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11/11/2010 Blood Smear & Differential Count

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Place a small drop of blood at one end of a clean glass slide.


While tilting a second glass slide tow ard the drop, slowly move it into the drop (left to right) until it contacts the blood.
Now quickly move the top slide back to the left, while maintaining the same angle betw een the tw o slides. The blood
should be drawn along as you move the top slide and a smear left behind on the bottom slide.
The slide must be stained with a Wright's or Giemsa's blood staining reagent before view ing under the microscope.
Wright/Giemsa is used to differentiate nuclear and cytoplasmic morphology of red and w hite blood cells as well as
parasites on blood smears. Wright/Giemsa stained blood smears are the basis for performing a clinical Differential Blood
Count. One limitation of the method is distinguishing B lymphocytes from T cells and natural killer cells; for this task, a
special immunochemical analysis such as flow cytometry has to be performed.
When performing a quality analysis of your slide, you can check that:

macroscopically, the blood film looks purplish. If blue, the w ash w ater was too alkaline; if pink to red, the water
was too acid.
macroscopically, the smear has a "feathered" edge (see picture below )
microscopically, red blood cells appear pinkish gray, platelets look deep pink, and w hite blood cells have purple-
blue nuclei and lighter cytoplasm.
can distinguish red blood cells; platelets; eosinophilic white blood cells (eosinophilic granules are bright purple-
red); neutrophilic w hite blood cells (neutrophilic granules are purple) and basophilic w hite blood cells (basophilic
granules are blue)

When counting 100 w hite blood cells, start at the X and move in the direction of the arrows. Only count cells in the
monolayer area in which the cells are evenly distributed and don't show artifact. First, use the high dry objective (40X)
to count the number of w hite blood cells per 5 to 10 random fields. The white blood cell count is normal if the average
number of white blood cells seen per 40X field averages between 2 and 7. Only five or more 40X objective fields are
necessary if a consistent number of cells is seen. A total of 10 fields may need to be counted if there are abnormalities.

Using the 100X objective, classify 100 white blood cells from the smear. The movement from the end of one vertical line
to the beginning of the next represents one 100X field. Moving farther than one field will result in reaching the thick
area in w hich the cells are overlapping. The initial vertical pattern may only be a few fields. As the width of the
monolayer area increases, the length of the vertical movement and the number of fields available for evaluation will
increase.

White Blood Cell Count with Differential:


The CBC (complete blood count) is a laboratory method that is based on the microscopic evaluation of a patient's cells
from a blood smear. The CBC information is also available from automated counting using equipment such as the
Coulter Counter, which measures cell presence and type by interruption of electrical current and electronic sizing. The
blood smears that were prepared on the first blood lab w ill be used to determine your differential white blood cell count.
The follow ing picture will help you identify the different w hite blood cell types.

Basophils (J) can increase in cases of leukemia, chronic inflammation, the


presence of a hypersensitivity reaction to food, or radiation therapy.
Decreased basophil levels can indicate acute infection, a response to an
overactive thyroid gland, and stress (normal=0-1% basophils).

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11/11/2010 Blood Smear & Differential Count
Eosinophils ( D) can increase in response to allergic disorders, inflammation
of the skin, and parasitic infections. They can also occur in response to some
infections or to various bone marrow malignancies. Decreased levels of
eosinophils can occur as a result of infection (normal=0-3% eosinophils).

Monocyte (F) levels can increase in response to infection of all kinds as well
as to inflammatory disorders. Decreased monocyte levels can indicate bone
marrow injury or failure and some forms of leukemia. (normal=3-7%
monocytes)

Lymphocytes (B, H) can increase in cases of bacterial or viral infection,


leukemia, cancer of the bone marrow , or radiation therapy. Decreased
lymphocyte levels can indicate diseases that affect the immune system, such
as lupus, and the later stages of HIV infection (normal=25-33%
lymphocytes).

Polymorphonuclear neutrophils (C, E) can increase in response to bacterial


infection or inflammatory disease. Severe elevations in neutrophils may be
caused by various bone marrow malignancies, such as chronic myelogenous
leukemia. Decreased neutrophil levels may be the result of severe infection
or other conditions, such as responses to various medications (normal=54-
62% PMNs). Band cells (I) are immature PMNs and generally constitute 3-5% of the overall white blood cell population.

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