Name: John Roter R.

Mendoza Date: September 9, 2021

Section: MMLS 3-4

Worksheet #3
Differential White Blood Cell Count

Differential white cell count is the determination of the percentage of each type of white blood
cells (WBCs) in the peripheral blood. It consists of the enumeration of the relative proportion of the
various types of WBCs as seen in stained blood smears.

I. Objectives:

1. Perform the differential count properly.

2. Evaluate normal peripheral blood smears.

3. Enumerate the causes of increased and decreased values of each type of WBC.

II. Materials:

1. Blood smear

2. Methanol

3. Eosin

4. Methylene Blue

5. Compound Light Microscope

6. Buffer solution pH7.2 / Aged distilled water

7. Differential counter

8. Cedarwood oil

9. Xylol

10. Xylol-alcohol

III. Methods:

STAINING JAR OR “DIP” METHOD

Staining:

1. Dip in solution 1 (methanol, the fixative) for 30 seconds

2. Dip in solution 2 (eosin, the acidic dye) for 6 seconds

3. Dip in solution 3 (methylene blue, the basic dye) for 4 seconds

4. Dip in buffer solution / aged distilled water for 45 seconds

Differential Counting:

1. Prepare stained blood smear.

2. Place one drop of cedarwood oil on the feathery edge of the stained blood smear.

3. Examine the smear using the LPO of the microscope. Focus on the area where the red blood cells are
not too overlapping or too scanty.

4. Shift to OIO. Using the strip differential method, count 100 white blood cells while differentiating
them.

NV in CU NV in SI
Image WBC Description
(%) (x10^9/L)

Nucleus is broken into

Neutrophil segments.
50-70% 0.50-0.70
segmenter Cytoplams contains small,
pinkish granules.

Nucleus is compact and

usually round.
Lymphocyte 18-42% 0.18-0.42
Cytoplasm is light blue and
scanty.
 Younger form of
Neutrophilic
neutrophil C, S, U or
band (Stab 0-5% 0-0.5
horseshoe-shaped
cell)
nucleus.

Nucleus is spongy and

sprawling with brain-like
convolutions.
Monocyte 2-11% 0.02-0.11
Cytoplasm is gray.
Vacuoles are sometimes
present.

Nucleus is usually bilobed.

Cytoplasm contains large,
Eosinophil 1-3% 0.01-0.03%
coarse, reddish or orange
granules

Nucleus is usually
indistinct and obscured by
the granules.
Basophil 0-2% 0-0.02%
Cytoplasm contains large
purplish-black or dark blue
granules

Types of Stains Used in Differential While Blood Cell Counting

1. Romanowsky stains

Romanowsky stain contains methylene blue or its oxidative product, such as azure B. It also
contains eosin B or eosin Y. The dyes produce multiple colors when used on cells and cellular
components. This is the reason why the stains are considered polychromatic.

a. Wright’s stain – most satisfactory in general routine hematologic studies

Composition:

Oxidized methylene blue

Eosin azures

b. Giemsa stain – excellent stain for the demonstration of inclusion bodies and intracellular parasites as
well as for staining WBCs.
 Composition:

Eosin Y with azure blue

Methylene blue in methanol with glycerin

c. Leishman, Jenner, and May-Grunwald – similar to Wright’s stain except for the method used to
oxidize methylene blue

2. Panoptic stains

Panoptic stains consist of Romanowsky stain and another dye.

a. Wright’s-Giemsa

b. Jenner-Giemsa

c. May-Grunwald-Giemsa

IV. Questions:

1. What are common causes of eosinophilia?

Eosinophilia occurs when a large number of eosinophils are recruited to a specific site in your body or
when the bone marrow produces too many eosinophils. This can be caused by a variety of factors,
including:

 Parasitic and fungal diseases

 Allergic reactions
 Adrenal conditions
 Skin disorders
 Toxins
 Autoimmune disorders
 Endocrine disorders
 Tumors

2. Differentiate Schilling’s hemogram from Arneth’s classification of neutrophilia.

Schilling’s hemogram
is a method of counting blood cells in which the polymorphonuclear neutrophils are separated into four
groups according to the number and the arrangement of the nuclear masses in each cell.

Arneth’s classification of neutrophilia

A classification of polymorphonuclear neutrophils based on the number of lobes (one to five) in the nucl
eus, termed stages one to five, respectively.

References:

https://medical-dictionary.thefreedictionary.com/Arneth+classification+of+neutrophils

https://my.clevelandclinic.org/health/diseases/17710-eosinophilia