CHAPTER 6

Quality control in Parasitology

Acknowledgement

 Addis Ababa university

 Jimma university
 Haramaya university
 University of Gondar
 American society of clinical pathology
 Centre for disease prevention and control- Ethiopia
Learning Objectives

Upon completion of this chapter the student will be able to:

 Describe quality control methods in
 Stool specimen collection, processing, examination,
reporting and disposal
 Blood specimen collection, processing, examination,
reporting and disposal
 Apply the knowledge of quality assurance in the routine
Parasitology laboratory
Introduction

 To ensure accurate and reliable results, quality control

must be applied to laboratory procedures for diagnosing
parasitic infections
 Controls must apply to collection of specimens, preparation
of reagents, performance of the techniques and
examination of the final preparation
Quality control for fecal examination

Collection of specimens:
 If fecal specimens are not properly collected and taken
care of before examination, they will be of little or no value
for accurate diagnosis
 This is especially true of protozoa
 Amebic trophozoites will begin to degenerate 1- 2 hours
after passage and alterations in appearance may result
in erroneous identification
Specimen collection…

 Flagellate trophozoites may also undergo changes that

would make differentiation difficult
 Cysts will deteriorate if fecal specimens are left standing
for many hours or overnight, especially if the
temperature is high
 Helminth eggs and larvae are less affected by the age
of the specimen than are protozoa
collection of …

 Nevertheless, changes may occur that would affect

identification
 Hookworm eggs, for example, may become
embryonated and larvae may hatch from the eggs
 Even Ascaris eggs may develop to multicellular stages
 In addition, larvae may degenerate in old stools making
it impossible to identify the species
Collection of …
 To ensure that good specimens are provided for
examination, pay attention to the following points:
 Use clean, dry containers for collecting feces
 Dirt will interfere with examinations and may
introduce free – living organisms from the soil that
would cause problems in identifying the species
 Urine and water will destroy trophozoites, if present
Collection of …
 Have the specimen brought to the laboratory as soon as
it is passed to prevent deterioration of protozoa and
alterations in the morphology of protozoa and helminths.
 Note the patient’s name and the date and time of
passage on the specimen
 Accept only freshly passed specimens for examination.
 Do not attempt to examine old specimens or those
contaminated with dirt, water, or urine.
Collection of …
 Instead, ask the patient to pass another specimen
 If specimens cannot be examined as soon as they
arrive, put them in a refrigerator (4 – 5O C) or in the
coolest, shadiest area in the laboratory. Do not leave
them in the sun
 Examine diarrheal specimens and those containing
blood and mucus immediately up on their receipt in the
laboratory.
Collection of …
 Some reagents will last indefinitely if kept properly
stoppered and out of direct sunlight.
 Examples are formalin solutions, isotonic saline,
fixatives, and alcohol solutions (unless evaporation
occurs)
 Other reagents may last for only a short time and are
ineffective if too old
 The “life” of each solution is indicated in the direction for
preparing it
Collection of …

1. Label all reagents with the date of preparation. Keep

records for each solution. Review these every week and
discard outdated solutions

2. Many of the solutions used in the method for trichrome

stain need to be changed at regular intervals. The intervals
are stated in the directions for the technique and must be
observed. Failure to do so will result in poor preparation
that are of no value for diagnosis
Performance of techniques
 No procedure used for examining fecal specimens is
100% effective – that is the procedures will not always
recover all the species present and, if a particular species
is present in only very low numbers, they may fail to
demonstrate them when used on a single specimen.
Because the techniques are not perfect, you should
perform them as carefully as possible for optimum results.
Also, be sure to use techniques that are appropriate for
the material you are examining.
Direct wet mounts

1. Be sure the density of the mount is correct. You should be

able to read small print through it (but not too clearly). If it
is too thick or too thin, observation of the elements in the
mount may be difficult

2. Be sure to prepare fresh iodine solutions every 10 – 14

days. Old iodine will not stain cysts properly. The iodine
must not be too strong or the fecal material may clump
and trap organisms in the clumps so that they cannot be
seen. If the iodine is too weak, it will not stain cysts
properly.
Concentration procedure
1. Select a fully representative sample of the stool for
concentration

2. Prepare well mixed suspensions of feces and water or

saline

3. Use the appropriate quantities of materials

4. Use the correct centrifuge speed and time

5. Prepare and examine mounts carefully as described for

direct wet mounts
Concentration procedure
6. Do not discard the tube containing the concentrated
material until you have completed your examination. You
may need to make another mount
Staining procedure
1. Select soft portions of the stool, and portions of mucus, if
present, for making smears for staining
2. Be sure that solutions are in good condition. Change them as
described in the procedures for staining. Keep the bottles of
stock solutions tightly closed and store away from direct
sunlight
3. Be sure to keep dishes covered. Keep the covers on except
when putting slides into, or taking them out of the dish.
Staining procedure…
 If the staining dishes are left uncovered, the solutions may
evaporate or debris may get into them, and adhere to the
preparation.
4. Use the correct quantity of mounting medium. Too much
medium may result in a thick layer through which it will be
difficult to focus, and you will not be able to see the smear
clearly. Too little medium will leave gaps under the
coverslip.
Staining procedure…
 If the smear was prepared from fresh, unpreserved feces, areas
where there are gaps may become unsatisfactory for diagnosis.
Gaps in the medium also make the mount difficult to examine
5. Always examine stained smears with the oil immersion
objective. Use the x10 objective to focus on the smear. If the
smear is not uniform, locate an area for examination where the
smear is not too thin or too thick and the staining is good.
 Then change to the oil immersion lens and examine for
protozoan trophozoites and/or cysts.
Stool Sample Collection…

 Do not accept stool samples collected in matchboxes,

newspapers or other containers that leak, health hazard!

 Ask the patient not to mix urine with the stool, destroys
trophozoites if present.
 A fresh specimen is required, tell the patient to bring the
sample within 1 hour of collection.
 Label the container clearly with the patient name and
laboratory number.
Sample Storage and Transportation

 Stool samples should be examined the same day.

 Liquid, watery or bloodstained stool should be examined
within one hour after collection.
 If you find eggs or cysts that you do not know you can
preserve the stool sample in 10% Formalin till an expert
comes or till the sample can be carried to the reference
laboratory.
Sample Storage and Transportation…
 Stool specimens for stool culture can not be preserved in
10% Formalin, they must be sent preserved in a special
culture media. (Stuart or Amies)
 Samples stained by modified Ziehl - Neelsen stain can be
stored in a box for future examination by a visiting expert
Waste Disposal and Precautions:
 Consider all stool samples as highly infectious.
 Avoid contact with bare fingers, wear gloves if possible or
handle with care.
 Do not reuse stool containers, burn stool sample containers
and wooden applicators.
 Soak glass slides in 5 % Phenol solution (e.g. Lysol) at
least over night.
 Cover slips break easily and may cause injuries, therefore
soak in a separate container in 5 % Phenol solution (e.g.
Lysol) at least over night.
Waste Disposal and Precautions…
 Chemical waste disposal
 Pour old and used chemicals into the sink and flush with
water if the sink is connected to a soak pit.
 Otherwise, pour chemicals directly into the soak pit. Ensure
that the soak pit is not near a natural water source.
 Formalin is irritating to the skin and the vapor should not be
inhaled.
Quality control for blood examination
Sample collection time:
 The number of certain parasites in the blood depends on the time of
collection.

Malaria

⇒ Highest number of parasites is found during fever attacks and before

the start of treatment

Microfilaria

⇒ W. bancrofti and Brugia malayi – take specimen at night between 10

p.m. – 2 a.m.
Sample collection time…
Smear for Malaria Parasites
 Follow proper collection procedures.
 Glass slides must be clean and free from grease.
 Thick films and thin films must be prepared properly.
 While drying protect blood films from dust, flies and insects.
 Do not dry exposed to direct sun light
 When fixing the thin film, be careful not to let methanol
touch the tick film.
Possible sources of error in the blood film
examination/ report
 Performance of a blood film examination on a poor quality
film achieves inaccurate results.
 A poor quality film caused by:
 Poor spreading technique
 Poor leukocytes distribution
 Too small a working area
 Red cell morphology altered by the spreading
process.
Possible sources …
 Use of the wrong anticoagulant, resulting in altered blood
cell size and/or morphology
 Patient sample mix up.
 Transcriptional error.
 Inadequate mixing of the sample with the anticoagulant,
resulting in the formation of small fibrin strands and a
decrease in the platelets count.
 Poor stain quality etc
Sample collection time…
Smear for Microfilaria
 Filaria are seldom found in the early and in the late stage
of the disease.
 The proper time of collection is important (10 p.m. to 2
a.m. )
 Un sheathed non-pathogenic filaria can be found any time
of the day.
 Patients with filaria in the blood show also eosinophilia in
the blood.
Summary
 Quality control procedures must apply to collection of
specimens, preparation of reagents, performance of the
techniques and examination of the final preparation
 If fecal specimens are not properly collected and taken care
of before examination, they will be of little or no value for
accurate diagnosis
 Performance of a blood film examination on a poor quality
film achieves inaccurate results.
 Filaria are seldom found in the early and in the late stage of
the disease. Hence, the proper time of collection is important
References
 Basics of Quality Assurance. WHO series 2, 1992
 Arneson, W and J Brickell: Clinical Chemistry: A Laboratory
Perspective first edition, FA Davis, 2007
 Becton Dickinson FACSCount System User’s Guide (For
Use with the BD FACS Count™ CD4/CD3 Reagent Kit)
 WHO. Basic Laboratory Methods in Medical Parasitology,
1991, Geneva.