SPECIMEN PROCESSING

Ova and Parasite Examination

What procedures constitute the Ova and Parasite (O&P) Examination?

The direct wet smear, concentration, and permanent stained smear constitute the routine O&P exam on fresh stool specimens.  If the specimens are submitted to the laboratory in stool preservatives, then the concentration and permanent stained smear should be performed.  If a laboratory indicates they provide an O&P examination, the CAP checklist indicates that the O&P examination must include the concentration and permanent stained smear.  Also, the CAP checklist requires the direct wet smear be performed on FRESH liquid or soft stool only (looking for motile trophozoites).  There is no need to perform a direct wet mount on fresh formed stool; the chances of seeing motile trophozoites is rare, since formed stool tend to contain only the cyst forms.

Why do you need to perform a permanent stained smear examination on every stool submitted for an O&P examination?

There is data to indicate that intestinal protozoa may or may not always be seen and identified from the concentration examination.  Since the permanent stained smear is designed to facilitate identification and/or confirmation of the intestinal protozoa, it is important that this procedure be performed on all stools for which the O&P has been ordered.  Also, since trophozoite stages will not be visible on the concentration examination (rare exceptions - trophozoites can be seen in concentration sediments prepared from SAF-preserved specimens), it is even more important to examine the permanent stained smear.  Even if organisms (trophozoites and/or cysts) are seen in the concentration wet mount, they may not be identified accurately and require confirmation from the examination of the permanent stained smear.  Remember, in patients who are symptomatic with diarrhea, they are more likely to have protozoan trophozoites in the stool, not the more resistant cyst form.  This approach is consistent with the O&P examination (CAP Checklist for lab inspections).

Why do you need to pour out some PVA onto paper towels prior to preparation of slides for permanent staining?

The reason this step has been included in processing directions is the following:  Many people were taking the PVA/stool right out of the vial and onto the slide – too much PVA was being carried over onto the slide.  It takes quite a bit of time for PVA to dry, thus the material often falls off during staining.  Also, the amount of PVA (plastic powder) it takes to glue the stool onto the slide is extremely small.  So, if you eliminate the excess PVA prior to making the smears, your slides will require less drying time, the stool will adhere to the glass, and you will get a better stained smear.  However, if the material (PVA/stool mixture) is taken right out of the vial onto the glass slide and this approach is working– there is no need to change protocols. 

Can you use concentrated sediment to prepare slides for permanent staining?

The main thing to remember is that a routine trichrome stain cannot be performed from the concentration sediment if the specimen has been rinsed using formalin, saline, or water.  One can perform a trichrome on SAF-preserved material, but not if the specimen has been rinsed with formalin, saline, or water.  If you centrifuge the fecal/fixative material (without adding any rinse reagents), then providing your fixative is compatible with the permanent stain, you can use some of the sediment for smear preparation prior to staining.  However, once you continue with the rinse steps, the final concentration sediment will often (again depending on the rinse fluids) not be compatible with trichrome staining.  If you want to use a single vial fixative system, then you can spin down the stool/fixative mixture (using no additional rinse reagents), prepare your slide for permanent staining from the sediment, then proceed with the regular rinses called for in the concentration procedure.