BLOOD SMEAR

I. Blood smear technique

- It is easiest to use microscope slides with a frosted end, so that identifying information can be written
there with pencil.

- Place a drop of blood approximately 4 mm in diameter on the slide (near the end if
one smear is to be made). See the drawing below.

- Spread the drop by using another slide (called here the “spreader”), placing the spreader at a 45° angle
and BACKING into the drop of blood. The spreader catches the drop and it spreads by capillary action
along its edge. To make a short smear, hold the spreader at a steeper angle, and to make a longer smear,
hold it closer to the drop. Now, push the spreader across the slide; this PULLS the blood across to make
the smear. Do not push the blood by having it ahead of the smearing slide! It should take about one
second to smear the drop. A smooth action is required, with the edge of the spreader held against the
slide.
 Coloring technique with May-
Grünwald Giemsa

1- Fixing
 pour on the slide 1 ml of May-Grünwald CONCENTRATED solution
to cover the blood smear – leave for 3 minutes, do not drop.

2- Staining with May-Grünwald solution

 cover the blood smear with May-Grünwald solution (diluted with

neutral water 1:1) – leave for 2 minutes.
 Practically you should add 1 ml of neutral distilled water over the
previously poured May-Grünwald concentrated solution
Wash the smear with neutral water.
3- Coloring with Giemsa solution

 Cover the smear with Giemsa solution (freshly diluted in neutral

water). From the concentrated Giemsa solurion we have to perform
a 5% Giemsa working solution – pour 1 ml over the smear and
leave for 25 minutes
 Washing with neutral water

4- Drying

 Drying in open air and then with filter paper

 RESULTS - Blood cells
 Erythrocytes
(red blood cells)
 Leucocytes
(white blood cells)
 Granulocytes
 Neutrophils
 Eosinophils
 Basophils
 Non-granulocytes
 Lymphocytes
 Monocytes
 Thrombocytes
(platelets)
 RBC– Red Blood Cell Count
Minimal Maximal
Units
value value
Male 4.2 5.7 x1012/L
Erythrocytes/
Female 3.5 5.1 or
Red blood cells
milions/mm3
(RBC) Infant/Child 3.8 5.5 or per l
26 130 x109/L
Reticulocytes Adult 0.5 1.5 % of RBC
Immature,
nucleated RBC Newborn 1.1 4.5 % of RBC
Infant 0.5 3.1 % of RB

1 ml = 1 cm3
1 l = 1 mm3
WBC – White Blood Cell Count

Neutrophil granulocytes, from 50-70%

Neutrophil band forms (immatures) 3-5%

Eosinophils, from 1 to 6 %

Basophils, from 0,5 to 1 %

Lymphocytes, from 25 to 35 %

Monocytes, from 3 to 10 %
WBC – White Blood Cell Count
WBC – White Blood Cell Count
Lower Upper
Test Patient type Unit
limit limit
Adult 4 9
White Blood Cell Count (WBC) Newborn 9 30 x109/L or x103/mm3 or x103/μL
1 year old 6 18
1.8 5 x109/L or x103/mm3 or x103/μL
Neutrophil granulocytes (A.K.A. grans, polys, Adult
PMNs, or segs) 50 70 % of WBC
Newborn 6 26 x109/L
0.7 x109/L
Neutrophilic band forms Adult
3 5 % of WBC
1.0 3.5 x109/L
Adult
Lymphocytes 20 35 % of WBC
Newborn 2 11 x109/L
0.1 0.8 x109/L
Adult
Monocytes 4 8 % of WBC
Newborn 0.4 3.1 x109/L
Mononuclear leukocytes 1.5 5 x109/L
Adult
(Lymphocytes + monocytes) 20 35 % of WBC

0.04 0.5 x109/L

Adult
Eosinophil granulocytes
1 6 % of WBC
Newborn 0.02 0.85 x109/L

40 300 x106/L
Adult
Basophil granulocytes
0.5 1 % of WBC
Newborn 0.64 x109/L
II. BLOOD SMEAR
MICROSCOPY

Cytological properties
 Erythrocytes
Red blood cells are the most common type of blood cell and the vertebrate
organism's principal means of delivering oxygen (O2) to the body tissues via the
blood flow through the circulatory system. They take up oxygen in the lungs or gills
and release it while squeezing through the body's capillaries.
These cells' cytoplasm is rich in haemoglobin, an iron-containing biomolecule that
can bind oxygen and is responsible for the blood's red color.
Binconcave shape
Oxygen carrier
Diameter : 7,5 µm

NO nucleus

Cytoplasm: without
granulations
 Granulocytes
Neutrophils (polymorphonuclear leukocyte)

Neutrophil granulocytes are the most

abundant type of white blood cells in mammals
and form an essential part of the innate
immune system.
Referred to as either neutrophils or
polymorphonuclear neutrophils (or PMNs),
- subdivided into
- segmented neutrophils (or segs) and
- banded neutrophils (or bands).

Neutrophils are normally found in the blood

stream. During the beginning (acute) phase of
inflammation, particularly as a result of
bacterial infection, environmental exposure,
and some cancers, neutrophils are one of the
first-responders of inflammatory cells to
 Phagocytes migrate towards the site of inflammation. They
Diameter: 15 µm
migrate through the blood vessels, then
Nucleus : polylobed (2 or 5 lobes – binded by
fine chromatin threads). through interstitial tissue, following chemical
Cytoplasm: signals
 2 granulations types: They are the predominant cells in pus,
 azurophilic (non specific)- less
specific – more frequent.
accounting for its whitish/yellowish appearance.
 Eosinophils
Components responsible for combating multicellular parasites and certain infections in
vertebrates. Along with mast cells, they also control mechanisms associated with
allergy and asthma.

Eosinophils are involved in:

- fighting viral infections, which is evident
from the abundance of RNases they
contain within their granules,
- fibrin removal during inflammation
- Eo are important mediators of allergic
responses and asthma pathogenesis and
are associated with disease severity
- fight helminth (worm) colonization and
may be slightly elevated in the presence
of certain parasites
- many other biological processes,
including postpubertal mammary gland
 Diameter: 15 µm development, oestrus cycling, allograft
rejection and neoplasia
 Nucleus: 2 lobes - antigen presentation to T cells
 Cytoplasm: large
granulations coloured in red
appear in many specific kinds of
inflammatory reactions
Diameter: 13 to 15 µm
Nucleus: 3 lobes, clover aspect or « S »
shaped, often masked by large granulations
Cytoplasm: large irregular granulations,
many basophiles (violet-blue).
Basophils: Basophils
- appear in many specific kinds of inflammatory
reactions, mainly allergies.
- contain anticoagulant heparin
- contain the vasodilator histamine
- they can be found in unusually high numbers at
sites of ectoparasite infection, e.g., ticks. Like
eosinophils, basophils play a role in both
parasitic infections and allergies.
- found in tissues where allergic reactions are
occurring and probably contribute to the
severity of these reactions. Basophils have
protein receptors on their cell surface that bind
IgE, an immunoglobulin involved in
macroparasite defense and allergy. It is the
bound IgE antibody that confers a selective
response of these cells to environmental
substances, for example, pollen proteins or
helminth antigens.
 Agranulocytes
Lymphocytes
The three major types of lymphocyte are
- T cells
- B cells and
- natural killer (NK) cells.

Natural killer cells

NK cells are a part of the innate immune system and play a
major role in defending the host from both tumors and virally
infected cells.
Immune function
T cell and B cell
T cells (thymus cells) and B cells (bursa-derived cells) are the Diameter: 10 to 15 µm
major cellular components of the adaptive immune response.
T cells are involved in cell-mediated immunity Nucleus: spherical
B cells are primarily responsible for humoral immunity (relating
to antibodies). The function of T cells and B cells is to Cytoplasm: contains some
recognize specific “non-self” antigens, during a process known azurophil granulations.
as antigen presentation. Once they have identified an invader,
the cells generate specific responses to eliminate specific
pathogens or pathogen infected cells. B cells respond to
pathogens by producing large quantities of antibodies which
then neutralize foreign objects like bacteria and viruses.
 Agranulocytes Immune function
Monocytes Diameter: 20 to 30 µm
Nucleus: irregular, kidney
shape
Cytoplasm: fine azurophil
granulations, many vacuoles.

Monocytes play multiple roles in

immune function.

Such roles include:

(1) replenish resident macrophages and
dendritic cells under normal states,
(2) in response to inflammation signals,
monocytes can move quickly
(approx. 8-12 hours) to sites of
infection in the tissues and
divide/differentiate into
macrophages and dendritic cells to
elicit an immune response. Half of
Monocyte under a light microscope (40x) from a
peripheral blood smear surrounded by red blood cells
them are stored in the spleen.
 Platelets (Thrombocytes)
 formation of blood clots
 2 to 3 µm
 140-400 x1000/L
 frequently agglutinated
 anucleated cells
PLATELETS are small, irregularly shaped clear
cell fragments (i.e. cells that do not have a
nucleus containing DNA), 2–3 µm in diameter
which are derived from fragmentation of
precursor megakaryocytes.

The average lifespan of a platelet is normally

just 5 to 9 days.

Platelets are a natural source of growth factors.

They circulate in the blood of mammals and are
involved in hemostasis, leading to the formation
of blood clots