fixatives.
Summary: Modified PVA (Mercury Substitutes)
Single-Vial Collection Systems (Other Than SAF)
Several manufacturers now have available single-vial stool collection systems, similar to the SAF or modified PVA methods. Some of these formulations are advertised as “ecologically friendly” and do not contain either mercury or formalin. From the single vial, both the concentration and permanent stained smear can be prepared. It is also possible to perform fecal immunoassays (EIA, FA, or the rapid-flow assay) with samples from some of these vials. Make sure to ask the manufacturer about all three assays (concentrate, permanent stained smear, and 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.
Summary: Single-Vial Collection Systems
Quality Control for Preservatives
Preservatives for fecal specimens are checked for quality control by the manufacturer prior to sale, generally with living protozoa. If you prepare your own fixatives, you can use the following approach 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, PVA, modified PVA, SAF, or the single-vial systems). However, this 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 to 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 mixture of feces and buffy coat in 10 ml of PVA, modified PVA, or SAF 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]).
6. Stain slides by normal staining procedure.
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. Record all quality control results. If the WBC morphology does not confirm good fixation, then describe the results and indicate what corrective action procedures were used (repeated the test, prepared new fixative).
Procedure Notes for Use of Preservatives (Stool Fixative Collection Vials)
1. Most of the commercially available kits have a “fill to” line on the vial label to indicate how much fecal material to add to ensure adequate preservation of the fecal material. 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 [permanent stained smear]) has always been the “gold standard,” laboratories are beginning to use other options such as the single-vial collection systems. Changes in the selection of fixatives are based on the following considerations.
a. Problems with disposal of mercury-based fixatives and the 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 immunoassay kits (EIA, FA, rapid cartridges [Giardia, Cryptosporidium, E. histolytica]) can be used with stool specimens preserved with that particular fixative
Procedure Limitations for Use of Preservatives (Stool Fixative Collection Vials)
1. Adequate fixation still depends on the following factors:
a. Meeting recommended time limits for lag time between passage of the specimen and fixation
b. Use of the correct ratio of fixative to specimen (3:1)
c. Thorough mixing of the fixative and specimen (once the specimen is received in the laboratory, any additional mixing will not counteract the lack of fixative-specimen mixing and contact prior to that time)
2. Unless the appropriate stain is used with each fixative, the final permanent stained smear may be difficult to examine (organisms may be hard to see and/or identify). Examples of appropriate combinations are
a. Schaudinn’s or PVA fixative with trichrome or iron hematoxylin stains
b. SAF fixative with iron hematoxylin stain
c. Single-vial mercuric chloride substitute systems with trichrome, iron hematoxylin, or company-developed proprietary stains matched to their specific fixatives
Collection of Blood
Collection and Processing
Depending on the parasite life cycle, a number of parasites may be recovered in a blood specimen, either whole blood, buffy coat preparations, or various types of concentrations. These parasites include Plasmodium, Babesia, and Trypanosoma spp., Leishmania donovani, and microfilariae. Although some organisms may be motile in fresh, whole blood, species are normally identified from the examination of permanent stained blood films, both thick and thin films (Table 3.4). Blood films can be prepared from fresh whole blood collected with no anticoagulants, anticoagulated blood, buffy coat cells, or sediment from the various concentration procedures (Table 3.5).
Blood can be collected from either finger stick or venipuncture. Venous blood should be collected in a tube containing EDTA. Multiple thick and thin blood films from the blood or buffy coat should be prepared and examined immediately after receipt of the blood by the laboratory, and multiple blood examinations should be performed before blood-borne parasite infection is ruled out. Unless you are positive that you will receive well-prepared thick and thin blood films from finger stick blood, request a tube of anticoagulated blood (EDTA anticoagulant [lavender top] is preferred). The tube should be filled with blood to provide the proper blood/anticoagulant ratio. For detection of stippling, the smears should be prepared within 1 h after the specimen is drawn. After that time, stippling may not be visible on stained films; however, the overall organism morphology is still acceptable. Most laboratories routinely use commercially available blood collection tubes; preparation of EDTA collection tubes in-house is neither necessary nor cost-effective. However, if the need should arise, EDTA (Sequestrene) can be prepared and tubed as follows: dissolve