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FECAL FIXATIVES USED IN DIAGNOSTIC PARASITOLOGY (INTESTINAL TRACT SPECIMENS)

FECAL FIXATIVES USED IN DIAGNOSTIC PARASITOLOGY (INTESTINAL TRACT SPECIMENS)

 

Fixative

Conc.

Permanent

Stained Smear

Trichrome, Iron-Hematoxylin, Special Stains/Coccidia and Microsporidia

Immunoassays (IAs)

Giardia lamblia

Cryptosporidium spp.

Comments

5%, 10% Formalin

Yes

NO

Yes

Concentrations and IAs (EIA, FA, Rapids)

5%, 10% Buffered Formalin

Yes

NO

Yes

Concentrations and IAs (EIA, FA, Rapids)

MIF

Yes

Polychrome IV Stain

ND

No published data

SAF

Yes

Iron-Hematoxylin best

Yes

Concentrations, permanent stains, and IAs (EIA, FA, Rapids)

Schaudinn’s (Hg base)

No PVA1

Rare

Yes

NO

Permanent stains; Hg interferes with IAs; primarily used with fresh stool specimens (no fixative collection vials)

Schaudinn’s (Hg base)

+ PVA1

Rare

Yes

NO

Permanent stains; Hg and PVA interfere with IAs; considered the “gold standard” fixative for permanent stains

Schaudinn’s (Cu base)

+ PVA2

Rare

Yes

NO

Permanent stains; PVA interferes with IAs; stains not as good as with Schaudinn’s fixative using Hg or Zn

Schaudinn’s (Zn base)

+ PVA3

Rare

Yes

NO

Permanent stains; PVA interferes with IAs; this is the same fixative as TOTAL-FIX without PVA (see below)

Ecofriendly

    ECOFIX (PVA)4

 

Rare

 

Yes

 

NO

Permanent stains; PVA interferes with IAs; works best with ECOSTAIN, Wheatley’s trichrome 2nd best

Universal Fixative5

Ecofriendly

      TOTAL-FIX™

 

Yes

 

 

Yes

 

 

Yes

 

No formalin, no mercury, no PVA; concentrations, permanent stains, special stains, fecal IAs

1These two fixatives use the mercuric chloride base in the Schaudinn’s fixative; this formulation is still considered to be the “gold standard” against which all other fixatives are evaluated (organism morphology after permanent staining).

2This modification uses a copper sulfate base rather than mercuric chloride; morphology of stained organisms not as good as with Hg or Zn.

3This modification (proprietary formula) uses a zinc base rather than mercuric chloride and works well with both trichrome and iron-hematoxylin.

4This fixative uses a combination of ingredients, but is prepared from a proprietary formula (contains PVA).

5This modification uses a combination of ingredients (including zinc), but is prepared from a proprietary formula.  The aim is to provide a “Universal Fixative” that can be used for the fecal concentration, permanent stained smear, and available immunoassays for Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica (or the Entamoeba histolytica/E. dispar group).  HOWEVER, currently fecal immunoassays for the Entamoeba histolytica/E. dispar group or Entamoeba histolytica (true pathogen) require fresh or frozen specimens; testing can also be performed from stool submitted in Cary-Blair transport medium. 

 

IA, immunoassay; Cu, copper; EIA, enzyme immunoassay; FA, fluorescent antibody;  Hg, mercury; MIF, merthiolate –iodine-formalin fixative; ND, no data; PVA, polyvinyl alcohol; Rapids, cartridge format membrane flow IAs; SAF, sodium acetate-acetic acid-formalin; Zn, zinc

 

COMMENTARY:

The most common collection option (original public health approach) is a two vial system:  one vial of 5% or 10% formalin or buffered formalin and one vial of fixative containing the plastic adhesive, polyvinyl alcohol (PVA).  The formalin vial is used for the concentration and fecal immunoassays, while the PVA vial is used for the permanent stained smear.  Regulations for formalin (see below) were originally developed for industry, not the clinical laboratory where amounts of formalin tend to be quite low.  However, a laboratory using any amount of formalin must be monitored (see below).

 

SEMI-UNIVERSAL FIXATIVES

Examples of a “Semi-universal Fixative” are:  SAF (no mercury or PVA; contains formalin) and ECOFIX (no mercury of formalin; contains PVA). 

 

UNIVERSAL FIXATIVE

Currently, TOTAL-FIX is the only fixative that contains NO formalin, NO PVA, or NO mercury.  TOTAL-FIX can be used without the addition of PVA to the fixative – adequate drying time for smears prior to staining is the most important step (minimum of 1 hr in 37ºC incubator – requires more time for thicker fecal smears).  This fixative can be used for the concentration, permanent stained smear, special stains for coccidia or microsporidia, and fecal immunoassays for Giardia and Cryptosporidium.

 

FORMALIN FIXATIVE

Formalin has been used for many years as an all‑purpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts, oocysts, and spores. Two concentrations are commonly used: 5%, which is recommended for preservation of protozoan cysts, and 10%, which is recommended for helminth eggs and larvae. Although 5% is often recommended for all‑purpose use, most commercial manufacturers provide 10%, which is more likely to kill all helminth eggs. To help maintain organism morphology, the formalin can be buffered with sodium phosphate buffers, i.e., neutral formalin. Selection of specific formalin formulations is at the user's discretion. Aqueous formalin will permit the examination of the specimen as a wet mount only, a technique much less accurate than a permanent stained smear for the identification of intestinal protozoa. However, the fecal immunoassays for Giardia lamblia and Cryptosporidium spp. can be performed from the aqueous formalin vial.  Fecal immunoassays for the Entamoeba histolytica/E. dispar group and Entamoeba histolytica are limited to fresh or frozen fecal specimens or Cary-Blair transport medium.  After centrifugation, special stains for the coccidia (modified acid-fast stains) and the microsporidia (modified trichrome stains) can be performed from the concentrate sediment obtained from formalin-preserved stool material.  Use of the sediment provides a more sensitive test.

 

OSHA REGULATIONS ON THE USE OF FORMALDEHYDE

Formaldehyde has been in use for over a century as a disinfectant and preservative, it is also found in a number of industrial products.  There is disagreement about the carcinogenic potential of lower levels of exposure and epidemiologic studies of the effects of formaldehyde exposure among humans have given inconsistent results.  Studies of industry workers with known exposure to formaldehyde report little evidence of increased cancer risk.  It also appears that persons with asthma respond no differently than healthy individuals following exposure to concentrations of formaldehyde up to 3.0 ppm.  OSHA requires all workers to be protected from dangerous levels of vapors and dust. Formaldehyde vapor is the most likely air contaminant to exceed the regulatory threshold in a laboratory, particularly in anatomic pathology. Current OSHA regulations require vapor levels not to exceed 0.75 ppm (measured as a time‑weighted average [TWA]) and 2.0 ppm (measured as a 15‑min short‑term exposure). OSHA requires monitoring for formaldehyde vapor wherever formaldehyde is used in the work place. The laboratory must have evidence at the time of inspection that formaldehyde vapor levels have been measured, and both 8‑h and 15‑min exposure must have been determined.

 

If each measurement is below the permissible exposure limit and the 8‑h measurement is below 0.5 ppm, no further monitoring is required as long as laboratory procedures remain constant. If the 0.5‑ppm 8‑h TWA or the 2.0‑ppm 15‑min level is exceeded, monitoring must be repeated semiannually. If either the 0.75‑ppm 8‑h TWA or the 2.0‑ppm 15‑min level is exceeded (VERY UNLIKELY in a routine microbiology laboratory setting), employees must be required to wear respirators. Accidental skin contact with aqueous formaldehyde must be prevented with the use of proper clothing and equipment (gloves, laboratory coats).

 

The amendments of 1992 add medical removal protection provisions to supplement the existing medical surveillance requirements for employees suffering significant eye, nose, or throat irritation and for those experiencing dermal irritation or sensitization from occupational exposure to formaldehyde.  In addition, these amendments establish specific hazard‑labeling requirements for all forms of formaldehyde, including mixtures and solutions composed of at least 0.1% formaldehyde in excess of 0.1 ppm.  Additional hazard labeling, including a warning label that formaldehyde presents a potential cancer hazard, is required where formaldehyde levels, under reasonably foreseeable conditions of use, may potentially exceed 0.5 ppm.  The final amendments also provide for annual training of all employees exposed to formaldehyde at levels of 0.1 ppm or higher.

 

Note The use of monitoring badges may not be a sensitive enough method to correctly measure the 15‑min exposure level. Contact the Occupational Health and Safety Office within your institution for monitoring options. Usually, the accepted method involves monitoring air flow in the specific area(s) within the laboratory where formaldehyde vapors are found.

 

POLYVINYL ALCOHOL (PVA) ADHESIVE (NOT A FIXATIVE)

Polyvinyl Alcohol (PVA) is a water-soluble synthetic polymer used as a viscosity-increasing agent in pharmaceuticals, an adhesive in parasitology fecal fixatives, and as a lubricant and protectant in ophthalmic preparations; PVA is also defined as a water-soluble polymer made by hydrolysis of a polyvinyl ester (such as polyvinyl acetate); used in adhesives, as textile and paper sizes, and for emulsifying, suspending, and thickening of solutions. PVA IS NOT A FIXATIVE, BUT AN ADHESIVE TO HELP GLUE THE STOOL MATERIAL ONTO THE SLIDE; this is the only purpose of PVA as an additive to parasitology fecal fixative formulations. 

 

PVA is a plastic resin that is normally incorporated into Schaudinn's fixative. Although some laboratories may perform a fecal concentration from a PVA‑preserved specimen, some parasites will not concentrate well, nor will some exhibit the typical morphology that would be seen in concentration sediment from a formalin‑based fixative. PVA fixative solution is highly recommended as a means of preserving cysts and trophozoites for later examination. The use of PVA fixative also permits specimens to be shipped (by regular mail service) from any location in the world to a laboratory for subsequent examination. PVA fixative is particularly useful for liquid specimens and should be used in the ratio of 3 parts PVA to 1 part fecal specimen.

 

NOTE:  Very detailed information on all fixative options can be found in:

Garcia, L.S. Diagnostic Medical Parasitology, 5th ed., ASM Press, Washington D.C., 2007.