Differential cell count

1. Author: Negash Alamin
Occupation: Clinical labscientist
E-mail: drnegash@gmail.com
Date: 24/12/17

2. Principle: A routine blood film is made and it is stained by the usual Giemsa stain and is
analyzed for blood cell morphology and the types of cells counted using modified battlement
method. The differential cell count aids as an additional means of monitoring peripheral cell
health and morbidity within the parameter of complete cell count. The counting methodology
proceeds via the modified battlement method as stated above and a total of 100 cells of each type
are counted and their percentage is calculated.
Materials:
1- Gloves as PPE.
2- Frosted slide.
3- Micropippette.
4- Anticoagulated venous blood.
5- Staining reagent like Giemsa stain.
6- Differential WBC counter or alternatively we can use a labeled paper with each type of
WBC and a pencil.
7- Compound microscope.
Procedure:
1- First prepare a blood film and air dry for staining.
2- Fix the smear with methyl alcohol (methanol) for 3-5 minutes.
3- Flood with the diluted1
stain and wait for 15 minutes.
4- Wash off with distilled water and examine with oil emersion.
5- Using the modified battlement method start examining the blood cell morphology at
the peripheral edge within the body of blood film by counting two fields parallel to
the head of the film and four fields at right angle to the head of the film.
6- In doing so remember that you are moving in a zigzag fashion from the head to the
tail counting all necessary 100 cells.
7- Finally you report your results in percentage of each WBC constituent.
1
The stock solution of Giemsa made up with 0.3 gm of Giemsa powder plus 25 ml of glycerol plus 25 ml of
methanol is diluted for working solution by mixing 1 ml of stock solution with 9 ml of distilled water. (see Monica
Cheesbrough book District laboratory Practice in Tropical countries page 404 reagent appendix for details of
preparation)

3. Important points to remember:
1- When performing the count you do not just look for Basophils, eosinophil,
neutrophils, monocytes and lymphocytes; but, for other pathological morphologies
and parasites as well.
2- Look for size and maturation of WBCs, and RBCs.
3- Look for Toxic granulation, inclusions, vacuolation, hyper segmentation, malaria and
other pathologies as well as abnormalities.
4- The absolute cell count of a single WBC can be counted and reported by multiplying
its percentage for relative counts: example 60 % (0.6) neutrophil count by absolute
WBC count 8.0 x /L which gives 4.8 x /L or 4,800 cells per microliter of
blood.

4. Table 1 Absolute WBC hematological reference values2
Total absolute WBC 4.5 -11.0 x /L Or 4,000 to 11,000/µL
Neutrophil 2.0 -7.5 x /L Or 2,000, to 7,500µL
Lymphocyte 1.3-3.5 x /L Or 1,300-3,500µL
Eosinophil 0-0.44 x L Or 0-440/µL
Basophil 0-0.10 x / L Or 0-100/µL
Monocyte 0.2-0.8 x /L Or 200-800/µL
Clinical significance: The common terms used in clinical settings to refer to the conditions
related with differential count and the increment or decrement of each WBC cell include
neutropenia, neutrophilia, lymphopenia, lymphocytosis, monocytopenia, monocytosis,
eosinophilia, and basophilia. These terms are medical terminologies whose suffix –penia indicate
decrement and –philia or –osis indicate increment of each type of cell. The laboratory scientist
will look for immature granulocytes (IG) like metamyelocytes, myelocytes, promyelocytes, and
blasts to assess the health of the bone marrow overall cell production cascade in the body; if too
much juvenile cells are present in the peripheral blood then there must be something pathology
in the immuno-hematological system and this is very agenda of differential count. That is to
observe cellular morphology like size comparison as in RDW, color, maturity and shape of every
type of cell in the blood.
Increase of neutrophils may occur due to:
 The routine culprit like infection with viruses, fungi, and bacteria.
 Tissue death or necrosis as in heart attack, and burns3
.
 Physiological stress and inflammation.
 Chronic leukemia
Decrease of Neutrophils
 Autoimmune disorder
 Chemotherapy
 Cancer that spreads to the bone marrow
2
Oxford medical dictionary: 2010
3
Labtestsonline.org

5.  Aplastic anemia4
Increase of lymphocytes
 Viral infections
 Lymphoma
Decrease of lymphocytes
 Autoimmune disorders
 Chemotherapy and radiation
Increase of monocytes
 Monocytic leukemia, chronic myelomonocytic leukemia, juvenile myelomonocytic
leukemia5
Decrease of monocytes
 Bone marrow damage, hairy cell leukemia.
.
4
Labtestsonline.org
5
Labtestsonline.org