Specimen Collection

and Processing

Charlie P. Cruz
Course Facilitator
 What’s Ahead
 Key Definitions
 Stool for Ova and Parasite Examination
 Stool Screening Methods
 Other Intestinal Specimens
 Other Specimens and Laboratory Techniques
 Immunologic Testing
 Reporting of Results and Quality Control
 Looking Back

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 Key Definitions
 Ova and parasites (O&P)
 Ova is the egg stage of select parasites.
 Parasites are the other morphologic forms that
may be present.
 Fixative
 Preserve morphology of protozoa
 Prevent further development of certain helminth
eggs and larvae
 Ratio of specimen to fixative imperative

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 Key Definitions
 Ocular micrometer
 Specially designed ocular piece equipped with a
measuring scale
 Must be calibrated before use
 Micron
 Unit measuring 0.001 (10-3 millimeter or 10-6
meter)

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 Key Definitions
 Direct wet mount/preparation
 Made by mixing unfixed stool with saline or iodine
 Put onto glass slide and coverslipped
 Used for the detection of motile protozoan
trophozoites
 Concentration techniques
 Used for the ability to detect small number of
parasites, including:
• Protozoan cysts
• Oocysts
• Helminth eggs and larvae
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 Key Definitions
 Concentrated wet preparations
 Parasites concentrated to allow the ability to
detect a small number of parasites
 Removes as much debris as possible
 Permanent stained smear
 Slide contains a fixed stool sample that has been
allowed to dry and subsequently stained.
 Slide is typically coverslipped and sealed, allowing
it to remain intact long term.

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Fecal Processing

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 Stool for Ova and Parasite
Examination
 Collection and transport
 Collection protocol
• 3 specimens collected every other day OR
• 3 specimens collected in 10 days
• Exception:
 Diagnosing amebiasis – collect up to 6 specimens in 14
days

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 Stool for Ova and Parasite
Examination
 Specimen requirements:
 2-5 grams of stool (size of walnut)
 Placed into a clean, airtight container
 Placed in preservative if expected delay between
collection and processing

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 Stool for Ova and Parasite
Examination
 Fixatives for preservation
 Formalin
• All-purpose preservative
• 5% solution for protozoan cysts
• 10% solution for helminth eggs and larvae
• Used for:
 Direct examination
 Concentration techniques
 NOT for permanent smears

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 Stool for Ova and Parasite
Examination
 Fixatives for preservation (continued)
 Polyvinyl alcohol (PVA)
• Plastic powder acts as an adhesive for stool.
• It is usually combined with Schaudinn solution, which
contains zinc sulfate, copper sulfate, or mercuric chloride
as a base.
• Used for:
 Permanent smears

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 Stool for Ova and Parasite
Examination
 Fixatives for preservation (continued)
 Sodium acetate formalin (SAF)
• Single vial system
• Mercury free (safer)
• Used with modified acid stain to detect coccidian oocysts
• Used for:
 Concentration techniques
 Permanent smears

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 Stool for Ova and Parasite
Examination
 Fixatives for preservation (continued)
 Modified PVA
• Contains copper or zinc sulfate
• Parasite identification more difficult without mercury-
based product
• Used for:
 Concentration techniques
 Permanent smears

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 Stool for Ova and Parasite
Examination
 Fixatives for preservation (continued)
 Alternative single-vial systems
• Non-toxic
• Free of formalin and mercury
• Parasite identification more difficult without mercury-
based product
• Used for:
 Concentration techniques
 Permanent smears

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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 Stool for Ova and Parasite
Examination
 Macroscopic examination
 Determine consistency and color
 Stool screened for gross abnormalities
 Stool needed:
• Fresh, unpreserved specimen

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 Stool for Ova and Parasite
Examination
 Microscopic examination (continued)
 3 distinct procedures:
• Direct wet preparation
• Concentrated technique
• Permanent smear
 Microscope considerations: ocular micrometer
• Measure objects observed microscopically accurately
• Use size in differentiating parasites from one another and
from artifacts
• Must be calibrated annually

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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 Stool for Ova and Parasite
Examination
 Direct wet preparation
 Saline
• 0.85% saline is put onto glass slide and mixed with a
small portion of unfixed stool.
• After coverslipping, 10X power is used to observe slide.
 Iodine
• Procedure is same as saline prep, except with Lugol’s or
D’Antoni’s iodine formula instead of saline.
• Formula enhances detail of protozoan cysts.
• Iodine kills trophozoites present.

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 Stool for Ova and Parasite
Examination
 Concentration methods
 Specimen is centrifuged and two techniques can
be used:
• Sedimentation or flotation
 Can detect small number of parasites present
 Removes as much debris as possible
 Protozoan trophozoites do not survive procedure
 Detects:
• Protozoan cysts
• Oocysts
• Helminth eggs and larvae

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 Stool for Ova and Parasite
Examination
 Concentrated wet preps
 Sedimentation
• Parasites are concentrated in sediment of tube following
centrifugation.
• Sediment is examined microscopically.
• Most say this method if preferred, more efficient and
easier than flotation.
 Flotation
• Parasites are less dense than solution they are in.
• During centrifugation they float to surface.
• Surface material is examined microscopically.

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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 Stool for Ova and Parasite
Examination
 Permanent stains
 Confirms the presence of protozoa cysts and
trophozoites
 Observe detailed features of protozoa by staining
intracellular organelles
 Only way to positively identify parasites
 Examples:
• Wheatley Trichrome
• Iron hematoxylin

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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Stool for Ova and Parasite
Examination

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 Stool Screening Methods
 Detection of a wide variety of parasites found
in stool specimens:
 Entamoeba histolytica
 Giardia intestinalis
 Cryptosporidium spp
 Examples:
 Enzyme immunoassay (EIA)
 Direct fluorescent antibody (DFA)
 Membrane flow cartridge techniques

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 Stool Screening Methods
 Pros:
 Highly sensitive and specific
 Not technically as demanding
 Cons:
 Can be labor intensive
 Requires an experienced microscopist
 Can only detect 1-2 pathogens at a time
 Requires provider or physician to have a good
idea of parasite present before ordering this type
of testing

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 Other Intestinal Specimens
Duodenal Material
 Collected using:
 Nasogastric intubation
 Enteric capsule test (Enterotest)
 Examine for:
 Giardia intestinalis trophozoites
 Cryptosporidium spp
 Isospora belli
 Strongyloides stercoralis
 Fasciola hepatica eggs
 Clonorchis sinensis eggs

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 Other Intestinal Specimens
Sigmoidoscopy Material
 Ulcers obtained by aspiration or scraping
should be examined by:
 Direct wet prep
 Permanent smear
 Examine for:
 Entamoeba histolytica
 Coccidian parasites
 Microsporidia spp.

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 Other Laboratory Techniques
Cellophane Tape Prep
 At night the adult female worm exits host
through rectum and deposits numerous eggs
in the perianal region.
 Specimen should be collected in morning
before defecating or showering.
 This can rule out pinworm infection after 5
days of collection.
 Examine for:
 Enterobius vermicularis eggs
 Taenia spp.
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Other Laboratory Techniques
Cellophane Tape Prep

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Other Laboratory Techniques
Cellophane Tape Prep

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 Other Specimens
Blood
 Collection and handling
 Aseptic technique must be used.
 Fingertip or earlobe blood collected in EDTA.
 Do not milk fingertip, as this could cause dilution
of parasite (hinders recovery).

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 Other Specimens
Blood
 Processing:
 Thick and thin blood smears
• Knott technique
• Buffy coat slides
 Stain using permanent smear technique
• Wright’s stain
• Giemsa stain
 Examine microscopically
 Set up and read cultures if necessary

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Giemsa-stained peripheral blood smear. Arrow A showing a classic, ring-shaped
trophozoite of Plasmodium falciparum. Arrow B showing a classic, headphone-
shaped trophozoite of P. falciparum. Arrow C showing two trophozoites of P.
falciparum within the same red blood cell.
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Other Specimens
Blood

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Other Specimens
Blood

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Other Specimens
Blood

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Other Specimens Blood

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 Other Specimens Blood
 Knott technique
 Concentrate blood specimens suspected of having
a low number of microfilariae
 Buffy coat slides
 Layer of WBCs between plasma and RBCs are
smeared onto slides to detect Leishmania and
Trypanosoma spp.
 Cultures
 Novy-MacNeal-Nicolle (NNN) media where
specimen is inoculated onto slant; held for 1
month and detects Leishmania and Trypanosoma
spp. Lorma Colleges, College of Medical Laboratory Science 54
 Other Specimens
Cerebrospinal Fluid and Other
Sterile Fluids
 Must be examined promptly to detect motility
of parasites
 Wet prep used in addition to Giemsa,
trichrome, or modified trichrome stain
 Examine for:
 Naegleria fowleri
 Acanthamoeba spp.
 Trypomastigote form of Trypanosoma spp.

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 Other Specimens
Tissue and Biopsy Specimens
 Surgical removal of specimens is necessary
to make preparations of histologic tissue
sections.
 Examine for:
 Leishmania spp.
 Toxoplasma gondii
 Trypanosoma spp.
 Trichinella spiralis
 Microsporidia spp.
 Entamoeba histolytica

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 Other Specimens
Sputum
 Early morning specimen is best without the
addition of saliva.
 Collect in a wide mouth container with screw
cap lid.
 Examined directly using:
 Wet prep or concentrated technique
 Using N-acetylcysteine
 Permanent smear may be performed.

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 Other Specimens
Sputum
 Examine for:
 Paragonimus westermani
 Strongyloides stercoralis
 Microsporidia spp.
 Entamoeba histolytica
 Entamoeba gingivalis
 Ascaris lumbricoides
 Hookworms

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 Other Specimens
Urine
 Urine is collected in clean container with
watertight lid and centrifuged.
 Sediment is observed microscopically.
 Examine for:
 Schistosoma haematobium
 Trichomonas vaginalis
 Microfilariae

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 Other Specimens
Genital Secretions
 Collected on a swab or in a collection cup
equipped with a lid
 Saline wet preparations – method of choice
 Permanent smears also acceptable
 Examine for:
 Trichomonas vaginalis

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 Other Specimens
Eye Specimens
 May test corneal scrapings that are kept
moist with saline, contact lenses, or contact
lens solution
 Can be cultured on media, transferred to
glass slides, and stained using calcofluor
white stain and fluorescent microscopy, or
processed using histologic methods

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 Other Specimens
Eye Specimens
 Examine for:
 Acanthamoeba keratitis
 Toxoplasma gondii
 Microsporidia spp.
 Loa loa

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 Other Specimens
Mouth Scrapings
 Placed in a clean, airtight container or on a
swab
 Extract material from swab or transfer out of
cup to make wet preps or permanent stains
 Examine for:
 Entamoeba gingivalis
 Trichomonas tenax

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 Other Specimens
Nasal Discharge
 Place in a clean, airtight container or on a
swab
 Extract material from swab or transfer out of
cup to make wet preps or permanent stains
 Examine for:
 Naegleria fowleri

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 Other Specimens
Skin Snips
 Procedure performed correctly if the patient
does not bleed during cutting
 Two methods can be used:
 Firm (scleral) punch into skin
 Razor blade, which makes small cut into skin
 Incubate skin for 30 minutes in saline
 Microscopically check for moving microfilariae
 Examine for:
• Onchocerca volvulus

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 Other Laboratory Techniques
Culture Methods
 Not a common means of detection for
recovery of parasites
 Type of testing performed by specialized
laboratories and research facilities
 Examine for:
 Entamoeba histolytica
 Trichomonas vaginalis
 Leishmania spp.
 Trypanosoma cruzi
 Toxoplasma gondii
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 Other Laboratory Techniques
Animal Inoculation
 Mice, guinea pigs, and hamsters are inoculated with
possible patient’s parasite.
 Specimens are collected using aseptic technique.
 When/if parasite is recovered from animal, patient is
considered positive.
 Testing occurs in facility equipped for animal testing.
 Examine for:
 Leishmania and Trypanosoma spp.
 Toxoplasma

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 Other Laboratory Techniques
Xenodiagnosis
 Used for diagnosis of Chagas’ disease
 Uninfected reduviid bug is allowed to take blood
meal from patient.
 Bug’s feces are examined for Trypanosoma cruzi.

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 Immunologic Testing
 Two methods commonly used:
 Antigen detection
• More reliable
• Positive test – active infection
 Antibody detection
• More complex
• Interpret cautiously; may not always indicate active
infection

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 Immunologic Testing

BF, Bentonite flocculation; CA, card agglutination; CF, complement fixation; DFA, direct fluorescent antibody;
EIA, enzyme immunoassay; IB, immunoblot; IHA, indirect hemagglutination; IFA, indirect fluorescent antibody;
LA, latex agglutination; PCR, polymerase chain reaction; Rapid, immunochromatographic cartridge

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 Immunologic Testing

BF, Bentonite flocculation; CA, card agglutination; CF, complement fixation; DFA, direct fluorescent antibody;
EIA, enzyme immunoassay; IB, immunoblot; IHA, indirect hemagglutination; IFA, indirect fluorescent antibody;
LA, latex agglutination; PCR, polymerase chain reaction; Rapid, immunochromatographic cartridge

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 Immunologic Testing

BF, Bentonite flocculation; CA, card agglutination; CF, complement fixation; DFA, direct fluorescent antibody;
EIA, enzyme immunoassay; IB, immunoblot; IHA, indirect hemagglutination; IFA, indirect fluorescent antibody;
LA, latex agglutination; PCR, polymerase chain reaction; Rapid, immunochromatographic cartridge

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Reporting of Results and Quality
Control
 Positive specimens should include the
following information in report:
 Scientific name of parasite
 Stage of parasite found
 Quantitation of other cells found (i.e., WBCs)
 Positive O&P specimens should include the
following phrase in report:
 “This procedure does not detect Cryptosporidium
spp, Cyclospora cayetanensis, or Microsporidia. It
will, however, recover Isospora belli oocysts.”

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Reporting of Results and Quality
Control
 Appropriate laboratory techniques that must
be present to assure proper results are being
released:
 Procedure manuals are up to date and available.
 Reference materials are up to date and available.
 Reagents and solutions are properly labeled.
 Centrifuges and ocular micrometer are calibrated.
 Controls are in date and used properly.
 Refrigerator and incubator temperatures are
recorded.
 Action plans are in place for out-of-control results.
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 Looking Back
 Accurate detection of parasites requires
appropriate specimen collection and
processing.
 Traditional parasitic testing involves stool
using the O&P, which includes a macroscopic
and microscopic evaluation, direct wet prep,
concentrated technique, and a permanent
stained smear.
 Other specimens may be necessary to detect
parasites (intestinal specimens, blood, tissue).
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 Looking Back
 Immunologic methods can be used in
situations where it is difficult or impossible to
determine parasitic involvement in a human.
 Quality assurance and quality control must be
followed at all times to ensure accurate
results are obtained.

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