MALARIA SPECIES IDENTIFICATION IN

THICK AND THIN BLOOD SMEARS

Advanced Quantitative Microscopy Refresher Workshop

Malabo, September 2019
Learning Objectives
• Know how to differentiate true parasites from
contaminants.
• Identify malaria parasites in thick and thin blood
smears.
• Learn how to differentiate between the different
species of malaria that infect humans.
Examples of Giemsa stained slides

Thick film Thin film

 Differences between smear types
Thick smear Thin smear
• Used to diagnose if a • Used to differentiate
patient is positive or between species
negative for malaria • If used for calculating
• More parasites can be parasite density, only a
seen on thick smears parasitemia percentage
• Better for calculating can be calculated
exact parasite densities
What cells are in the blood?
 Causes of False Positive
1. Inability to recognize blood components
(WBCs and Platelets).
2. Inability to recognize some bacteria, spores,
vegetable cells.
3. Inability to recognize debris or dust particles.
4. Inability to recognize crystals (Giemsa’ stain
and buffer salt precipitates).
Artifact

• Artifacts
• chromatin not red
• cytoplasm not true blue
• only chromatin without cytoplasm
 True Parasite
• 100% typical parasite
• purple red chromatin dot
• blue cytoplasm
• brown-black/yellowish green pigment
• distinct morphology

NB: Chromatin, cytoplasm and other features should be

in the correct form in relation to the species and age of
the parasite observed.
Malaria: Ring form trophozoite

Look for:
• A red nucleus
• Blue cytoplasm
• Malarial pigment
Taxonomi Classification

Plasmodium falciparum
Plasmodium vivax
Plasmodium malariae P. falciparum
Plasmodium ovale
P. malariae

P. vivax
P. ovale
How to differentiate between malaria species

• What is the appearance of the parasites?

• What is the size and shape of surrounding red
blood cells?
• Are there dots/stippling present?
• Are pigments present?
• Which developmental stages are seen?
• What is the appearance of the gametocytes?
 Diagnostic points for P. falciparum

• Rings appear fine and delicate with scanty cytoplasm

• Small chromatin dots
• Red blood cells are not enlarged
• May have several rings in one cell
 Diagnostic points for P. falciparum

• Presence of marginal or appliqué forms 

• Some rings may have two chromatin dots
Ring form of Plasmodium falciparum

red nucleus
blue cytoplasm

Rings appear fine and delicate with scanty

cytoplasm
Small chromatin dots
 Ring form of Plasmodium falciparum

red nucleus

blue cytoplasm

Rings appear fine and delicate with scanty cytoplasm and

small chromatin dots
 Diagnostic points for P. falciparum

Thick film Thin film

• Maurer's dots may be present. 

 Diagnostic points for P. falciparum

Schizonts Gametocytes

• It is unusual to P. falciparum schizonts in peripheral blood smears. 

• Gametocytes have a characteristic crescent shape appearance.
• However, gametocytes do not usually appear in the blood during
the first four weeks of infection. 
 Schizonts pigments of P. falciparum in
cerebrum cells

• Development of all phases after the trophozoite stage occurs in the

capillaries of the viscera.
Summary of diagnostic points for
P. falciparum
• The surrounding red blood cells are not enlarged. 
• Rings appear fine and delicate and there may be several in
one cell.
• Some rings may have two chromatin dots. 
• Marginal or appliqué forms may be seen.
• It is unusual to see schizonts in peripheral blood smears. 
• Gametocytes have a characteristic crescent shape
appearance, but are not usually present in the blood
during the first four weeks of infection. 
• Maurer's dots may be present.
 Diagnostic points for P. vivax

vacuole

• The young ring stage usually has a large chromatin dot with a thick
cytoplasmic circle around the vacuole.
• The trophozoites usually have multi-shaped, irregular, amoeboid cytoplasm.
• Red blood cells containing parasites are usually enlarged.
• Schuffner's dots are frequently present in the red blood cells. 
 Diagnostic points for P. vivax

• The mature ring forms tend to be large

and coarse.
• Schizonts are frequently present. 
 Diagnostic points for P. vivax

trophozoite

gametocyte

• The infected cells are larger than a normal red blood cell. 
• The gametocyte stage does not have a vacuole.
 P. vivax gametocytes

• No vacuole
• Round or oval homogenous cytoplasm
• Diffuse light brown pigments
• Large red chromatin
Summary of diagnostic points for
P. vivax
• The young ring stage usually has a large chromatin dot with a
thick cytoplasmic circle around the vacuole.
• The trophozoites usually have multi-shaped, irregular, amoeboid
cytoplasm.
• Red blood cells containing parasites are usually enlarged.
• Schuffner's dots are frequently present in the red blood cells. 
• The mature ring forms tend to be large and coarse.
• Schizonts are frequently present. 
• Gametocytes:
– Round or oval homogenous cytoplasm
– Diffuse light brown pigments
– Large red chromatin
 Diagnostic points for P. malariae

band form

• Ring forms may have a square appearance.

• Band forms are characteristic for this species.
 Diagnostic points for P. malariae

• The chromatin of growing trophozoites sometimes may

be hidden by coarse, dark brown pigments.
 Diagnostic points for P. malariae

• Mature schizonts may have a classic “daisy head appearance” with up to ten merozoites
forming the “petals”.
• Red blood cells are not enlarged.
P. malariae schizonts
 P. malariae gametocyte

• Same size as a normal red blood cell

• Round in shape
• Scattered pigment throughout the cytoplasm
Summary of diagnostic points for
P. malariae
• Ring forms may have a square appearance.
• Band forms are characteristic of this species.
• Mature schizonts may have a typical “daisy head
appearance”.
• Red blood cells are not enlarged.
• Gametocytes are
– the same size as normal red blood cells
– round in shape
– scattered pigment throughout the cytoplasm
 Diagnostic points for P. ovale

• Infected cells are slightly larger than normal and have an

oval or irregular frayed edge (tear drop or ghost shape).
 Diagnostic points for P. ovale

• James’ dots are present in all stages. The dots may be

larger and darker than Schuffner’s dots.
 Diagnostic points for P. ovale
schizont

merozoites

• P. ovale schizonts are usually smaller than P. vivax

schizonts.
• 75% of the cell is occupied by 8 to 12 merozoites in
irregular clusters.
 Diagnostic points for P. ovale

• Gametocytes have compact cytoplasm with dark-

brown pigments.
• Gametocytes also have a teardrop or ghost shape.
Summary of diagnostic points for
P. ovale
• Infected cells are slightly larger than normal and have an
oval or irregular frayed edge (tear drop or ghost shape).
• James’ dots are present in all stages. The dots may be larger
and darker than Schuffner’s dots.
• P. ovale schizonts are usually smaller and more compact
than P. vivax schizonts.
• 75% of the cell is occupied by 8 to 12 merozoites in
irregular clusters.
• Gametocytes have compact cytoplasm with dark-brown
pigments.
• Gametocytes also have a teardrop or ghost shape.
THANK YOU...