MALARIA PARASITE READING

AND COUNTING TECHNIQUES

Advanced Quantitative Microscopy Training Workshop

Jakarta, October 2019
 Learning Objective

• Explain the importance of reading with passes method

and Earle-Perez method of parasite counting
• Know counting techniques applicable to thick film
• Know conversion of parasite counts to parasite density
per microliter.
• Know standard formats of reporting parasite counts.
 Background

• Accurate identification and quantification of

malaria parasites are critical for measuring clinical
trial outcomes.

• Positive and negative diagnosis is usually sufficient

for the assessment of therapeutic outcome, but
vaccine or prophylactic drug trials require
measuring density of infection as a primary
endpoint.
DEFINITION

• Reading – is a systematic method of scanning

parasites in a blood film.

• Counting – is a systematic method of

enumerating parasites in a blood film.
Quality checks before counting

• Looking for presence of debris/artifacts

• Quality of staining

• Thickness and area of film (at least 90%intact)

• Lyses and dehaemoglobinization of red cells

 Counting Techniques

• Thick Film Techniques

WHO Plus System
Parasite Counts against WBCs
Earle-Perez Method
(Parasite counts per HPF)

• Thin Film Technique

Counting parasitized RBCs

• Conversion of parasite counts to parasite densities per μL

• In determinations of the parasite density
from the thick film, the methods using
the white cell count, the ‘per HPF’
method or the Earle-Perez method may
be used (Earle & Perez, 1932).
• Accuracy with the Earle-Perez method
may not differ greatly from counting
against a known white cell count on a
thick film (Bowers et al., 2009).
• The Earle-Perez (1932) method is a method for determining
parasite density from the thick film, which can be used
when actual WBC counts are not available (WHO-WPRO,
2010c; Laurens et al., 2012). However, results can be similar
to calculation against a known white cell count, and either
method is acceptable (Bowers et al., 2009).

• In the Earle-Perez method, a known volume of blood (5 µL)

is spread evenly over a rectangle of 6 × 15 mm. The thick
film is examined using an ocular fitted with a 6 × 6 grid
reticule by moving across the width of the thick film, and
the parasite density is estimated based on the number of
parasites seen per ‘band’, i.e. calculated volume of blood,
examined across the width of the film.
• The World Health Organization recommends
counting about 0.5 microliters of blood in clinical
trials of malaria vaccines (Consensus SOP for
Malaria Microscopy in the Context of Clinical
Challenge Trials, 2010).

• This volume is approximately the same as the

volume counted in anti-malaria drug clinical trials,
which was the result of counting 200 high-power
fields of a circular thick smear with a diameter of
1.0-1.6 centimeters.
Thick Film Technique : Parasite Counts/HPF
(Earle-Perez Method)

• Gives approximate density of parasites per ul

of blood
• Thick smears are prepared as a standard 10
uL smear evenly spread.
• Size of thick film 10mm x 20mm = 200 mm2
area; therefore the depth of the smear is
10mm3/(20mm x 10mm) or 0.05 mm.
 Counts Number of Parasites Per Pass

• Perform quality check as mentioned earlier

• Start from one the middle
• Examine the smear systematically
(Vertically)
• Count number of parasites found per pass.
• Report as NO PARASITE SEEN (NPS) if 6
passes are scanned.
For a microscope with a field of diameter 0.22 mm, each
traverse (pass) through 1 cm of the film includes a
volume of :

0.22mm x 10 mm x 0.05 mm = 0.11 microliters

Therefore, 5 passes include a volume of

0.11 microliters x 5 = 0.55 microliters
AREA, VOLUME & DEPTH OF BLOOD SMEAR

1) Smear dimension is
1cm (W) x 2cm (L) equivalent to
10mm(w)x 20mm (L)
2) Area of the blood smear
10mm x 20mm = 200mm2
3) Volume used to make smear is 10µL =
10mm3
4) Depth of Blood smear is:
Volume used 10mm3 = 0.05mm
Area of smear 200mm2
Microscope with 0.18 mm HPF:
• For a microscope with a field of diameter 0.18 mm, each
traverse (pass) through 1 cm of the film includes a
volume of 0.18mm x 10 mm x 0.05 mm = 0.09 microliters
• Therefore, 6 passes include a volume of 0.54 microliters

Microscope with 0.22 mm HPF:

• For a microscope with a field of diameter 0.22 mm, each
pass through 1 cm of the film includes a volume of 0.22
mm x 10 mm x 0.05 mm = 0.11 microliter
• Therefore, 5 passes include a volume of 0.55 microliters
SLIDE EXAMINATION (PASSES)
VOLUME OF 1 PASS

• Volume of 1 Pass is:

= FOV x Width x Depth
= 0.2mm x 10mm x
0.05mm
= 0.1mm3
Thick Smear
COUNTING METHOD
Conversion to parasite counts per ul

Example:
Microscope with 0.22 mm HPF
Read 5 passes.
Found 10 PF parasites.

Therefore 10 parasites/ 0.55 uL

Equal to 18 parasites/microliters
HOW MANY PASSES IS YOUR MICROSCOPE?

• Objective is to calculate how many passes using our Microscope

• So using a Microscope with an HPF of 0.2mm, how many passes
should we read?
• Consensus SOP requires that we read 0.5 (±0.05) µL
• 1 Pass = 0.1 µL
• X Passes = 0.5 µL
• X Pass = 1 Pass x 0.5 µL
0.1µL
X Passes = 5
• 
Calculating Passes & Volume

• For a microscope with a field

of diameter 0.18 mm, each
traverse (pass) through 1 cm
of the film includes a volume
of :

• 0.18mm x 10 mm x 0.05 mm =
0.09 microliters

• Therefore, 6 passes include a

volume of
• 0.09 microliters x 6 = 0.54
microliters
Calculating Passes & Volume
Microscope with 0.18 mm
HPF:
• For a microscope with a
field of diameter 0.18 mm,
each traverse (pass) through
1 cm of the film includes a
volume of 0.18mm x 10
mm x 0.05 mm = 0.09
microliters
• Therefore, 6 passes include
a volume of 0.54 microliters
Microscope with 0.22 mm
HPF:
• For a microscope with a
field of diameter 0.22 mm,
each pass through 1 cm of
the film includes a volume
of 0.22 mm x 10 mm x
0.05 mm = 0.11 microliter
• Therefore, 5 passes include
a volume of 0.55
microliters
Measuring the Width of the High-Power Field

• The best way to determine the field of view of a microscope is to

consult the manufacturer’s description.
• The field of view of several microscope models that are often used
in clinical trials is shown below:

Manufacturer, model Width of high-power field in millimeters

• Olympus BX40 0.22
• Olympus CH-2 (CH-T) 0.18
• Olympus BX41 0.22
• Olympus BX60 0.22
• Nikon 0.22
MICROSCOPE
S FOV
•Manufacturer, model
Width of high-power field
in millimeters
• Olympus BX40
0.22
• Olympus CH-2 (CH-T)
0.18
• Olympus BX41
0.22
• Olympus BX60
0.22
• Nikon 0.22
Reference
• Basic Malaria Microscopy. Part 1. Learner’s Guide. 2nd ed. World Health
Organization, Geneva, 2010. p. 16.
• UWF-C-003 WRAIR SOP of Competency Assessment for Plasmodium
falciparum slide reading, 2008.
• Consensus SOP for Malaria Microscopy in the Context of Clinical
Challenge Trials. Appendix 1 of WHO Guidelines for Design and Conduct
of Clinical Malaria Challenge Trials. 2010.
• WHO: Microscopy for the detection, identification and quantification of
malaria parasites on stained thick and thin blood films in research
settings: procedure: methods manual. ISBN 978 92 4 154921 9.
• WHO: Research Malaria Microscopy Standards Working Group :
Microscopy for the detection, identification and quantification of malaria
parasites on stained thick and thin films. Geneva, 2015
THANK YOU...