Staining and Screening Flashcards
Immunoassay
Types include DFA, EIA, IFA
TechLab ELISA kit discriminates pathogenic E. histolytica from non-pathogenic E. dispar
Several companies with immunoassay products for detecting Giardia
Patient population
Low-income communities with less facilities more likely to become infected; mostly traditional microscopic examination (O & P) ordered.
Lab statistics
Labs establish their own procedures of sample collection, handling, test methods etc. based on their previous data of a geographic area.
Physician sophistication and ordering practices
Based on patient travel history and symptomatology, some doctors want a definitive diagnosis by immunoassays or molecular tests.
Expertise of laboratory staff
O & P examination as well as molecular tests demand training and skill. Therefore, labs are selective in performing diagnostic procedures.
Wet mount
Made by mixing with saline, will show movement of the parasites (this is done to see the motility)
Specimen Collection
3 specimens on 3 different days over a 10 day period, parasites will shed in showers and may not appear from one day to another
Specimen Processing
Liquid stools: examine within 30 minutes.
Soft stools: examine within an hour.
Formed stools: refrigerate and examine within 24 hours.
If the examination cannot be done in a timely manner, preserve the specimen.
Formalin
An all-purpose fixative for protozoa and helminths
- 5% conc. preserves protozoan cysts
- 10% conc. preserves helminths eggs and larvae
Easy to prepare/long shelf life
Preserves specimens for up to several years
Not appropriate for permanent stains, makes the stain faint
Not ideal for trophozoites
Morphologic details of cysts and eggs fade with time (helminths and protozoa)
Polyvinyl alcohol (PVA)
Water-soluble synthetic polymer with excellent film-forming emulsifying, and adhesive properties.
‘Gold standard’ fixative esp. when combined with Schaudinn’s solution which contains mercuric chloride, absolute alcohol and acetic acid.
PVA-Schaudinn’s solution easier to mix with specimen, has longer shelf life.
Can be used for concentration smears and permanent stains.
Mercuric chloride is highly poisonous, can cause serious health problems.
Sodium acetate-acetic acid-formalin (SAF)
Inexpensive, easy to prepare/longer shelf life.
Buffering effect to decrease the distortion of protozoa.
Provides good results with iron-hematoxylin permanent stain.
Does not have good adhesive properties, vial system uses Albumin/glycerin used to “glue” the specimen to the slide.
Quite dilute, greater chances of missing the parasites.
Merthiolate-iodine-formalin (MIF)
Used to preserve trophozoites, cysts, larvae and eggs.
Can be used for wet mount or concentration procedures.
However not used for permanently stained smears.
2 vial system
Vial of 10% formalin + a vial of PVA with Schaudinn’s fixative
Excellent preservation of morphology
Contains mercuric chloride
OR
Vial of 10% formalin +a vial of PVA without mercuric chloride. Zinc better than copper.
Morphology may be inconsistent.
Sedimentation
(Formalin-ethyl acetate concentration) More commonly used than flotation Usually done for wet mounts More debris but more parasites recovered, except some trophozoites Examine anytime
Flotation
(Zinc sulfate flotation and Sheather sugar flotation)
Ideal for Cryptosporidium and Isospora oocyst recovery
Less debris but some helminth ova don’t float, sink to the bottom instead
Walls of eggs and cysts may collapse
Must examine within an hour
Direct Saline Mounts
Saline mounts can be used to detect motile trophozoites from fresh stool only.
Iodine enhances detection and differentiation, Use Lugol’s or D’Antoni’s iodine
Trichrome
Trophs and cysts stain blue-green or purple with nucleus bright red to red-purple against a light green background
WBCs, yeast, and other artifacts may be confused with trophs and cysts
Helminth eggs and larvae do not stain well
Iron-Hematoxylin
Long staining time
Cytoplasm stain blue to purple, nucleus dark blue to dark purple against light blue background
Color contrast not as good as with Trichrome
Modified IH/Kinyoun allows detection of acid-fast organisms, e.g. Cryptosporidium
Duodenal aspirates
Strongyloides stercoralis, Giardia lamblia
Direct exam (endoscopy)
Entero-Test
Culture for Strongyloides
Sigmoidoscopy specimens
Entamoeba histolytica
Urine
Schistosoma hematobium & Enterobius vermicularis ova
Cellophane tape
Enterobius vermicularis ova
Vaginal/urethral discharge
Trichomonas vaginalis trophozoites
Direct exam
Culture (In-Pouch, PEM)
Genzyme Trichomonas Rapid Test
Sputum
Paragonimus westermani eggs Entamoeba histolytica (from pulmonary abscess)
CSF
Naegleria and Acanthamoeba
