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 5 g of EDTA in 100 ml of distilled water, aliquot 0.4 ml into tubes, and evaporate the water. This amount of anticoagulant is sufficient for 10 ml of blood. One can also use 20 mg of EDTA (dry) per tube (20 mg/10 ml of blood).
The time when the specimen was drawn should be clearly indicated on the tube of blood and also on the result report. The physician will then be able to correlate the results with any fever pattern or other symptoms that the patient may have. There should also be some comments on the test result report that is sent back to the physician that one negative specimen does not rule out the possibility of a parasitic infection.
STAT Test Requests and Risk Management Issues
All requests for malaria diagnosis are considered STAT requests, and specimens should be collected, processed, examined, and reported accordingly. All requests for examination of blood films should include a possible diagnosis of malaria; thus, these requests are always considered STAT. Not only the blood collection should be considered STAT, but also the processing and examination of both thick and thin blood films should be performed immediately on receipt of the blood (Table 3.6). Immunologically naive individuals with no prior exposure to malaria often present to the emergency room or clinic with symptoms such as fever and malaise and a relevant travel history to an area of the world where malaria is endemic. These patients often have vague symptoms, but have the potential to become very ill with malaria, even with a low parasitemia (0.0005% to 0.1%).
Collection of Specimens from Other Body Sites
Although clinical specimens for examination can be obtained from many other body sites, these specimens and appropriate diagnostic methods are not as commonly performed as those used for the routine stool specimen. The majority of specimens from other body sites (Table 3.7) would be submitted as fresh specimens for further testing. More information is given in the discussion of specific procedures in Section 5.
*Dientamoeba fragilis trophozoites can be found in formed stool specimens.
Suggested Reading
Beaver, P. C., R. C. Jung, and E. W. Cupp. 1984. Clinical Parasitology, 9th ed. Lea & Febiger, Philadelphia, PA.
Brooke, M. M., and M. Goldman. 1949. Polyvinyl alcohol-fixative as a preservative and adhesive for protozoa in dysenteric stools and other liquid material. J. Lab. Clin. Med. 34:1554–1560.
