CHAPTER 2 (SPECIMEN COLLECTION AND PROCESSING) Flashcards
Specimen Collection and Processisng
the most common procedure performed in the area of parasitology is the examination of
stool specimen for ova and
parasites
There are two general components associated with O and P
macroscopic and microscopic examination
The microscopic examination consists of
three possible components
collection, transport, and fixatives for preservation.
Morphologic forms of protozoa and helminths may be detected from a properly collected and prepared stool specimen. When present, the protozoan forms known as
trophozoites and cysts
eggs, larvae, proglottids are collectively known as
Helminth stages
The typical stool collection protocol consists
of
three specimens, one specimen collected
every other day or a total of three collected in 10 days.
One exception of the typical stool collection protocol is in the diagnosis of
amebiasis in which up to six specimens
in 14 days is acceptable
Certain medications and substances may
interfere with the detection of parasites. Stool samples from patients whose therapy includes
barium, bismuth, or mineral oil
patients who have taken antibiotics
or antimalarial medications
How many days should pass before collecting stool sample from patients who have treatments that contain barium, bismuth, or mineral oil?
5 to 7
Collection of specimens
from patients who have taken antibiotics
or antimalarial medications should be delayed for?
2 weeks
Stool specimens should be collected in a
clean, watertight container with a tight-fitting lid
The acceptable amount of stool required for parasite study is
2 to 5 g
should not be allowed to contaminate
the stool specimen because it has been
known to destroy some parasites
Urine
Stool should not be retrieved from ________ water because free-living protozoa and nematodes may be confused with human parasites
toilet bowl
water may destroy select parasites, such as
schistosome eggs and amebic trophozoites.
Toilet paper in the stool specimen may?
mask parasites or make examination of the sample difficult.
The specimen container should be labeled
with
the patient’s name and identification number,
the physician’s name, and the date and time of sample collection.
The specimen should be placed
into _____________ for transport to the
laboratory.
a zip lock plastic bag
When handling all specimens, __________ should be worn at all times.
gloves and a
protective coat
__________ should also be used in laboratories,
when present.
Biohazard hoods
Universal precautions, as
outlined by the _________ for handling blood and body fluids, should be exhibited and enforced at all times.
Occupational Safety and Health
Administration (OSHA)
Another important consideration in testing
fecal specimens for parasites is the
time frame from sample collection to receipt and examination in the laboratory
______________ is sensitive to environmental changes and, on release from the body, disintegrates rapidly thus a fresh sample is required
trophozoite stage
fresh specimen of protozoan trophozoites is required in order to?
demonstrate the motility
it is recommended
that liquid specimens be examined
within _______ because trophozoites are usually found in this type of stool
30 mins of passage
In keeping with stool consistency, semiformed specimens may yield a mixture of protozoan cysts and trophozoites and should be evaluated within?
1 hour of passage
Formed stool specimens are not likely
to contain trophozoites; therefore, they can be held for
24 hours following collection
The specimen can be preserved by placing it directly into a ______ at the time it is collected or on ______
fixative
receipt in the laboratory
How many stool samples should be collected when following the typical O&P collection protocol?
A. 1
B. 2
C. 3
D. 4
are substances that preserve the morphology of protozoa and prevent further development of certain helminth eggs and larvae
Fixatives
whatever fixative is used, the recommended ratio is
three parts fixative to one part stool
3:1
The specimen must be
fixed in the preservative for at least ______ before processing
30 minutes
has been used for many years as an all-purpose fixative for the recovery of protozoa and helminths.
Formalin
Two concentrations
of formalin are commonly used; a
5% and 10%
5% concentration ideally preserves
protozoan cysts
10% concentration preserves
helminth eggs and larvae
Formalin may be routinely used for
direct examinations
and concentration procedures
Formalin is not used for?
permanent smears
There are three primary advantages for the use of formalin
(1)it is easy to prepare
(2) it preserves specimens for up to several years
(3) it has a long shelf life
disadvantages of formalin
is that
it does not preserve parasite morphology
adequately for permanent smears.
trophozoites usually cannot be recovered and morphologic details of cysts and eggs may fade with time
requirements to use formalin under OSHA regulations
Monitoring of vapors, use of protective clothing, and a comprehensive, written chemical hygiene plan (CHP)
Concentration, Permanent stain, and Antigen Tests of 10% formalin
(+) (-) (+)
Concentration, Permanent stain, and Antigen Tests of SAF
(+) (+) iron hematoxylin (+)
Concentration, Permanent stain, and Antigen Tests of PVA
(±) (+) trichrome or iron hematoxylin (-)
Concentration, Permanent stain, and Antigen Tests of Modified PVA (zinc)
(±) (+) trichrome or iron hematoxylin (±)
Concentration, Permanent stain, and Antigen Tests of Modified PVA (zinc) Single-vial system
(+) (+) trichrome or iron hematoxylin (±)
comprised of a plastic powder that acts as an
adhesive for the stool specimen when preparing slides for staining
Polyvinyl Alcohol
PVA is most often combined
with
Schaudinn solution, which usually contains
zinc sulfate, copper sulfate, or mercuric chloride as a base
What can PVA detect
Trophozoites and cysts of the protozoa, as well as most helminth eggs
The greatest advantage of PVA is
preparation of a permanent stained smear
The biggest disadvantage of the use of PVA
is that
Schaudinn solution contains mercuric chloride.
This preservative can be used for performing concentration techniques and permanent stained smears. Some laboratories have adopted this fixative because it only requires a single vial and it is mercury-free.
Sodium Acetate Formalin
Advantages of SAF
SAF is
easy to prepare
long shelf life
used for preparing smears for staining with the modified acid-fast stain to detect coccidian oocysts.
SAF also has disadvantages because
adhesive is not good
protozoa morphology not as clear
limited choice of permanent stains
what is added to aid SAF for it to adhere
albumin
Other alternatives to mercury-based PVA are the use of substitute compounds containing copper sulfate or zinc sulfate.
Modified Polyvinyl Alcohol
advantage and disadvantage of Modified Polyvinyl Alcohol
can be used for concentration methods
and permanent stained smears
substitute products do not provide the same
quality of preservation for adequate protozoan morphology on a permanent stained slide as the mercury-based fixatives
Several manufacturers have developed alternative nontoxic fixatives. These single-vial fixatives are free of formalin and mercury and can be used for concentration techniques and permanent stained smears.
Alternative Single-Vial Systems.
disadvantage of Alternative Single-Vial Systems.
do not provide the same quality of preservation as mercury-based fixatives and organism identification will be more difficult from permanent stained slides.
advantages of single vial systems
can also be used for performing fecal immunoassays
What is the purpose of fixatives for the collection of
stool samples?
A. Enhance the motility of protozoa.
B. Stain the cytoplasmic inclusions of protozoa.
C. Preserve the morphology of protozoa and prevent
further development of helminths.
D. All of the above.
Once a stool specimen has been received in the laboratory, the analytic phase of laboratory testing, also referred to __________ begins
processing
Stool specimens submitted for parasitic study should first be examined ________ to determine the consistency and color of the sample. The specimen should be screened and examined for the presence of gross abnormalities.
macroscopically
Macroscopic examination stool samples should be?
fresh, unpreserved stool specimen
In such situations where macroscopic examination is not possible what is recommended at the time of specimen collection.
a notation of the gross appearance, either on the actual specimen container or on the requisition form
What type of parasites are potentially present in
Liquid Stools?
Fully Formed Stools?
Both?
Liquid Stools - Protozoan trophozoites
Fully Formed Stools - Protozoan cysts
Both - Helminth eggs and larvae
What physical characteristics of a stool indicates the patient’s condition such as whether a patient has recently had a special procedure or if the patient is on antibiotic therapy?
Color of the stool
Range of colors of stool
Black to Green to Clay
Brown is normal
Unusual colors = particular medication
Gross abnormalities possibly found in stool
include?
adult worms, proglottids, pus, and
mucus
First step of macroscopic examination of stools
surface of the stool should be examined for parasites, such as pinworms, tapeworm proglottids, and adult worms
Second step of macroscopic examination of stools
sample should then be broken up and examined once more for macroscopic parasites, especially adult helminths.
(a wooden applicator stick works nicely for this task)
What to do if adult worms are found in the sample
sample should be carefully washed through a wire screen
Other macroscopic abnormalities in the specimen may have parasitic indications. For example,
blood and/or mucus in loose or liquid stool may suggest the presence of
amebic ulcerations in the
large intestine
Bright red blood on the surface
of a formed stool is usually associated with
irritation and bleeding
Which of the following characteristics is observed during the macroscopic examination of stool specimens?
A. Consistency
B. Color
C. Adult worms
D. All of the above
Macroscopic Examination of
Stool Specimens: Possible
Descriptive Terms
Consistency Terms (IN ORDER)
Hard
Soft
Mushy
Loose
Diarrheic
Watery, liquid
Formed
Semiformed
Macroscopic Examination of
Stool Specimens: Possible
Descriptive Terms
Possible Colors (IN ORDER)
Dark brown
Black
Brown
Pale brown
Clay
Yellow
Red-brown
Green, other
Macroscopic Examination of
Stool Specimens: Possible
Descriptive Terms
Gross Appearance Terms (IN ORDER)
Conspicuously fibrous
Fiber scanty to moderate
Colloidal (homogeneous)
Scanty mucus
Much mucus
Mucus with scanty blood
Other (e.g., blood, barium)
To detect the presence of parasites in a stool specimen, ______are performed.
microscopic examinations
The microscopic examination of stool for ova and parasites which uses fresh samples involves three distinct procedures which are
direct wet preparations (preps)
concentrated technique
resulting in concentrated wet preparations
permanently stained smear
If the specimen is received in fixative what step in the microscopic examination can be eliminated?
direct wet preparation
Which of these procedures is involved in the microscopic
examination of stool specimens for parasites?
A. Performing a concentration technique
B. Determining specimen consistency
C. Examining sample for gross abnormalities
D. Analyzing sample for color
After the three steps in the microscopic examination, what is done next?
concentrate and permanent stain techniques are performed
The most important piece of equipment
in the parasitology laboratory is the
microscope
Because size is an important diagnostic
feature in parasitology, it is necessary for
the microscope to contain a specially designed ocular piece equipped with a measuring scale known as an
ocular micrometer
The diagnostic stages of parasites detected
microscopically are measured in units known
as
microns
μ or μm
0.001 [10−3] millimeter
10^−6 meter
An important step in order to ensure accurate measurements of the microscope and what tool it uses
calibration
stage micrometer
defined as a slide made by mixing a small portion of unfixed stool with saline or
iodine and subsequent examination of the resultant mixture under the microscope
direct wet preparation
/direct wet mount
Direct wet preparations is used to?
detect the presence of motile protozoan trophozoites.
Other parasite
stages that might be observed in a direct wet
preparation include
protozoan cysts, oocysts, helminth eggs, and larvae.
First step in direct saline wet preparation
placing a drop of 0.85% saline on a glass slide
and mixing with a small portion of unfixed stool using a wooden applicator stick or another mixing tool.
Second step in direct saline wet preparation
A 22-mm square cover slip is placed on the slide and the preparation is examined microscopically in a systematic fashion
Third step in direct saline wet preparation
The entire cover glass should be scanned using the low power (10×) objective on the microscope, and the power should only be increased when a suspicious object requires further investigation.
Performing this procedure allows for the ability to observe greater detail using the 100× objective.
oil immersion
is not usually recommended on
wet preparations unless the cover slip is sealed to the slide.
oil immersion
How to seal cover slip to the slide
A temporary seal can be prepared
using a hot paraffin-petroleum jelly (Vaseline)
mixture around the edges of the cover slip
may be made
to enhance the detail of protozoan cysts
direct iodine wet preparation
what iodine formulas are used for a direct iodine wet preparation
Lugol’s or D’Antoni’s formula)
Which are killed when using a direct iodine wet preparation?
trophozoites
What are the proper adjustments for the microscope in order to view even transparent protozoa?
light should be reduced using the
iris diaphragm - (provide contrast between the cellular elements)
Lowering the condenser - (allow transparent
structures to be seen)
The direct wet preparation can be eliminated from the O&P examination if the specimen is received in a fixative.
A. True
B. False
provide the ability to detect small numbers of
parasites that might not be detected using direct wet preparations
Concentration techniques
(or methods? idk same same maybe)
The purpose of concentration
is to
aggregate parasites
remove as much debris
Concentration techniques examination allows
the laboratory technician to detect
protozoan cysts, oocysts, helminth eggs, and larvae
do not usually survive concentration techniques
Protozoan trophozoites
There are two types of concentration methods available
sedimentation and flotation
Most experts recommend what concentration technique be used, because it is more efficient and easier to perform accurately.
sedimentation
The most widely used sedimentation
technique is the
formalin–ethyl acetate sedimentation
procedure
The advantage of formalin–ethyl acetate sedimentation procedure is that it
good recovery of most parasites
and is easy to perform
The disadvantage of formalin–ethyl acetate sedimentation procedure is that it
preparation contains
more fecal debris than a flotation technique
is also based on differences
in specific gravity between the sample
debris, which in this case is heavy and sinks to
the bottom of the test tube, and potential parasites, which are lighter and float toward the top of the tube
Zinc Sulfate Flotation Technique
Zinc Sulfate Flotation Technique uses? (include specific gravity)
zinc sulfate with a specific gravity of 1.18 to 1.20
The advantage of Zinc Sulfate Flotation Technique
more fecal debris is removed
and it yields a cleaner preparation
The disadvantage
of Zinc Sulfate Flotation Technique
some helminth eggs are very dense and will not float
It is recommended
that if laboratories perform zinc sulfate flotation technique that they
examine saline and iodine preps made from the sediment microscopically, as well as the surface film (direct wet preparation samples)
Which of the following parasitic stages is not usually
detected after using a concentration technique?
(Objective 2-9)
A. Protozoan cysts
B. Protozoan trophozoites
C. Helminth eggs
D. Helminth larvae
B. Protozoan trophozoites
defined as a microscope slide that
contains a fixed sample that has been allowed to dry and subsequently stained
permanent stained smear
The final procedure in the O&P examination is the preparation and examination of a
permanent stained smear
Permanent staining samples is a critical portion of the O&P examination because it is designed to
confirm the presence of protozoa cysts and/or trophozoites
Example of protozoa that only possess a trophozoite stage and will not be detected in the concentrated wet mount preparation that is why permanent stains are important
Dientamoeba fragilis
The permanent stained smear is not the
method of choice for the identification of _______ because these parasites often
stain too dark or appear distorted
helminth eggs or larvae
helminth eggs or larvae are best detected and identifies using what technique?
concentration technique
The sample of choice for such stains is a
thinly prepared slide of see-through thickness
made from a
PVA-preserved sample
Specimens fixed with SAF may also be used in permanent stain techniques, but the choice of stain is limited to
iron hematoxylin
Slides can also be prepared from a
fresh stool specimen but?
Should not be dried and fixed immediately
Example of a fixative used to samples with fresh stool in permanent stain techniques
Shaudinn fixative
How many fields are reviewed before the slide can be considered negative in permanent stain techniques?
300 fields
Two common stains used for routine O&P
testing include
trichrome (Wheatley modification)
iron hematoxylin
are not part of the routine O&P examination
and must be specifically requested
Specialized stains
These
specialized stains include what stains?
modified acid-fast
and modified trichrome stains.
What structure or material appear LIGHT PINK OR BLUE-GREEN on Trichrome Stain
Cytoplasm of Entamoeba histolytica trophozoites and cysts
What structure or material appear PURPLE TINT on Trichrome Stain
Cytoplasm of Entamoeba coli cysts
What structure or material appear BRIGHT RED TO RED-PURPLE on Trichrome Stain
Nuclear karyosomes
What structure or material appear LIGHT GREEN on Trichrome Stain
Degenerated parasites
What structure or material appear GREEN on Trichrome Stain
Background
The most widely used
permanent stain is the
Wheatley trichrome stain
may be used instead of the trichrome technique. Historically, this procedure was considered to be time-consuming. However, a shorter technique using this stain is now available
iron hematoxylin
stain
The iron hematoxylin stain
reveals excellent morphology of the
intestinal protozoa
Appearance of Select
Protozoan Structures and
Background Material on
IRON HEMATOXYLIN Stain
BLUE TO PURPLE
Protozoa cytoplasm
Appearance of Select
Protozoan Structures and
Background Material on
IRON HEMATOXYLIN Stain
DARK BLUE TO DARK PURPLE
Protozoa Nuclear Material
Appearance of Select
Protozoan Structures and
Background Material on
IRON HEMATOXYLIN Stain
LIGHT BLUE (sometimes with pink tint)
Debris and Background Material
Appearance of Select
Protozoan Structures, Yeast,
and Background Material
on MODIFIED ACID-FAST STAIN
PINK TO RED
Oocysts of Cryptosporidium and Isospora
Appearance of Select
Protozoan Structures, Yeast,
and Background Material
on MODIFIED ACID-FAST STAIN
VARIABLE; CLEAR TO PINK RED
Oocysts of Cyclospora
Appearance of Select
Protozoan Structures, Yeast,
and Background Material
on MODIFIED ACID-FAST STAIN
BLUE
Yeast
Appearance of Select
Protozoan Structures, Yeast,
and Background Material
on MODIFIED ACID-FAST STAIN
BLUE OR LIGHT RED
Background Material
Disadvantage of specialized stains
do not detect oocysts of the coccidian parasites or spores of microsporidia
has become an important permanent
stain procedure for the detection of the oocysts of Cryptosporidium, as well as those of Isospora and Cyclospora
modified acid-fast stain
this allows for the detection of acid-fast
parasites in addition to the other protozoa normally recovered using the iron hematoxylin stain
a carbol fuchsin step incorporated to a modified iron hematoxylin stain
a carbol fuchsin step incorporated to a modified iron hematoxylin stain uses what samples
SAF-preserved fecal samples
Although the spores of microsporidia will also stain with the acid-fast technique, their small size (1 to 2 μm) makes it difficult to identify them without the use of special stains. What modified stain are available to demonstrate these parasites
Modified Trichrome Stains
Appearance of Microsporidia on MODIFIED TRICHROME STAIN
PINK TO RED WITH CLEAR INTERIOR
Spores of microsporidia
Appearance of Microsporidia on MODIFIED TRICHROME STAIN
RED HORIZONTAL OR DIAGONAL BAR
Polar Tubule
Appearance of Microsporidia on MODIFIED TRICHROME STAIN
PINK TO RED
Bacteria, Yeast, Debris
Appearance of Microsporidia on MODIFIED TRICHROME STAIN
GREEN
Background
These methods can be obtained as kits that contain monoclonal antibody. This commercial antibody is used to detect
antigens in patient specimens with the main purpose of ease and speed.
rapid methods, or stool-screening methods.
Current available assays of stool screening methods include
enzyme immunoassay (EIA), direct fluorescent antibody (DFA), and membrane flow cartridge techniques.
The antigen detection methods on stool-screening methods are commercially available for specific intestinal protozoa, including
Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp.
What is one advantage of the stool screening method?
A. It is highly sensitive and specific.
B. It can detect all parasites.
C. It can be performed on fresh or preserved
specimens.
D. It is labor-intensive.
A. It is highly sensitive and specific.
The permanent stained smear is critical for detection
of helminth eggs and larvae.
A. True
B. False
When reporting a positive specimen, the
report should state
scientific name
stage of development of the parasite
also helpful:
presence of certain cells (White blood cells should be reported semiquantitatively)
The results of the O&P procedure should
include a comment indicating that
this procedure does not detect Cryptosporidium spp., Cyclospora
cayetanensis, and microsporidia; it will recover the oocysts of Isospora belli
The results of fecal immunoassays should indicate the
specific parasite(s) that is (are) tested for in the assay
In general, for most parasites, quantitation is
highly required
true or false
false - not indicated
Situations in which quantitation of parasite is important are as follows
Blastocystis hominis
helminth eggs
Trichuris trichiura
Clonorchis sinensis
Schistosoma spp.
Plasmodium spp.
Babesia spp.
These are crystals which are reported and quantitated when found
Charcot-Leyden crystals
Which one of these parasites should be quantitated
in the parasitology report?
A. Giardia intestinalis
B. Entamoeba coli
C. Trichomonas vaginalis
D. Blastocystis hominis
D. Blastocystis hominis
