SPECIMEN COLLECTION

Intestinal Tract

What has been the “gold standard” for stool collection systems?

The standard two-vial set has been used for many years and consists of one vial of 10% formalin from which the concentration is performed and one vial of PVA fixative (base fixative can be mercury or non-mercury to which has been added the plastic resin powder – polyvinyl alcohol, PVA) from which the permanent stained smear is prepared.

What is the basis for the recommendation that three stools should be collected on alternate days, rather than three days in a row or three in one day?

Intestinal protozoa are shed in the stool on a cyclic basis.  It is generally accepted that this time frame is approximately 10 days, so if specimens are collected too close together, sampling may occur at a low point in the cycle and the organisms may be missed.  By collecting specimens over a wider time frame, days with heavier shedding may be sampled, as well.  

Is it acceptable to accept three stools collected three days in a row?

Yes, but chances of sampling during the total time frame for shedding (10 days) might be more likely to include days of heavy shedding.

Is it ever appropriate to accept three stools in one day from the same patient?

Generally, the answer is no with rare exceptions.  If a patient has severe diarrhea or dysentery, there is a large dilution factor that may make finding any organisms present more difficult.  Therefore, after consultation with the physician, more than one stool could be examined within any one day.

Is it good laboratory practice to accept 2 (rather than 3) stools for the O&P examination?

Although the examination of 3 stool specimens provides a more statistically accurate result, many laboratories feel the percentage of organism recovery with the examination of 2 stools is acceptable.

Do you have to collect the stool specimen any differently if you are looking for the coccidia (Cryptosporidium spp., Cyclospora cayetanensis, Isospora belli)?

Fresh stool or specimens preserved in routine stool fixatives (formalin-based) can be used for diagnostic procedures for the identification of the coccidial oocysts (special modified acid-fast stains for the coccidia or fecal immunoassays for Cryptosporidium spp.).  Some of the single vial collection options are also acceptable.

Do you have to collect the stool specimen any differently if you are looking for microsporidia?

Fresh stool or specimens preserved in routine stool fixatives (formalin-based) can be used for diagnostic procedures (Modified Trichrome stains) for the identification of the microsporidial spores. Some of the single vial collection options are also acceptable.

Fixatives

What are the pros and cons of using stool fixatives?

Although it would be helpful to see motile organisms in the direct saline wet preparation, often the trophozoite forms have already begun to disintegrate by the time the stool specimen reaches the laboratory.  In order to reduce the lag time between passage of the specimen and submission/arrival in the laboratory, stool fixatives are recommended.  The benefits of fixation and preservation of organism morphology far outweigh the benefits of receiving a fresh, unpreserved stool.

When selecting a single vial stool fixative, what questions should be asked?

Can one perform a concentration, permanent stained smear and/or immunoassay procedures from the specimen received in that vial?  What stains work best with that particular fixative?  Another issue for consideration involves the types and numbers of parasites that might be missed using this approach. 

What are the advantages and disadvantages of using 5% vrs. 10% formalin?

Although 5% formalin is thought to provide a more “gentle” fixation of protozoa, this concentration formalin will not always kill all helminth eggs.  The 10% formalin will kill most helminth eggs (exception:  Ascaris lumbricoides), but may be more damaging to protozoa.  In reality, most people probably can’t tell the difference by examining clinical specimens preserved in either 5% or 10% formalin. 

What is the difference between buffered and nonbuffered formalin?

Buffered formalin tends to produce fewer osmotic changes in the organisms during fixation; however, on a day-to-day basis, it may be difficult to detect morphologic differences from buffered or unbuffered formalin.

Should the laboratory be concerned about the amount of formalin used within the parasitology lab?  Also see a complete discussion under (8) Concentration Section.

The amount of formalin used in diagnostic parasitology testing is very minimal.  The regulations governing formalin use were developed for industry, where large amounts of formalin are sometimes used.  However, the regulations do indicate that any place using formalin must be monitored.  Once you have been monitored and your records are on file, there is no need to be rechecked again (unless something dramatically changes in terms of your formalin volume use).  We have never heard of any microbiology laboratory coming even close to the limits, so formalin use within diagnostic parasitology is perfectly acceptable.

What is PVA? 

PVA stands for polyvinyl alcohol, a plastic powder/resin that is incorporated into the fixative (Schaudinn’s fixative) and serves as an adhesive to “glue” the stool material onto the slide.  PVA itself has no preservation capability.

What are some good stool fixative/stain combinations?

The “gold standard” has been 1 vial of 10% formalin, from which the concentration is performed.  The second vial has been with mercuric-chloride based PVA, from which the permanent stained smear is prepared (trichrome or iron hematoxylin).  Other options in terms of overall quality are:

a. SAF and iron hematoxylin stain
b. UNIFIX or Z-PVA (Medical Chemical Corp.) and trichrome stain
c. ECOFIX and ECOSTAIN (Meridian Bioscience)
d. SAF and trichrome stain

There may be other options available – check the published literature.