Guidelines on

Malaria

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Contents
Introduction to Malaria.....................................................................................................................................1
History............................................................................................................................................................2
Malaria Etiology.............................................................................................................................................4
Life Cycle and Mode of Transmission of Plasmodium...................................................................................4
Malaria Parasitesh..........................................................................................................................................6
Incubation Period..........................................................................................................................................6
Uncomplicated Malaria.................................................................................................................................7
Complicated (Severe Malaria).......................................................................................................................7
Malaria Reinfection.......................................................................................................................................8
Other Manifestations of Malaria...................................................................................................................9
Microscopy.....................................................................................................................................................9
Advantages................................................................................................................................................9
Disadvantages..........................................................................................................................................10
Rapid Diagnostic Test...................................................................................................................................10
Technique.................................................................................................................................................10
Advantages..............................................................................................................................................10
Disadvantages..........................................................................................................................................10
Microscope: Types, Care and Handling...........................................................................................................11
Microscope..................................................................................................................................................11
Types of Microscope....................................................................................................................................11
Simple microscope...................................................................................................................................11
Compound Microscope............................................................................................................................11
Electron Microscope................................................................................................................................12
Care and Handling of Microscope................................................................................................................13
Cleaning a Microscope.................................................................................................................................13
Anti-mold strips.......................................................................................................................................13
Microscope Cleaning Process..................................................................................................................13
Steps in Cleaning Eye Piece......................................................................................................................13
Cleaning Objectives..................................................................................................................................13
Cleaning the Microscope Stage...............................................................................................................14
Cleaning the Microscope Body................................................................................................................14
Cleaning the Condenser...........................................................................................................................14
Laboratory Safety............................................................................................................................................15
Introduction.................................................................................................................................................15

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 Why Safety is Important?........................................................................................................................15
General Safety Guidelines...........................................................................................................................15
Specimen Collection, Smear Preparation, Fixation and Staining (Pre-Examination Process)........................16
Blood Sample Collection..............................................................................................................................16
Capillary Blood Collection............................................................................................................................16
Venous Blood...............................................................................................................................................16
Blood Film Preparation................................................................................................................................17
Types of Blood Films................................................................................................................................17
Qualities of good thick and thin films......................................................................................................18
Common Mistakes in Making Blood Films..............................................................................................18
Fixation of Blood Film..............................................................................................................................19
Staining of Blood Film..............................................................................................................................19
Microscopic Examination and Species Identification......................................................................................21
Examining Blood Films for Malaria Parasites..............................................................................................21
Systematic Approach of Examining Thick and Thin Blood Films.................................................................21
Examining the Thick Film.........................................................................................................................21
Examining the Thin Film..........................................................................................................................22
Identification of Malaria Parasite Species, Other Blood Parasites and Artifacts........................................22
A. Morphology, stages and distinctive diagnostic features of the plasmodium.....................................22
species......................................................................................................................................................22
The schizonts stage......................................................................................................................................23
The gametocyte stage..................................................................................................................................25
Diagnostic Points of Malaria Parasites........................................................................................................26
For P. falciparum......................................................................................................................................26
For P. vivax...............................................................................................................................................27
For P. ovale..............................................................................................................................................28
For P. malariae.........................................................................................................................................29
Number of Parasites/μl Of Blood (thick film):.........................................................................................30
Proportion of Parasitized Erythrocytes/Total RBC Counts (thin film):....................................................30
Number of Parasites/μl Of Blood (thin film):..........................................................................................31
Semi Quantitative Count (thick film).......................................................................................................32
Diagnostic Quality Control Depends Upon The:..........................................................................................32
Document and Record Keeping.......................................................................................................................33
Essential Elements of Recording and Reporting..........................................................................................33
Laboratory Request and Report Forms.......................................................................................................33
Microscopy Report.......................................................................................................................................33

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 The microscopy report should include the following information:........................................................33
Entry of Data into the Laboratory Register.................................................................................................34
Supply and Logistics Management in Malaria Laboratory Diagnosis.............................................................35
Supply List for Malaria Diagnosis................................................................................................................35
Equipment and Materials:.......................................................................................................................35
Consumables............................................................................................................................................35
1. Blood lancets........................................................................................................................................35
Reagents...................................................................................................................................................36
Documents and records...........................................................................................................................36
Supply list of Malaria RDT........................................................................................................................36
Quality Assurance of Malaria Laboratory Diagnosis.......................................................................................37
What is Quality Assurance?.........................................................................................................................37
Professional Ethics and Good Laboratory Practices........................................................................................38
Scenario........................................................................................................................................................38
Reference.........................................................................................................................................................39

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Introduction to Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by protists (a
type of microorganism) of the genus Plasmodium. It begins with a bite from an
infected female mosquito, which introduces the protists via its saliva into the circulatory system, and
ultimately to the liver where they mature and reproduce. The disease causes symptoms that typically
include fever and headache, which in severe cases can progress to
coma or death. Malaria is widespread in tropical and subtropical regions in a broad band around the
equator, including much of Sub-Saharan Africa, Asia, and the Americas.
The term malaria originates from Medieval Italian: mala aria — "bad air"; the disease was formerly
called ague or marsh fever due to its association with swamps and marshland.
Malaria was once common in most of Europe and North America, where it is no longer endemic,
though imported cases do occur.
Other Plasmodium species cause infections in certain animals. Several mammals, birds and reptiles
have their own form of malaria.

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History
References to the unique periodic fevers of malaria are found throughout recorded history,
beginning in 2700 BC in China. Malaria may have contributed to the decline of the Roman
Empire, and was so pervasive in Rome that it was known as the "Roman fever".
1820 Quinine first purified from tree bark. For many years prior, the ground bark had
been used to treat malaria.
1880 Charles Louis Alphonse Laveran first identifies the malaria parasite. He is
awarded the 1907 Nobel Prize for the discovery.
1898 Sir Ronald Ross demonstrates that mosquitoes transmit malaria. He wins the
1902 Nobel Prize for this work.
1934 Hans Andersag in Germany discovers the Anti-malarial drug Chloroquine,
which is not widely used until after World War II.
1939 Paul Hermann Muller in Switzerland tests the insecticide DDT. He wins the
Nobel Prize for this work in 1948.
1952 Malaria is eliminated in the United States.
1955 World Health Organization (WHO) launches Global Malaria Eradication
Campaign, which excludes sub-Saharan Africa and is eventually abandoned.
1957 First documented case of resistance to Chloroquine is reported.
1976 William Trager and JB Jensen grow parasite in culture for the first time, opening
the way for drug discovery and vaccine research.
1989 The U.S. Food and Drug Administration approves the use of the anti-malaria
drugMefloquine hydrochloride, registered as Lariam® by Hoffman-LaRoche.
1992 Malaria vaccine candidate RTS,S, developed by GlaxoSmithKline and the
Walter Reed Army Institute of Research, enters clinical trials.
1996 Insecticide-treated bednets are proven to reduce overall childhood mortality by
20 percent in large, multi-country African study.
1998 Roll Back Malaria Partnership (RBM) launched by WHO, UNICEF, UNDP and
World Bank with goal of halving malaria incidence and mortality by 2010.
WHO adopts home management strategy for malaria whereby trained
community volunteers provide antimalarials in remote African communities.
2000 The U.N. General Assembly adopts the Millennium Development Goals, setting
a target to halt and begin reversing malaria incidence by 2015.
2001 WHO prequalifies first fixed-dose Artemisinin combination therapy (ACT), sold
by Novartis as Coartem® and recommends ACT as first-line malaria treatment.
2002 The Global Fund to Fight AIDS, Tuberculosis and Malaria is established and is
led by UCSF’s Sir Richard Feachem.
Genome sequencing of Anopheles gambiae (mosquito) and Plasmodium
falciparum (parasite) completed.
2005 World Health Assembly adopts target of 80 percent worldwide coverage of
insecticide nets and ACTs by 2010.
2007 UCSF study shows combination malaria therapy effective in treating African
children
World Malaria Forum convenes in Seattle, hosted by Bill and Melinda Gates
Foundation.
2008 The Global Health Group at UCSF comes forward with the first high-level
strategy for the eventual achievement of malaria eradication. This strategy has since
been widely adopted.
United Nations adopt April 25 as World Malaria Day.
Rectal application of the inexpensive antimalarial drug artesunate proven to

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save the lives of young children with severe malaria.
Representatives of nations around the world meet in New York and endorse
the Global Malaria Action Plan (GMAP), which lays out a vision for reducing
malaria in the short term and eventually eradicating it when new tools become
available.
2009 Global health experts at UCSF release new guidance on malaria elimination
2010 UCSF study examines progress in meeting international health goals
UCSF leads Lancet series on malaria elimination
UCSF experts outline new strategy to eliminate malaria
2011 UCSF taps Sepúlveda to lead global health efforts

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Malaria Etiology
Malaria is a disease caused by obligate intra-cellular protozoan blood parasites of the Plasmodium
species and transmitted to humans by the bite of infected female Anopheles mosquitoes. Malaria
trophozoites may also be transmitted through blood transfusion and trans-parentally (congenital
malaria). The life cycle follows three stages: the exoerythrocytic, erythrocytic and sporogonic cycle.
There are approximately 156 named species of Plasmodium which infect various species of
vertebrates. There are four different human malaria species (P. falciparum, P. vivax, P. malariae and P.
ovale), of which P. falciparum and P. vivax are the most prevalent and P. falciparum the most
dangerous. Recently, a new malaria parasite species named P. knowlesi is identified in Asia affecting
both humans and animals. Malaria
can be very severe and can lead to death if left untreated. Malaria parasite is transmitted from an
infected person to another by the bite of a female anopheline
mosquito. This can occur only after the parasite has been inside the mosquito for at least a week.
Female mosquitoes take blood meals to carry out egg production, and such blood meals are the link
between the human and the mosquito hosts in the parasite life
cycle. The successful development of the malaria parasite in the mosquito (from the “gametocyte”
stage to the “sporozoites” stage) depends on several factors. The
most important is ambient temperature and humidity (higher temperatures accelerate the parasite
growth in the mosquito) and whether
the Anopheles survives long enough to allow the parasite to complete its cycle in the mosquito host
(“sporogonic” or “extrinsic” cycle, duration 10 to 18 days).
Differently from the human mosquito host does not suffer noticeably from the presence of the
parasites.
Life Cycle and Mode of Transmission of Plasmodium
The natural ecology of malaria involves malaria parasites infecting successively two types of hosts: humans
(intermediate host) and female Anopheles mosquitoes (definitive host). In humans, the parasites grow and
multiply first in the liver cells and then in the red cells of the blood. In the blood, successive broods of
parasites grow inside the red cells and destroy them, releasing daughter parasites (“merozoite”) that
continue the cycle by invading other red cells. The blood stage parasites are those that cause the symptoms
of malaria. When certain forms of blood stage parasites (“gametocytes”) infective stage to mosquito are
picked up by a female Anopheles mosquito during a blood meal, they start another, different cycle of
growth and multiplication in the mosquito. After 10-18 days, the parasites are found (as “sporozoites”) in the
mosquito’s salivary glands. When the Anopheles mosquito takes a blood meal on another human, the
sporozoites (infective stage to human being) are injected with the mosquito’s saliva and start another
human infection when they parasitize the liver cells. Thus, the mosquito carries the disease from one human
to another (acting as a

“vector”). Differently from the human host, the mosquito vector does not suffer from the presence of
the parasites.

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Malaria Parasitesh
Among the parasites of the genus Plasmodium four species have been identified which can cause
disease in humans:
 Plasmodium falciparum
 Plasmodium vivax
 Plasmodium ovale
 Plasmodium malariae
 Plasmodium knowlesi.

Incubation Period
Following the infective bite by the Anopheles mosquito, a period of time (the “incubation period”)
goes by before the first symptoms appear. The incubation period in most cases varies from 7 to 30
days. The shorter periods are observed most frequently with P. falciparum and the longer ones with
P. malariae. Anti-malarial drugs taken for prophylaxis by travelers can delay the appearance of
malaria symptoms by weeks or months, long after the traveler has left the malariaendemic area. (This
can happen particularly with P. vivax and P. ovale, both of which can produce dormant liver stage
parasites; the liver stages may reactivate
and cause disease months after the infective mosquito bite.) Such long delays between exposure and
development of symptoms can result in misdiagnosis or delayed diagnosis because of reduced clinical
suspicion by the health-care provider. Returned travelers should always remind their health-care
providers of any travel in areas where malaria occurs during the past 12 months.

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Uncomplicated Malaria
Uncomplicated malaria: - is characterized by fever and other features including
chills, profuse sweating, muscle pains, joint pains, headache, abdominal pain,
diarrhea, nausea, vomiting, loss of appetite, irritability, and refusal to feed (in
infants). These features may occur singly or in combination and are due to the
presence of asexual forms of the parasites in the blood. Classically (but infrequently
observed) the attacks occur every second day with the “tertian” parasites (P.
falciparum, P. vivax, and P. ovale) and every third day with the “quartan” parasite
(P. malariae).

More commonly, the patient presents with a combination of the following symptoms:
In countries where cases of malaria are infrequent, these symptoms may be attributed to influenza, a
cold, or other common infections, especially if malaria is not suspected. Conversely, in countries
where malaria is frequent, residents often recognize the symptoms as malaria and treat themselves
without seeking diagnostic confirmation (“presumptive treatment”).
The classical (but rarely observed) malaria attack lasts 6-10 hours. It consists of:
• A cold stage (sensation of cold, shivering)
• A hot stage (fever, headaches, vomiting; seizures in young children)
• A sweating stage (sweats, return to normal temperature, tiredness).
Physical findings may include:
• Elevated temperatures
• Perspiration
• Weakness
• Enlarged spleen
• Mild jaundice
• Enlargement of the liver
• Increased respiratory rate

Complicated (Severe Malaria)

Severe and complicated malaria is a life-threatening condition, defined as the detection of P.
falciparum in peripheral blood together with any of the following clinical or laboratory features
(singly or in combination):
• Inability to or difficulty in sitting upright; standing or walking without support; or
inability to feed (in an infant)
• Alteration in the level of consciousness (ranging from drowsiness to deep coma)
• Cerebral malaria (unarousable coma not attributable to any other cause, other neurological signs)
• Respiratory distress
• Multiple generalized convulsions (2 or more episodes within a 24-hour period)
• Circulatory collapse (shock, septicemia)
• Pulmonary oedema
• Abnormal bleeding (Disseminated Intravascular Coagulation – DIC)
• Jaundice
• Hemoglobinuria (black water fever)
• Acute renal failure – presenting as oliguria (passing scanty urine) or anuria (not passing urine)
• Severe anemia (haemoglobin <5g/dl or hematocrit < 15%)
• Hypoglycemia (blood glucose level < 2.2 mmol/l)
• Hyperparasitaemia (parasitaemia of >200,000/μl - in patients from high transmission areas; or
100,000/μl in patients from low transmission areas)
• hyperlactatemia (whole blood lactate >5 mmol/l)
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Examples of illnesses that may present with symptoms and signs similar to malaria include:
• Meningitis
• Otitis media
• Pharyngo-tonsillitis
• Pneumonia
• Acute gastroenteritis
• Typhoid fever
• Urinary tract infection
• Viral infections (e.g. mumps, measles)
• Hepatitis
Complicated malaria occurs when infections are complicated by serious organ failures or
abnormalities in the patient’s blood or metabolism. The manifestations of severe malaria include.
• Cerebral malaria, with abnormal behavior, impairment of consciousness, seizures, coma, or other
neurologic abnormalities
• Severe anemia due to hemolysis (destruction of the red blood cells)
• Hemoglobinuria (hemoglobin in the urine) due to hemolysis
• Acute respiratory distress syndrome (ARDS), an inflammatory reaction in the lungs that inhibits
oxygen exchange, which may occur even after the parasite counts have decreased in response to
treatment
• Abnormalities in blood coagulation
• Low blood pressure caused by cardiovascular collapse
• Acute kidney failure
• Hyper parasitemia, where more than 5% of the red blood cells are infected by malaria parasites
• Metabolic acidosis (excessive acidity in the blood and tissue fluids), often in association with
hypoglycemia
• Hypoglycemia (low blood glucose). Hypoglycemia may also occur in pregnant women with
uncomplicated malaria, or after treatment with quinine.
Severe malaria is a medical emergency and should be treated urgently and aggressively.
Malaria Reinfection
is a new infection that follows a primary infection; can be distinguished from recrudescence by the
parasite genotype, which is often (but not always) different from that which caused the initial
infection.

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Other Manifestations of Malaria
• Neurologic defects may occasionally persist following cerebral malaria, especially in children. Such
defects include trouble with movements (ataxia), palsies, speech difficulties, deafness, and blindness.
• Recurrent infections with P. falciparum may result in severe anemia. This occurs especially in young
children in tropical Africa with frequent infections that are inadequately treated.
• Malaria during pregnancy (especially P. falciparum) may cause severe disease in the mother, and
may lead to premature delivery or delivery of a low-birth-weight baby.
• On rare occasions, P. vivax malaria can cause rupture of the spleen.
• Nephrotic syndrome (a chronic, severe kidney disease) can result from chronic or repeated
infections with P. malariae.
• Hyper reactive malarial splenomegaly (also called “tropical splenomegaly syndrome”) occurs
infrequently and is attributed to an abnormal immune response to repeated malarial infections. The
disease is marked by a very enlarged spleen and liver, abnormal immunologic findings, anemia, and a
susceptibility to other infections (such as skin or respiratory infections.
Microscopy
Malaria parasites can be detected, identified and quantified by examining stained blood film under
the microscope. This technique remains the gold standard for laboratory confirmation of malaria.
However, it depends on the quality of the reagents, of the microscope, and on the experience of the
laboratory personnel. When used stained thick and thin blood film, Microscopy remains the “gold
standard” for laboratory confirmation of malaria parasites. It can detect to the lowest 5 parasites per
micro liter of blood. This test should be performed immediately when ordered by a health-care
provider. They should not be saved for the most qualified staff to perform or batched for
convenience. In addition, these tests should not be sent out to reference laboratories with results
available only days to weeks later. It is vital that health-care providers receive results from these tests
within hours in order to appropriately treat their patients infected with malaria.
Advantages
Microscopy is an established, relatively simple technique that is familiar to most laboratorians. Any
laboratory that can perform routine hematology tests is equipped to perform a thin and thick malaria
smear. Within a few hours of collecting the blood, the microscopy test can provide valuable
information. First and foremost, it can determine that malaria parasites are present in the patient’s
blood. Once the diagnosis is established – usually by detecting parasites in the thick smear – the
laboratorian can examine the thin smear to determine the malaria species and the parasitemia, or the
percentage of the patient’s red blood cells that are infected with malaria parasites. The thin and thick
smears are able to provide all 3 of these vital pieces of information to the doctor to guide the initial
treatment decisions that need to be made acutely.

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Disadvantages
Microscopy results are only as reliable as the laboratories performing the tests. Those laboratorians
who does not perform this test regularly, and may not be maintaining optimal proficiency.
Rapid Diagnostic Test
RDTs detect antigens (proteins produced by malaria parasite) in the blood of a patient with malaria .
Various test kits are available to detect antigens derived from malaria parasites. Such immunologic
(“immunochromatographic”) tests most often use a dipstick or cassette format, and provide results in
2-15 minutes. These “Rapid Diagnostic Tests” (RDTs) offer a useful alternative to microscopy in
situations where reliable microscopic diagnosis is not available. Malaria RDTs are currently used in
some clinical settings and programs.
A Rapid Diagnostic Test (RDT) is an alternate way of quickly establishing the diagnosis of malaria
infection by detecting specific malaria antigens in a person’s blood.
Technique
A blood specimen collected from the patient is applied to the sample pad on the test card along with
certain reagents. After 15 minutes, the presence of specific bands in the test card window indicate
whether the patient is infected with Plasmodium falciparum or one of the other 3 species of human
malaria. It is recommended that the laboratory maintain a supply of blood containing P. falciparum
for use as a positive control.
Advantages
High-quality malaria microscopy is not always immediately available in every clinical setting where
patients might seek medical attention. Although this practice is discouraged, many healthcare settings
either save blood samples for malaria microscopy until a qualified person is available to perform the
test, or send the blood samples to commercial or reference laboratories. These practices have
resulted in long delays in diagnosis. The laboratories associated with these healthcare settings may
now use an RDT to more rapidly determine if their patients are infected with malaria.
Disadvantages
The use of the RDT does not eliminate the need for malaria microscopy. The RDT may not be able to
detect some infections with lower numbers of malaria parasites circulating in the patient’s
bloodstream. Also, there is insufficient data available to determine the ability of this test to detect the
2 less common species of malaria, P. ovale and P. malariae. Therefore, all negative RDTs must be
followed by microscopy to confirm the result.

In addition, all positive RDTs also should be followed by microscopy. The currently approved RDT
detects 2 different malaria antigens; one is specific for P. falciparum and the other is found in all 4
human species of malaria. Thus, microscopy is needed to determine the species of malaria that was
detected by the RDT. In addition, microscopy is needed to quantify the proportion of red blood cells
that are infected, which is an important prognostic indicator.

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Microscope: Types, Care and Handling
Microscope
Microscope is an instrument used to see objects that are too small to be seen by the naked eye.
Types of Microscope
There are three types of microscopes
1. Simple Microscope.
2. Compound Microscope.
3. Electron Microscope
Simple microscope
Simple microscope the simple microscope is an ordinary magnifying glass which may have a
magnification of 5x, 10x, 20x or more.

Compound Microscope
A compound microscope has a much higher magnification than the simple microscope. The typical
compound light microscope is capable of increasing our ability to see details 1000 times enlarged, so
that objects as small as 0.1 micrometer (μm) or 100 nanometers (nm) can be seen. This microscope
uses at least two lenses positioned at different places. A magnified image of the object is first
produced by one lens and this image is further enlarged by a second lens to give a more highly
magnified object. These two lenses are placed one at the end of each tube. The first lens which is
near to the object is known as the objective lens. While the second lens which is near the eye is
known as the eyepiece lens.

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Electron Microscope
Electron microscopes use a beam of electrons rather than visible light to illuminate
the sample. They focus the electron beam using electromagnetic coils instead of
glass lenses (as a light microscope does) because electrons can’t pass through
glass.

A. The transmission electron microscope (TEM) was the first electron microscope to be developed. It
works by shooting a beam of electrons at a thin slice of a sample and detecting those electrons that
make it through to the other side. The TEM lets us look in very high resolution at a thin section of a
sample (and is therefore analogous to the compound light microscope). This makes it particularly
good for learning about how components inside a cell, such as organelles, are structured.

B. The scanning electron microscope (SEM) lets us see the surface of threedimensional objects in high
resolution. It works by scanning the surface of an object with a focused beam of electrons and
detecting electrons that are reflected from and knocked off the sample surface. At low
magnifications, entire objects (such as insects) viewed on the SEM can be in focus at the same time.
That’s why the SEM is so good at generating three-dimensional images of lice, flies, snowflakes and so
on.

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Care and Handling of Microscope
Good working knowledge and proper care of the microscope are critical to good diagnostic work.
There are only a few absolute rules to observe in caring for the6 microscopes you will use. Taken care
of, these instruments will last many decades and continue to work well. Please report any
malfunctions immediately.
• Always use two hands to carry the microscope - one on the arm and one under the base. Never
carry the microscope upside down, for the ocular can and will fall out.
• Never expose it to sharp knocks, vibrations, moisture, dust or direct sunlight.
• Use lens paper to clean all lenses before and after using the oil immersion lens. Other papers are
too impure and will scratch the optical coating on the lenses. Also, do not use any liquids when
cleaning the lenses – use lens paper only!
• Always use the proper focusing technique to avoid ramming the objective lens into a slide - this can
break the objective lens and/or ruin an expensive slide.
• Always turn off the light when not in use.
• Always carefully place the wire out of harm’s way. Wires looped in the leg spaces invite a major
microscope disaster. Try sliding the wire down through the drawer handles beside your bench space.
• Always replace the cover on the microscope when you put it away

Cleaning a Microscope
Anti-mold strips
Anti-mold strips can be also applied to prevent mold. Replace these strips every 3 years. Always keep
the four optical parts of the microscope clean (see figure 11.1of the original note). Remove dust
attached to the microscope with a blower or other towels/tissue paper.
Microscope Cleaning Process
• Cleaning the eye piece
• Cleaning the objectives
• Cleaning the microscope stage
• Cleaning the microscope body
• Cleaning the condenser
• Cleaning Microscope Eyepiece Lenses
Steps in Cleaning Eye Piece
• Blow to remove dust before wiping lens
• Clean the eyepieces with a cotton swab moistened with lens cleaning solution
• Clean in a circular motion inside out
• Wipe the eyepieces dry with lens paper
• Repeat cleaning and drying if required
Cleaning Objectives
• Objectives are cleaned while attached to the microscope
• Moisten the lens paper with the cleaning solution
• Wipe gently the objective in a circular motion from the inside out
• Wipe with a dry tissue or lens cleaning paper
• Objectives should never be removed from the nosepiece

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Cleaning the Microscope Stage
• Wipe the microscope stage using the cleaning solution on a soft cloth
• Thoroughly dry the stage
• Repeat the above steps, if required
Cleaning the Microscope Body
• Unplug the microscope from the power source
• Moisten the cotton pad with a mild cleaning agent (please give examples)
• Wipe the microscope body to remove dust, dirt and oil
• Repeat steps1–3, if required
Cleaning the Condenser
• Unplug the microscope from the power source
• Clean the condenser lens and auxiliary lens using lint-free cotton swabs moistened with lens
cleaning solution
• Wipe with dry swabs

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Laboratory Safety
Introduction
Safety is one of the 12 quality system essentials which is important in order to protect the lives of
employees and patients, to protect laboratory equipment and facilities, and to protect the
environment.
Why Safety is Important?
 Coming in contact with human blood or blood products is potentially hazardous
 Good Safety practice protectant us Safety involves taking precautions to protect you and the
client against infection. What Else Needs protection?
 Good safety practice also protects Other people who may come in contact with testing by-
products
 It Protect integrity of test products Protect environment from hazardous Materials
General Safety Guidelines
Standard operating procedures (SOPs) that cover all steps should be clearly written
and carried out which also ensures safety measures. Generally, the following safety
precautions should be implemented at all times.
 Wear a laboratory coat when in the working room and remove any protective clothing before
leaving the laboratory.
 Wear gloves when taking and handling blood specimens.
 Do not touch your eyes, nose or other exposed membranes or the skin with gloved hands.
 Change gloves between patients and remove the gloves before touching objects and surfaces
e.g. door handles and other objects not usually touched by gloved hands; wash your hands
and put on new gloves.
 Cover broken skin with water resistant wound covers before wearing gloves
 Wash your hands with soap and water immediately after any contamination and after the
work is completed. If gloves are worn, wash your hands with soap and water after removing
gloves.
 Puncture wounds, cuts and skin contaminated by spills or splashes of blood should be
thoroughly washed with soap and water. Bleeding from the wound should be encouraged.
 Dispose of used lancets in a sharps container.
 Disinfect work surface areas when blood collection procedures are completed and at the end
of each working day.
 Do not eat, drink or smoke in the working area.
 For use on all surfaces, use 0.5% solution of bleach.
 Prepare fresh working solutions of bleach daily.
 Carefully handle all chemicals and reagents according to accepted standards

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Specimen Collection, Smear Preparation, Fixation
and Staining (Pre-Examination Process)
Blood Sample Collection
Whenever possible, specimens should be collected for laboratory examination before treatment is
initiated. When malaria is suspected, blood smears should be obtained and examined without delay.
Blood sample for malaria parasite diagnosis can be collected either from the capillaries or the veins
Capillary Blood Collection
Capillary blood is obtained by finger stick and is preferred to venous blood. Blood obtained by
pricking a fingertip is the ideal sample because the density of developed trophozoites or schizonts is
greater in blood from capillary-rich areas. For adults, the best site to prick is the lateral side of the
third or fourth finger of the non-dominant hand (left hand unless the patient is left-handed) and the
big toe and/or the heel is preferred for infants. The skin area to be punctured should be warm so that
blood flow will be adequate. Depending on the physical settings and the patient’s condition, warming
the hand with warm water, covering the hand with a hot, wet towel or briskly rubbing the hand may
be used to warm the hands prior to the finger prick. Label the frosted end of the slide with the patient
ID number and date. Apply gentle pressure again (do not squeeze the finger too tightly) to transfer
more blood and collect two or three larger drops (approximately 6 μl) on the slide, about 1 cm from
the drop intended for the thin film or 1 cm from the end of the slide.
Venous Blood
Venous blood is obtained by venipuncture. It is not collected for routine use in malaria laboratory
diagnosis. The venipuncture procedure is complex, requiring
both knowledge and skill to perform. Each phlebotomist generally establishes a method that is
comfortable for her or him. Several essential steps are required for
every successful collection procedure and venipuncture site selection. Although the larger and fuller
median cubital and cephalic veins of the arm are used most
frequently, the basilic vein on the dorsum of the arm or dorsal hand veins are also acceptable for
venipuncture. Palpate and trace the path of veins with the index
finger. Arteries pulsate, are most elastic, and have a thick wall. Thrombosed veins lack resilience, feel
cord-like, and roll easily.
If superficial veins are not readily apparent, you can force blood into the vein by massaging the arm
from wrist to elbow, tap the site with index and second finger, apply a warm, damp wash cloth to the
site for 5 minutes, or lower the extremity over the bedside to allow the veins to fill. Foot veins are a
last option because of the higher probability of complications. One should recognize complications
associated with the phlebotomy procedure, assess the need for recollection and/or rejection of
sample and perform proper labeling of the specimen.
Venous blood samples provide sufficient material for performing a variety of diagnostic tests,
including concentration procedures (filariasis, trypanosomiasis). However, in some parasitic diseases
(e.g., for diagnosis of malaria in particular), anticoagulants in the venous blood specimen can interfere
with parasite morphology and staining characteristics; this problem can be further compounded by
excessive delays prior to making the smears. In such cases, capillary blood samples are preferable.
Note: Capillary blood is always preferred for malaria diagnosis than venous blood

19
Blood Film Preparation
Types of Blood Films
Two types of blood films, thick and thin, are used in the microscopic diagnosis of malaria. Both thick
and thin films should be prepared and examined in all cases of suspected malaria. For routine malaria
microscopy, thin and thick blood films are prepared on the same slide.
54
Thick Blood Film
Thick blood film consists of a thick layer of lysed erythrocytes. The blood elements (including
parasites, if any) are more concentrated (~30x) than in an equal area of a thin smear, allowing a
greater volume of blood to be examined. Because a larger volume (6 μl) of blood is examined in the
thick film, it is much better than the thin film for detection of low levels of parasitemia and
reappearance of circulating parasites during infection, recrudescence or relapse. Thick film is
therefore the most suitable method for the rapid detection of the parasite, but it does not permit an
optimal review of parasite morphology for species identification. If the thick smear is positive for
malaria parasites, the thin smear should be used for species identification. Thus, the thick films are
performed to detect and quantify (parasite density) malaria parasites in routine malaria microscopic
diagnosis.

Thin Blood Film

Thin blood film consists of blood spread in a layer such that the thickness decreases progressively
toward the feathered edge of microscopic slide. In the feathered edge, the red blood cells should be
in a single layer, not touching one another. Thin blood smear should be fixed with methanol so that
the parasites are found intact inside the RBCs. The morphological identification of the parasite to the
species level is much easier and provides greater specificity than the thick film examination.

However, low-density infections can be missed and require more time to read. Thin blood film is
used to assist in the identification of the malaria species after the parasites have been seen in the
thick film. Both thick and thin films must be thoroughly dry . Allow the slide with the thin and thick
films to dry inside a folder rack in a flat, level position (which allows the thick film to dry with even
thickness), protected from flies, dust and extreme heat. Insufficiently dried blood film (and/or blood
films that are too thick) can detach from the slides during staining. Thin smears will dry and be ready
to fix and stain in about 15 minutes. Thick smears will dry in a minimum of 30 minutes at room
temperature. You can accelerate the drying by using a fan or hair dryer (set on cool). Do not dry in an
incubator or by exposure to heat or sunlight as this will fix the blood cells and interfere with
lysing the red blood cells prior to staining.
55

20
Qualities of good thick and thin films
A thin smear should
• Be uniformly spread over the slide
• Be thin enough so that it is tongue shaped
• Consist of a single layer of RBCs with a feathered end
A thick smear should
• Be 10 mm away from the edge of the slide
• Be round in shape with a diameter of about 10-12 mm
• Have a thickness containing 10 layers of RBCs
• Have 10-12 WBCs visible per oil immersion field of microscope

Common Mistakes in Making Blood Films

(a) Badly positioned blood films
Care should be taken that the blood films are correctly smeared on the slide. If they are not, it may be
difficult to examine the thick film. Also portions of the films may even be rubbed off during the
staining or drying process.
56
(b) Too much volume of blood
After staining films made with too much blood, the background of the thick film will be too blue.
There will be too many white blood cells per thick film field, and these could obscure or cover up any
malaria parasites that are present. If the thin film is too thick, red blood cells will be on top of one
another and it will be impossible to examine them properly after fixation.

(c) Too little volume of blood:

If too little blood is used to make the films, there will not be enough white cells in the thick film field
and you will not be able to examine enough blood in the standard examination. The thin film may be
too small for use as a label for patient identification.

(d) Blood films spread on a greasy slide:

On a greasy slide blood films will spread unevenly, making the examination very difficult. Some of the
thick film will probably come off the slide during the staining process
57
(e) Chipped edge of spreader slide
When the edge of the spreader slide is chipped, the thin film spreads unevenly, is streaky and has
many ‘tails.’ The spreading of the thick film may also be affected.

(f) Thin film too large, thick film in the wrong place
If the thin film is too large, the thick film will be out of place and may be so near the edge of the slide
that it cannot be seen through the microscope. During staining or drying, portions of the thick film
will probably be scraped off by the edges of the staining trough or drying rack. It may be very difficult
or impossible to position the thick film on the microscope stage for examination.

21
Fixation of Blood Film
Fixation is used to preserve cellular and parasitic morphology. It is done when the blood films are
completely dried. Absolute methanol is a recommended fixative solution.

Note: Auto-fixation may also occur spontaneously with time if thin films not fixed immediately after
being dried (7 to 15 days, varying with humidity and temperature of the atmosphere)

Staining of Blood Film

Principles of Romanowsky Stains
Blood cells and parasites are stained by Romanowsky stains. Romanowsky stains comprise two
staining components: azures (oxidation products of methylene blue)
and eosin. Examples of Romanowsky stains include Field’s stain, Giemsa’s stain, Leishman’s stain and
Wright’s stain. Giemsa stain is regarded as the best stain for malaria microscopy. Field’s stain is useful
in health facilities with a low patient workload as it is rapid, economical and easy to use. All
Romanowsky stains can be used to stain thick and thin blood films once the staining principles are
understood.

Giemsa Stain
The Giemsa stain must be diluted for use with water buffered to a pH 7.2, depending on the specific
technique used. The stain should be tested for proper staining reaction before use. The stock is
stable, but it must be protected from moisture because of the staining reaction. Giemsa stain will not
function as expected if stock is mixed with even small amounts of water or moisture solution during
its preparation or storage.
To control the quality of Giemsa stain for proper staining results, a known positive smear should be
included with each new batch of working Giemsa stain. Control slides may be prepared from a
patient’s blood and stored for future use. From a patient known to have a malaria infection, a blood
sample is collected in an EDTA (ethylene diamine tetra acetic acid) or citrated blood tube if it requires
multiple blood film preparations or needs further diagnosis at a molecular level. An ideal quality
control blood film should have at least one parasite in every 2–3 fields on a thin blood smear. Make as
many thin smears as possible, preferably within one hour of drawing the blood from the patient.
Label the outside of the slide box with the species, date and ‘Giemsa control slides.’ The slides can be
stored at room temperature but will last longer if stored at -20°C or -70°C. Just before use, remove
the slide from the box and allow the condensation to evaporate; label the slide with the date and ‘+
control.’ The smear can then be stained and examined to check that the working solution of Giemsa
stain is of good quality.

Principle of Giemsa Stain

A properly stained blood film is critical for malaria diagnosis, especially for precise identification of
malaria species. Use of Giemsa stain is the recommended and most reliable procedure for staining
thick and thin blood films. Giemsa solution is composed of eosin and methylene blue (azure). The
eosin component stains the parasite nucleus red, while the methylene blue component stains the
cytoplasm blue.

22
Preparation of Giemsa Stock Stain
To make about 500 ml of Geimsa stock solution, we need:
• Giemsa Powder ------------------------------------------------------------- 3.8g
• Absolute methanol---------------------------------------------------------- 250ml
• Glycerol---------------------------------------------------------------------- 250ml
Field’s Stain
Field’s stain is useful for rapid detection of malaria parasites particularly for thick films. However,
Schuffner’s dots are not always stained with this procedure. It is made up of Field’s stain A and Field’s
stain B as both are used in the staining procedure.

Buffer Solution for Malaria Staining

A phosphate buffer solution, correctly balanced to pH 7.2, is essential for Giemsa and Field’s staining
for malaria parasites. Check the pH level using narrow-range pH papers or a pH meter and store the
buffer solution at room temperature. The buffer is stable for several months. To check its quality, the
pH of buffered water should be checked, and appropriate correcting fluid should be added.

Buffer Tablets
Buffer tablets that produce a solution of pH 7.2 when dissolved are readily available from laboratory
suppliers but are rather expensive. Per the manufacturer’s instruction, different grams of the buffer
tablet are dissolved in a defined volume of distilled water and used to prepare a Geimsa working
solution.

Quality Control of Buffered Water

Prepare a buffer reagent carefully; weighing accurately the dry chemicals and checking the pH level.
Alternatively, use buffer tablets. Store buffered reagents at 2- 8ºC in a tightly stoppered (preferably
plastic) bottle; when in use, avoid leaving the reagents exposed to sunlight (which encourages the
growth of algae) and check for contamination (cloudiness) at regular intervals.

23
Microscopic Examination and Species Identification
Examining Blood Films for Malaria Parasites
Microscopic examination of Giemsa stained thick and thin blood film is the most acceptable method
for detecting malaria parasites. This technique requires trained and experienced laboratory personnel
and equipment such as a microscope. The thick film used for rapid detection of malaria parasites and
enhances sensitivity for the detection of low levels of parasitemia. On the other hand, the thin film is
used to confirm plasmodium species and also to assist in the identification of mixed infections. Both
thick and thin films should initially be examined completely with low power magnification to avoid
missing large organism such as Microfilaria and Trypanosomes.

Systematic Approach of Examining Thick and Thin Blood Films

Examining the Thick Film
Thick films are performed to detect parasites and measure parasite density (quantification), and can
be used to monitor response to treatment. Parasites are quantified by counting ring forms
(trophozoites) against white blood cells. The results are expressed as parasite count per 200 white
blood cells (WBC) or parasite count per microliter of blood, assuming a total white blood cell count of
8000/μl if a measured white blood cell count is not available. This method of quantitation is useful in
low and moderate parasitaemia. Examination of a thick film requires observation of 100 good fields.
The blood film can be pronounced negative only after no parasites have been found in at least 100
fields. If parasites are found, a further 100 fields should be examined before a final species
identification is made.
This ensures that there is little possibility of a mixed infection being overlooked. Since the
erythrocytes have been lysed and the parasites are more concentrated, the thick film is useful for
screening of parasites and detecting mixed infections.

Procedure for examination of thick blood film

1. Place the stained slide on the mechanical stage.
2. Scan the entire film at a low magnification.
3. Place a drop of immersion oil on the middle of the thick film.
4. Examine the film using the 100×.
5. Select an area that is well-stained, free of stain precipitate, and well populated WBCs (10-20
WBCs/field).
6. Move the blood film following the pattern shown in the diagram.
7. If you see parasites, make a tentative species determination on the
thick film and then examine the thin film to confirm the species
present.

24
Examining the Thin Film
Thin films are useful for species identification of plasmodium species. Examination of thin film greatly
assists in the identification of mixed infections and is also used for quantification purposes.
Examination of thin films is recommended in the following circumstances:

• When the thick film is too small, has become auto fixed, or is not examinable for other reasons.
• When it is necessary to confirm the identification of species.

Procedure for examination of thick blood film

1. Place a drop of immersion oil on the edge of the middle of the film.
2. Carefully examine the film using the 100×.
3. If doubtful diagnosis examine more fields

Identification of Malaria Parasite Species, Other Blood Parasites and

Artifacts
A. Morphology, stages and distinctive diagnostic features of the
plasmodium
species
The simplest guide of distinguishing between the four species of malaria is the effect of the parasite
on infected red blood cells. Diagnostic features to be considered include the size of the red blood cell
(whether it is enlarged or not) and presence or absence of Schuffner’s dots or Maurer’s dots (also
known as Maurer’s clefts) within the cell. Schuffner’s dot refers to a hematological finding that is
associated with malaria, exclusively found in Plasmodium ovale and Plasmodium vivax. Plasmodium
vivax induces morphologic alterations in infected host erythrocytes that are visible by light
microscopy in Romanowsky-stained blood smears as multiple brick-red dots. These morphologic
changes, referred to as Schuffner’s dots, are important in the identification of this species of malarial
parasite. Maurer's dots are any of the fine granular precipitates or irregular cytoplasmic particles
usually present in red blood cells infected with the trophozoites of Plasmodium falciparum.

25
Malaria parasites pass through a number of developmental stages. In all stages, the structural
features of the parasite stain the same color using Romanowsky stains, as follows:
• Chromatin (part of the parasite nucleus) is usually round in shape and stains red.
• Cytoplasm occurs in a number of forms, from a ring shape to a totally irregular shape. It is always
stained blue, although the shade of blue may vary between malaria species.
• Malaria pigments stain in various shades from yellow-gold through brown to black.
• Stippling stains in shades of pink which vary among different species.

Identification of Malaria Parasite Species and Stages

The trophozoites stage
This stage is the most commonly seen. It is often called the ring stage (although sometimes it takes
the form of an incomplete ring).

Because the trophozoites stage is a growing stage, the parasite within the red blood cell may vary in
size from small to quite large. Pigment appears as the parasite grows. Malaria pigment is a byproduct
of the growth or metabolism of the parasite. It does not stain, but has a color of its own, which may
range from pale yellow to dark brown or black.

The schizonts stage

At the schizonts stage the malaria parasite starts to reproduce. This reproduction is referred to as
asexual because the parasite is neither male nor female but reproduces itself by simple division.
There are several obvious phases in this stage, ranging from parasites with two chromatin pieces to
parasites with a number of chromatin dots and definite cytoplasm. These are seen clearly in the
diagram below.

26
Note: The process of forming schizonts, which takes place in the liver and
in blood, is referred to as schizogony.

27
The gametocyte stage
The gametocyte stage is the sexual stage, in which parasites develop into either male or female forms
in humans for the preparation of the next stages, which takes place in the mid-gut of the female
Anopheles mosquito. Gametocytes may be either banana-shaped (P. falciparum) or round (other
species), and are either male (microgametocytes) or female (macro gametocytes).

Appearance of different malaria parasite species in thick blood films

In Thick Blood Films
The staining process ruptures non-nucleated red cells but keeps white cells, nucleated red cells and
parasite structures intact. Parasites and white blood cells can therefore be seen, although they may
appear smaller and less welldefined than in thin blood films.
In thick blood films, the fine rings of cytoplasm of trophozoites may appear incomplete or broken. The
absence of red blood cells makes Schuffner’s dots (usually seen in Plasmodium ovale or vivax) more
difficult to see. The “ghosts” of red cells may be seen surrounding the parasites in the thinner part of
the film.
In Thin Blood Films
Malaria parasites appear more well-defined in thin blood films, although it may be more difficult to
detect parasites in blood with low parasitaemia. Some species of malaria parasites have an effect on
the appearance of red blood cells in thin blood films: for example, enlargement of red cells in P. vivax
infection; or oval red cells in P. ovale infection. Staining may also reveal Schüffner’s dots, or Maurer’s
clefts within the cells.
67

28
Diagnostic Points of Malaria Parasites
For P. falciparum
• Red Cells are not enlarged
• Maurer’s dots may be present
• No Schüffner’s dots
• Rings appear fine and delicate, there may be several in one cell
• Some rings with 2 chromatin dots
• Presence of marginal or appliqué forms
• Gametocytes have a crescent/banana shape
• Usually trophozoites and gametocytes are seen
• Schizonts are rarely seen

29
For P. vivax
• Red cells are usually enlarged
• Schüffner’s dots are frequently present in the red cells as shown above
• The mature ring forms tend to be large and coarse
• Trophozoites schizonts and gametocytes
• Developing forms are frequently Present

30
For P. ovale
• Red cells are enlarged
• Comet forms are common (top right)
• Rings are large and coarse
• Schüffner’s dots, when crescent, may be prominent
• Mature schizonts similar to those of P. malariae but larger and more coarse

31
For P. malariae
• RBCs are not enlarged
• Ring forms may have a square-like appearance
• Band forms are a characteristic of this species
• Mature schizonts may have a typical daisy head appearance with up to ten merozoites

32
Number of Parasites/μl Of Blood (thick film):
Accurate parasite density estimation based on parasites per micro liter or white cell count is
necessary when parasite density determination is important for clinical decision-making (for example
in severe malaria or where monitoring of treatment efficacy is required) and in clinical trials. It is
recommended in routine practice that parasite quantitation be performed against 200 or 500 WBCs.
If, after counting 20080 WBCs, 100 or more parasites are found, record the results in terms of the
number of parasites per 200 WBCs. If less than 100 parasites are found after counting 200 WBCs,
parasite quantification should be continued until 500 WBCs are counted.
(This gives a probability <5% of chance variation greater than 25% of true parasite density using a
x100 oil immersion objective and an eyepiece with a field number
of 18). All parasites in the final field are counted, even if the count exceeds 500 WBCs. To determine
parasite density, the parasite count is adjusted against the true WBC count where available. If
unavailable, a common practice is to assume a WBC count of 8000/μL.

Proportion of Parasitized Erythrocytes/Total RBC Counts (thin film):

This method will indicate the percentage of erythrocytes that are infected by malaria parasites. The
percentage of infected red cell is determined by counting the number of red cells and that of
parasitized red cells. This method of quantification is useful in high parasitemia. Since it takes almost
10 times as long to examine a thin film as to examine a thick film, routine examination of thin films is
not recommended. The number of parasitized erythrocytes (asexual forms) present in 25 microscopic
fields is counted and divided by the total number of erythrocytes present in these fields (about
5,000), and multiplied by 100.

In this example 2% of the RBCs are infected with asexual forms of malaria parasites

33
Number of Parasites/μl Of Blood (thin film):
This method requires the preliminary determination of the number of erythrocytes present in the
average microscopic field. The number of asexual parasites is counted in at least 25 microscopic
fields. The number of RBCs in the average microscopic field is about 200, so total RBCs counted in 25
fields is roughly 200*25 = 5000. If the hemogram is not available, RBCs/μL is assumed at 5,000,000 for
males and 4,500,000 for females.

34
Semi Quantitative Count (thick film)
This method has been used for a long period of time but currently it is not recommended because it is
less accurate.
Reporting:
• + 1-10 asexual parasites / 100 thick film fields
• ++ 11-100 asexual parasites / 100 thick film fields
• +++ 1-10 asexual parasites / single thick film field
• ++++ > 10 asexual parasites / single thick film field
Reporting Blood Film Results
Reporting of malaria positive blood film should include the species, stage and, if necessary, the
density of the parasite. Species, stage and approximate number of parasites
1. P. falciparum
o Ring only + Number (per μl)
o Ring and gametocyte
o If schizonts seen, report immediately and mark with a red pen!
2. P. vivax, P. malariae, and P. ovale – all stages can be found.
3. Mixed infection – report appropriately
4. No hemoparasites found (NHPF) or Negative – if you do not find malaria and other hemo-parasite
after examination of a minimum of 100 oil immersion fields.
82
Diagnostic Quality Control Depends Upon The:
• Compliance with standards
• Availability of supplies, equipment, infrastructure
• Condition of the microscope
• Training of lab personnel and Regular supervision
• Quality of reagents and stains and Cleanliness
• Work load, Technical ability and type of techniques used

35
Document and Record Keeping
Essential Elements of Recording and Reporting
Accurate document and record keeping in the malaria laboratory is essential.
Proper recording and reporting of patient data used to:
• Determine work load
• Planning of resources
• Compile important epidemiological information for malaria prevention and
control program.
Laboratory Request and Report Forms
In many countries, the laboratory request form and the microscopy report form are combined into a
single sheet of paper. This enables better tracking of reports and reduces the time to transcribe the
patient- and sample-related information on separate report forms and hence reduces transcription
errors. A Laboratory request form must be submitted with the patient information and it must match
the information on the slides exactly. If the form is incomplete but the patient is available, ask the
patient for the required information.
A completed Laboratory Request Form should contain the following information:
• Name of the health facility
• Date (day, month, year)
• Patient’s name, address, age and sex
• Type of specimen
• Patient ID/ Specimen ID number (laboratory serial number)
• Clinical impression
• Signature of person requesting the exam
Microscopy Report
After the blood film has been read, immediately write the result in the result form. Check that the
specimen ID (laboratory serial numbers) on the slide matches that on the laboratory request form.
Subsequently, write the results in the Laboratory Register, again checking to make sure that the
laboratory serial number matches exactly.
The microscopy report should include the following information:
• Specimen ID number
• Date and time of specimen Collection
• Date of specimen analysis (day, month, year)
• Blood film result (presence of parasite, species, stage and parasite count/density against WBC)
• If necessary (high parasitemia) percentage of infected RBC in thin films
• Name and signature of laboratory technician performed the test
• Name and signature of laboratory technician reviewed the result
Once completed, the microscopy report should be made available as soon as possible. Send the
report to the referring health facility or clinician. Ensure that the health facility or clinician receives
the result.

36
Entry of Data into the Laboratory Register
Laboratory Register Book: The laboratory register book is a record book maintained by the laboratory
personnel responsible for blood film examination of patients with suspected malaria. The health
facility laboratory register must include the following data for each patient with suspected malaria:
• Laboratory serial number
• Date of specimen collection
• Date of specimen testing (day, month, year)
• Patient’s name, sex, age, and address
• Type of test
• Test result(s)
• Signature of person responsible for tests

37
Supply and Logistics Management in Malaria
Laboratory Diagnosis
For successful supply Management, the system must fulfill the Six rights of supply chain management.
• The right products
• In the right quantity
• with the right quality
• On the right Place
• At the right time
• By the right cost.

Supply List for Malaria Diagnosis

Equipment and Materials:
1. Slide box
2. Staining jar (to hold 20 slides, placed back to back)/rack.
3. Drying rack
4. Forceps
5. Measuring cylinder
6. Slide boxes
7. Binocular microscope- Microscope spare bulb: 1 per microscope for 1 year, Microscope
8. Spare mirror, fuses, eyepiece, and oil immersion objective: 1 per microscope for 10 years
9. Tally counter(s)
10. Funnel
11. Dropper (with rubber bulb)
12. PH meter
13. Timer
14. Beam balance
15. brown bottle
16. spatula/spoon
Consumables
1. Blood lancets
2. Absorbent cotton wool/cotton
3. Alcohol (70% ethanol)
4. Disposable gloves
5. Clean glass slides
6. Pencil/pen/marker
7. Sharps container
8. Biohazard containers
9. Distilled water/ buffered water
10. Lens paper
11. Immersion oil (Type A)
12. Lens cleaning solution
13. Filter paper (e.g., What man #1)
14. Measuring cylinder, capacity 100-500 ml (depending on the number of slides to be stained)
15. Measuring cylinder, capacity 10-25 ml (depending on the amount of stock stain to be measured)

38
16. PH tab
17. Beads
18. QC slides
19. Ethanol
20. Plaster
21. Weighting paper
Reagents
1. Absolute methanol
2. Giemsa powder
3. Glycerol
4. 10% Giemsa working solutions
5. Giemsa stock solution
Documents and records
1. Laboratory Registration book
2. Laboratory test request & result reporting form
3. Material safety data sheet (MSDS)
4. Bin card
5. Stock card
6. SOP
7. Job aid
8. Different formats
Supply list of Malaria RDT
1. RDT Device
2. Sterile Blood Lancet
3. Alcohol Swab
4. Sample transfer Pipette
5. Buffer
6. Glove
7. Timer
8. Sharp container
9. Biohazard bag
10. Labeling pencil / Pen/ permanent marker
11. Waste Bin

39
Quality Assurance of Malaria Laboratory Diagnosis
What is Quality Assurance?
Quality Assurance (QA) is a system designed to improve the reliability and efficiency of laboratory
services, which are critical to the success of malaria control programs. All parts of the testing system
must be monitored to ensure the quality of the overall process, to detect and reduce errors, and to
improve consistency between testing sites. To ensure reliability and to reduce errors, a quality system
must address all parts of laboratory testing.
The components of a quality assurance program for laboratory diagnosis of malaria are the following:

Quality Control (QC): QC is systematic internal monitoring of work practices, technical procedures,
equipment and materials, including the quality of stains. External Quality Assessment (EQA): EQA is a
schematic assessment by an external entity of a laboratory’s performance in testing of known and
standardized but undisclosed content and comparing the results with those of other participating
laboratories to assess laboratory practices and identify problems and weaknesses. EQA includes
onsite evaluation of laboratories, proficiency panel tests and blinded smear rechecking.

Quality Improvement (QI): QI is a process by which the components of blood film microscopy
diagnostic services are analyzed with the aim of identifying and permanently correcting any
deficiencies. Data collection, data analysis, and creative problem solving are skills used in this process
Generally quality assurance has three phases: pre-analytical, analytical and post analytical phases as
summarized in the figure below.

40
Professional Ethics and Good Laboratory Practices
Scenario
A SMALL TRUTH MAKES LIFE 100%
IF
ABCDEFGHIJKLMNOPQRSTUVWXYZ
IS EQUAL TO
12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Luck: L+U+C+K = (12+21+3+11 = 47%
Love: L+O+V+E = 12+15+22+5 = 54%
Money: M+O+N+E+Y= 13+15+14+5+25 = 72%
Behavior: B+E+H+A+V+I+O+R = 2+5+8+1+22+9+15+18 = 80%
Leadership: L+E+A+D+E+R+S+H+I+P= 12+5+1+4+5+18+19+9+16 = 89%
• Then what makes 100%? Is it Knowledge? ... NO!!
K+N+O+W+L+E+D+G+E =11+14+15+23+12+5+4+7+5 = 96%
Hard Work? ... NO!!! H+A+R+D+W+O+R+K= 8+1+18+4+23+15+18+11 = 98%
• Every problem has a solution, only if we perhaps change our attitude.
• To go to the top, to that 100%, what we really need to go further... a bit more...
ATTITUDE A+T+T+I+T+U+D+E; 1+20+20+9+20+21+4+5 = 100%

41
Reference
The appearance of Plasmodium stages in the different malaria species are
shown.www.rph.wa.gov.au/malaria/diagnosis.html

http://wwwn.cdc.gov/mpep/labquality.aspx accessed on 04/09/2012

CDC. DPDx: Laboratory Identification of Parasites of Public Health Concern/Parasites and
Parasitic Diseases; Blood-borne Parasites: Malaria http://www.dpd.cdc.gov/dpdx, accessed
on March 15, 2009.

Chansuda W, Mazie JB, et.al. (2007). Review of Malaria Diagnostic Tools: Microscopy
and Rapid Diagnostic Test (RDT) American journal of tropical medicine and hygiene, 77,
pp.

Cheesbrough M (1998). District laboratory Practice in Tropical Countries. Part 1,

Cambridge University Press, UK.239-258.

Chotivanich K, Silamut K, Day N (2007). A Short review of methods; Laboratory

Diagnosis of malaria infection, New Zealand Journal of medical laboratory science

EHNRI (2008). Acid fast bacilli sputum smear microscopy training module.

EHNRI (2008). National AFB participant manual (Draft), April 2008.

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