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OBJECTIVES

After the practical session, student should:


1. Familiar with different methods of counting different leukocytes
2. Understand the importance of performing DC
3. Able to perform DC
4. Comprehend the reasons for neutrophila, neutropenia, eosinophilia, eosinopenia,
basophilia, basopenia, monocytosis and monocytopenia
5. Know the normal percentage of each leukocyte.

PRINCIPLE
White blood cell is very important in immunity to fight against infection. White cell differential
count / differential leukocyte count or DC Count is a test to determine the percentage distribution
of leukocytes in peripheral blood. It enables us to determine the number of each type of white
blood cell present in the blood after a thin blood film is stained with Romanowsky stain. Then,
the stained blood film is examined under oil immersion objective. The usage of automated
counter assists and provides advantages such as speed and accuracy over manual techniques.
However manual techniques of DC Count is still very important. The absolute number of each
type of white blood cell helps to diagnose conditions that affect white blood cells.

REAGENTS AND APPARATUS


1. A well stained smear
2. Microscope
3. Immersion oil
4. Differential cell counter

PROCEDURE
Examination of Peripheral Blood Smear

(a) Observation under low power (10X or 20X)


Observations under this low power was for scanning purpose (overview for the whole picture)
1. A well-stained slide was placed (see Figure 1) on the stage of microscope and focused
using the low-power objective.
2. A check was done to see if there are good counting areas that are free of ragged edges and
3.
4.
5.
6.

cell clumps.
The WBC distribution was checked.
The staining quality of the smear was checked.
The presence of large platelets, platelet clumps or fibrin strands were checked.
The number of WBC was estimated.

(b) Observation under low power (10X or 20X)


1. Platelet estimates was done under 100X with RBCs bare touching, approximately 200
RBCs.
2. To count the cells a zig zag method was used (see Figure 2). This method of counting
was done by going back and forth lengthwise or sidewise.
3. At least ten fields were counted. On average there are 8-20 platelets per field.
4. The number of WBCs was estimated.
5. The percentage of each type of WBC was recorded directly from the counter.

RESULT
White Cell Differential Count or DC Count

Absolute values

Percentage
Neutrophil
41%
41
Lymphocytes
36
36%
Monocytes
3
3%
Eusinophils
19
19%
Basophil
1
1%
Table 1: The absolute values and percentage of each type of white blood cell (WBC)

DISCUSSION
Based on white blood cell differential count conducted under oil immersion objective.
The absolute number of neutrophils, lymphocytes, monocytes, eosinophil and basophils obtained
from the count are 41, 36, 3, 19 and 1 respectively. The normal percentage of neutrophils is
between 40 to 80%. The slide observed has a normal percentage of neutrophils. The normal
percentage of lymphocytes is within 20 to 40%. The percentage of observed lymphocytes from
the slide is 36% which is normal. The normal percentage of monocytes is 2 to 10%. The
percentage of monocytes from observed slide is within normal range which is 3%. Next is the
eosinophils normal percentage is range from1 to 6%. The observed eosinophil is 19% which
means the number of eosinophils in the blood is abnormal, indicates eosinophilia. Lastly is
basophils which normal percentage is less than 1 to 2%. The percentage of basophil on the
observed slide is 1% which is within the normal range.
Neutrophilia is a condition where there is an increased in absolute number of mature
neutrophils in blood. It is usually occurred during pregnancy, due to acute infections,
corticosteroid therapy and acute blood loss. Inflammation and corticosteroid therapy causes
increased release of neutrophils from bone marrow (Individual WBC, n.d.). The neutrophils of
individuals with neutrophilia due to severe bacterial infections show heavy cytoplasmic
granulation (Bain, Bates, Laffan & Lewis, 2012).
On the other hand, neutropenia is a decreased absolute number of mature neutrophils and
can occur due to opposite mechanisms to those that cause neutrophilia. The most common cause
is inflammation which causes increased migration into tissue particularly when there is evidence
of neutrophil immaturity (Individual WBC, n.d.). Consumption of certain drugs and having

congenital defect also leads to neutropenia by decreasing the production of neutrophils in bone
marrow.
Eosinophilia is a condition of abnormal increased of absolute number of eosinophils in
blood. This condition occurred mostly due to parasitic infections, an allergic reaction and cancer
which cause increased production and release of eosinophils (Mayo Clinic Staff, 2016).
Eosinopenia is lower than normal number of eosinophils in peripheral blood. It is a rare
abnormality that is associated with certain bacterial and viral infections, with corticosteroids and
adrenergic agents, and with stress (Polosa, Prosperini, Pintaldi, Rey & Colombrita, 1997). There are
also cases of congenital eosinopenia but it is relatively uncommon (Bashir, 2004). Eosinopenia may also
be associated with asthma, allergic reactions, urticaria, immunoglobulin deficiencies, and autoimmune
cases (Polosa et al., 1997).
Basophilia is a condition of abnormal high number of basophils in peripheral blood. Basophils are
type of white blood cell that play an important role in immunity and allergic reactions. Individuals with
hyperthyroidism and myeloproliferative disorder has marked increased in basophil number (Territo, n.d.).
Basophilia also can occur due to rare allergic reactions and infections.
In contrast, basopenia is a condition of abnormal low number of basophils in peripheral blood.
The condition is a response to thyrotoxicosis, acute hypersensitivity reactions, and infections (Territo,
n.d.). Khurana (2014) states that corticosteroid therapy also another cause of basopenia.
Monocytosis is a rise in blood monocytes above 800/L. As monocytes share a common
committed stem cell with neutrophils therefore monocytosis can occur associated with neutropenia during
bacterial infection. The causes of monocytosis include bacterial infections such as tuberculosis and
syphilis, viral infections, inflammation and paraneoplastic reactions. Besides that, monocytosis also occur
due to recovery from acute marrow injury (Individual WBC, n.d.).
Monocytopenia refers to condition where there is decreased of monocytes circulating in blood. It
is an uncommon condition and has no clinical significant (Individual WBC, n.d.). however, this condition
can be seen in hypoplastic bone marrow (Khurana, 2014).

CONCLUSION

DC count allow to determine conditions that occur to the white blood cells. The observed stained
peripheral blood smear shows condition of eosinophilia. There is an obvious increased of percentage of
eosinophil number compared to normal range, which is 19%. The normal range of eosinophils only
between 1 to 6%. This condition can be due to allergic reaction, parasitic infections, inflammation or
certain cancer that occur to the patient.
QUESTIONS
1. Procedure on how to prepare
a. Wrights stain
The materials needed to prepare Wrights stain includes capillary or venous blood,
Wrights stain powder, methanol and EDTA as anticoagulant. To prepare Wrights stain,
1.
Weigh the Wright powder and transfer it to a dry brown bottle.
2.
Add a few glass beads to the brown bottle. These glass beads assist in dissolving
the dye.
3.
Using a dry cylinder, measure the methanol and add this to the stain.
4.
Mix well at intervals until the powder is completely dissolved.
5.
Warm the solution in a 37C in water bath to help the dye to dissolve.
6.
Label the bottle containing the Wrights stain with Flammable and Toxic.
7.
Store it at room temperature in the dark. When kept tightly stoppered, the stain is
stable for several weeks. Moisture must not be allowed to enter the stain (Preparation of
Wrights stain for blood film staining, n.d.).
b. Sorensons Phosphate Buffer
Sorensons phosphate buffer is a physiological buffer. It is a mix of two stock solutions to
get desired pH. To prepare 2M Sorensons phosphate solution with pH 7.4, we need to
prepare two solutions at appropriate volume by:1.First, add 27.6 g sodium phosphate, monobasic to 1000 ml ddH2O in a 1500 ml beaker.
2.Add 28.4 g dibasic sodium phosphate into a 1500 ml beaker containing 1000 ml ddH2O
and stir.
3.Keep each solution at room temperature for up to 1 year.
4.Mix the two solutions in the following ratios: 19 ml of solution monobasic buffer and
81.0 ml of dibasic buffer with 100 ml of ddH2O. This gives a final volume of 200 ml of a
0.1 M solution (Supplementary Method, n.d.).

REFERENCES
Bain, B. J., Bates, I., Laffan, M. A., & Lewis, S. M. (2012). Dacie and Lewis - Practical
Haematology (Eleventh Edition). China: Churchill Livingstone.
Bashir, S. (2004). Eosinophils and Their Abnormalities. Indian Journal for the Practising
Doctor, 1(1).

Individual WBC (n.d.). Retrieved November 14, 2016, from


http://www.eclinpath.com/hematology/leukogram-changes/leukocytes/
Khurana, I. (2014). Medical Physiology for Undergraduate Students. Rohtak, India: Elsevier
Health Sciences.
Mayo Clinic Staff (2016). Eosinophilia. Retrieved November 4, 2016, from,
http://www.mayoclinic.org/symptoms/eosinophilia/basics/definition/sym-20050752
Territo, M. (n.d.). Basophilic Disorders. Retrieved November 4, 2016, from
http://www.msdmanuals.com/home/blood-disorders/white-blood-celldisorders/basophilic-disorders