1 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
White blood cell differential
Prepared by;
Hakami, Hana A
Viewed by;
Dr.Naseem Siddiqui
2 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
First: Blood Smear preparation
Introduction :
Giemsa stain is a classical blood film stain for
peripheral blood smears and bone marrow
specimens. Erythrocytes stain pink, platelets show
a light pale pink, lymphocyte cytoplasm stains sky
blue, monocyte cytoplasm stains pale blue, and
leukocyte nuclear chromatin stains magenta.
Blood Smear preparation:
Purpose :
To prepare Blood Smear and stain it with giemsa stain.
Materials :
Alcohol swab , Lancet , Ethanol , Giemsa Stain , Slides , cover slides ,
Pasteur pipette , Gloves , Light microscope , Sharp container
Procedure :
In clean aseptic work area :
1.
2.
3.
4.

Obtain 2 clean microscope slides, alcohol wipes, and lancet.
Clean a finger with an alcohol and puncture with lancet.
Place a small drop of blood at the end of one slide.
Use the second slide to make a thin blood film as directed below:
Place the second slide at a 30 degree angle and touch the slide
with the blood drop.
 Move the spreader slide to touch the blood drop allowing the
drop to spread by capillary action along the edge of the slide.
3 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
 Immediately pull/push the slide away from the blood drop,
making a thin smear that should dry quickly as you move away
from the drop.
 A perfect smear will have a "feathered" edge and separated RBCs
when you view it with the microscope.
5. Place thoroughly dried smear on horizontal staining rack.
6. Flood smear with Fixative (Ethanol or Methanol) for 10 seconds,
(fixes cells to slide/prepares cells for dyes) , drain .
7. Flood smear with Giemsa stain for 30 minutes.
8. Rinse the smear with distilled water for 1 minute.
9. Air dry and examine under the microscope.
10.After certain from quality stain, cover the smear with cover slide
by DPX.
results :
Red blood cells stain pink.
Platelets show a light pale pink.
White blood cells stains blue and them nuclear chromatin stains
magenta .
4 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
Second: Differential White Blood Cells Count
Introduction :
There are five distinctly different kinds of white blood cells,
lymphocytes, monocytes, neutrophils, eosinophils and basophils. Some
have the ability to change with needs and situations in the body. So, for
example, there are different monocytes found in different tissues, and
different types of lymphocytes with different roles in fighting infections.
These cells can leave the bloodstream, sliding out through the vessel
walls and attacking invaders at the site of an infection.
Neutrophil
Monocyte
Lymphocyte
Eosinophil
Basophil
White blood cells, or leukocytes, are classified into two main
groups: granulocytes and nongranulocytes (also known as
agranulocytes).
The granulocytes, which include neutrophils, eosinophils, and
basophils, have granules in their cell cytoplasm. Neutrophils,
eosinophils, and basophils also have a multilobed nucleus. As a result
they are also called polymorphonuclear leukocytes or "polys." The nuclei
of neutrophils also appear to be segmented, so they may also be called
segmented neutrophils or "segs."
The nongranuloctye white blood cells, lymphocytes and monocytes,
do not have granules and have nonlobular nuclei. They are sometimes
referred to as mononuclear leukocytes.
The lifespan of white blood cells ranges from 13 to 20 days, after
which time they are destroyed in the lymphatic system. When immature
WBCs are first released from the bone marrow into the peripheral
blood, they are called "bands" or "stabs." Leukocytes fight infection
through a process known as phagocytosis. During phagocytosis, the
leukocytes surround and destroy foreign organisms. White blood cells
5 Human Physiology-I (PHSL 205) Lab
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Hakami,Hana A- 2010/2011
also produce, transport, and distribute antibodies as part of the body's
immune response.
Two measurements of white blood cells are commonly done in a CBC:
1. the total number of white blood cells in a microliter (1x10-9 liters)
of blood, reported as an absolute number of "X" thousands of
white blood cells, and
2. the percentage of each of the five types of white blood cells. This
test is known as a differential or "diff" and is reported in
percentages.
Normal values for total WBC and differential in adult males and
females are:
1.
2.
3.
o
o
o
4.
o
o
Total WBC: 4,500 - 10,000
Bands or stabs: 3 - 5 %
Granulocytes (or polymorphonuclears)
Neutrophils (or segs): 50 - 70% relative value (2500-7000 absolute
value)
Eosinophils: 1 - 3% relative value (100-300 absolute value)
Basophils: 0.4% - 1% relative value (40-100 absolute value)
Agranulocytes (or mononuclears)
Lymphocytes: 25 - 35% relative value (1700-3500 absolute value)
Moncytes: 4 - 6% relative value (200-600 absolute value)
Each differential always adds up to 100%. To make an accurate
assessment, consider both relative and absolute values. For example; a
relative value of 70% neutrophils may seem within normal limits;
however, if the total WBC is 20,000, the absolute value (70% x 20,000)
would be an abnormally high count of 14,000.
Differential White Blood Cells Count method:
Purpose :
To determine the white blood cells (WBCs) concentration (% &
cells/ml) in blood smear.
6 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
Materials :
Gloves , Blood smear slide , Pasteur pipettes , Cedar wood oil , Light
Microscope , Tally Counter
Procedure :
In clean aseptic work area :
1. Examine blood smear slide on microscope (oil lens x100).
2. Began to count WBCs by tally counter
from edge of slide toward another edge
until 100 cells (total all types of WBCs ;
Neutrophils, Lymphocytes, Monocytes,
Eosinophils and Basophis).
3. Found percentage for each type of WBCs.
results :
Write result and study it :
WBC
Lymphocyte
Monocyte
Neutrophil
Eosinophil
Basophil
Percent
%
%
%
%
%
7 Human Physiology-I (PHSL 205) Lab
3rd Lab
Hakami,Hana A- 2010/2011
Attachment:
Type
Diagram
Approx. %
in adults
Diameter
(μm)
54–62%
10–12
Main targets
+bacteria
Neutrophil
Nucleus
Granules
Lifetime
multilobed
fine,
faintly
pink (H&E
Stain)
bi-lobed
full of
pinkorange
(H&E
Stain)
large blue
6 hours–
few days
(days in
spleen
and
other
tissue)
8–12
days
(circulate
for 4–5
hours)
a few
hours to
a few
days
weeks to
years
+fungi
Eosinophil
1–6%
10–12
+larger parasites
+modulate allergic
inflammatory responses
Basophil
<1%
12–15
Lymphocyte
25–33%
7–8
+release histamine for
inflammatory responses
bi-lobed
or trilobed
+B cells: releases antibodies
and assists activation of T cells
deeply
staining,
eccentric
+T cells:
-
-
-
Monocyte
2–10%
14–17
NK-cells
and
Cytotoxic
(CD8+) Tcells
Th (T helper) cells:
activate and regulate T
and B cells
CD8+ cytotoxic T cells:
virus-infected and
tumor cells.
γδ T cells:
Regulatory (suppressor)
T cells: Returns the
functioning of the
immune system to
normal operation after
infection; prevents
autoimmunity
+Natural killer cells: virusinfected and tumor cells.
Monocytes migrate from the
bloodstream to other tissues
and differentiate into tissue
resident macrophages or
dendritic cells.
kidney
shaped
none
hours to
days