the concentration and permanent stained smear can be prepared (Fig. 2.7 through 2.9). It is also possible to perform fecal immunoassay procedures from some of these vials. Make sure to ask the manufacturer about all three capabilities (concentration, permanent stained smear, fecal immunoassay procedures) and for specific information indicating that there are no formula components that would interfere with any of the three methods. Like the zinc substitutes, these formulas are proprietary.
Figure 2.7 ECOFIX is an example of a fixative that represents the single-vial collection system. This fixative has been coupled with its own stain, the ECOSTAIN (11). Remember to inquire about the compatibility of all single-vial systems with the immunoassay procedures; not all single-vial preservatives are compatible with all fecal immunoassays. doi:10.1128/9781555819002.ch2.f2.7
Figure 2.8 ProtoFix is a single-vial collection option. It is always important to review peer-reviewed literature regarding the results of new products compared with those previously in use. doi:10.1128/9781555819002.ch2.f2.8
Figure 2.9 UNIFIX is a single-vial collection system; information on this product can be found at the company website (www.med-chem.com). This fixative works well with trichrome stain. doi:10.1128/9781555819002.ch2.f2.9
Universal Fixative (TOTAL-FIX)
The TOTAL-FIX stool collection kit is a single-vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, most fecal immunoassays, and some molecular methods can be performed from a TOTAL-FIX preserved specimen (Fig 2.10). TOTAL-FIX is a mercury-, formalin-, and PVA-free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories. www.med-chem.com (accessed 4/17/13). TOTAL-FIX is similar to Unifix and Zinc PVA (Z-PVA), commonly used fixatives that have been commercially available and used in many laboratories since 1992.
Figure 2.10 TOTAL-FIX is a single-vial collection system; the fixative contains no mercury, no formalin, and no PVA. It is considered a Universal Fixative and can be used for concentrations, permanent stained smears, fecal immunoassays, special stains, and some molecular methods. doi:10.1128/9781555819002.ch2.f2.10
Quality Control for Stool Fixatives
Fixatives for fecal specimens are checked for quality control by the manufacturer before sale, generally with the use of living protozoa. If you prepare your own fixatives, the following approach can be used for quality control. The specimen used for quality control presented below is designed to be used with fixatives from which permanent stained smears will be prepared (Schaudinn’s fluid, Schaudinn’s fluid containing PVA, copper- or zinc-based fixative containing PVA, SAF, MIF, and Universal Fixatives). However, the same quality control specimen can also be used in a concentration; the white blood cells (WBCs) can be seen in the concentrate sediment (sedimentation concentration) or in the surface film (flotation concentration).
1. Obtain a fresh, anticoagulated blood specimen, centrifuge, and obtain a buffy coat sample (try and find a specimen with a high WBC count).
2. Mix approximately 2 g of soft, fresh fecal specimen (normal stool, containing no parasites) with several drops of the buffy coat cells.
3. Prepare several fecal smears, and fix immediately in Schaudinn’s fluid to be quality controlled.
4. Mix the remaining feces-buffy coat mixture in 10 ml of fixatives with or without PVA, SAF, MIF, or Universal Fixative preservative to be quality controlled.
5. Allow 30 min for fixation, and then prepare several fecal smears. Allow to dry thoroughly (60 min at room temperature or 30 to 60 min in an incubator [approximately 35°C]). Do not use a heat block.
6. Stain the slides by the normal staining procedure (trichrome, iron hematoxylin).
7. After staining, if the WBCs appear well fixed and display typical morphology and color, one can assume that any intestinal protozoa placed in the same lot number of preservative would also be well fixed, provided that the fecal sample was fresh and fixed within the recommended time limits.
8. The bulk quality control specimen can be concentrated as for a normal patient specimen. If the fixative is performing correctly, the WBCs will be visible in the concentration sediment or surface film (depending on the method used).
9. Record all quality control results. If the WBC morphology does not confirm good fixation, describe the results and indicate what corrective actions were used (repeated the test, prepared new fixative).
Procedure Notes for Use of Preservatives
1. Most of the commercially available kits have a “fill to” line on the vial label to indicate how much fecal material should be added to ensure adequate preservation of the fecal material (ratio of 1 part stool to 3 parts fixative). However, patients often overfill the vials; remember to open the vials with the vials turned away from your face. There may be excess gas in the vials that may create aerosols once the vial lids are opened.
2. Although the two-vial system (one vial of 5 or 10% buffered formalin [concentration] and one vial of PVA fixative [permanent stained smear]) has always been the “gold standard,” many laboratories are now using other options, including the single-vial collection systems and the Universal Fixative. Changes in the selection of fixatives are based on the following considerations:
A. Problems with disposal of mercury-based fixatives (availability of high-temperature incineration facilities and cost) and lack of multilaboratory contracts for disposal of such products
B. The cost of a two-vial system compared with the cost of a single collection vial
C. Selection of specific stains (trichrome, iron hematoxylin) to use with specific fixatives
D. Whether the newer fecal immunoassay kits can be used with stool specimens preserved in that particular fixative
Procedure Limitations for Use of Preservatives
1. Adequate fixation still depends on the following parameters:
A. Meeting recommended time limits for lag time between passage of the specimen and fixation
B. Use of the correct ratio of specimen to fixative (1:3)
C. Thorough mixing of the fixative and specimen (once the specimen is received in the laboratory, any additional mixing at that time will not counteract the earlier lack of fixative-specimen mixing and contact)
2. Unless the appropriate stain is used with each fixative, the final permanent stained smear may be difficult to examine (organisms hard to see and/or identify). Examples of appropriate combinations are as follows:
A. Schaudinn’s or PVA fixative with trichrome or iron hematoxylin stain
B.