood cells.

All abnormal blood smears should be examined by another trained person for confirmation of the results.

Normal Differential Values.

  • Banded neutrophil: 0 to 6 percent.
  • Segmented neutrophil: 40 to 75 percent.
  • Eosinophils: 1 to 7 percent.
  • Basophils: 0 to 2 percent.
  • Lymphocytes: 22 to 40 percent.
  • Monocytes: 1 to 10 percent.

EXAMINATION OF BLOOD MARROW SMEARS

Nucleated blood cells are counted and classified from a bone marrow smear stained with a Romanowsky stain containing both Wright and Giemsa stains.

Procedure

  1. Using oil immersion magnification, count and classify 300 to 500 nucleated cells.
  2. Classify all blood cells according to cell type and various stages of maturation.
  3. Calculate myeloid-erythroid ratio by dividing the number of nucleated erythrocytes into the number of granulocytic (myeloid) cells.
  4. A peripheral blood evaluation usually accompanies the bone marrow reports. This evaluation usually includes an erythrocyte count, leukocyte count, differential count, hemoglobin, hematocrit, and a reticulocyte count.

The differential cell count on a bone marrow smear is carried out by a hematologist, pathologist, or trained technician.

Since interpretation of findings in bone marrow examinations is very difficult, it is of utmost importance that the smears and stains are carefully prepared using scrupulously clean equipment.

The laboratory technician is usually responsible for preparing bone marrow smears, staining the smears, checking the quality of the stained smear, and coverslipping the slides.

Normal Values.

Leukocytes.

  • Myeloblast: 0 to 1 percent.
  • Promyelocytes: 2 to 5 percent.
  • Neutrophilic myelocytes: 5 to 19 percent.
  • Neutrophilic metamyelocytes: 13 to 22 percent.
  • Neutrophilic bands: 17 to 33 percent.
  • Neutrophilic segmented cells: 3 to 11 percent.
  • Eosinophilic cells: 0 to 3 percent.
  • Basophilic cells: 0 to 1 percent.
  • Lymphocytes: 5 to 15 percent.
  • Monocytes: 0 to 2 percent.
  • Plasmocytes: 0 to 1 percent.

Erythrocytes.

  • Rubriblasts: 0 to 1 percent.
  • Prorubricytes: 1 to 4 percent.
  • Rubricytes: 3 to 10 percent.
  • Metarubricytes: 5 to 25 percent.
  • Megakaryocytes. 0 to 3 percent.
  • Myeloid-Erythroid Ratio (M:E). 3-4:1.

Section II. ERYTHROCYTE INDICES AND FRAGILITY TESTS

 

David L. Heiserman, Editor

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Revised: June 06, 2015