of leishmaniasis: review of alternatives to conventional microscopy ICT for detection of anti-rK-39 antibodies Concentration procedures Cytocentrifugation technique Knott concentration procedure Membrane filtration technique Gradient centrifugation technique Triple-centrifugation method for trypanosomes Special stain for microfilarial sheath Delafield’s hematoxylin
Depending on the 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 species, Leishmania donovani, and microfilariae. Although some organisms are motile in fresh, whole blood, species identification is normally accomplished from the examination of both thick and thin permanent stained blood films. Blood films can be prepared from fresh, whole blood collected with no anticoagulants, from anticoagulated blood, or from sediment from the various concentration procedures. Although for many years Giemsa stain has been the stain of choice, the parasites can also be seen on blood films stained with Wright’s stain, a Wright/Giemsa combination stain, or one of the more rapid stains such as Diff-Quik (American Scientific Products, McGaw Park, IL), Wright’s Dip Stat stain (Medical Chemical Corp., Torrance, CA), or Field’s stain. Delafield’s hematoxylin stain is often used to stain the microfilarial sheath; in some cases, Giemsa stain does not provide sufficient stain quality to allow differentiation of the microfilariae. A complete discussion of the proper way to examine a blood film is presented later in this chapter. This information is important and particularly relevant when one is examining proficiency testing blood films in the absence of clinical information about patient history and possible etiologic agents.
It is important to remember that standard precautions should be used at all times when blood or body fluids are handled (1). Remember that all requests for malaria diagnosis are considered STAT requests, and specimens should be ordered, collected, processed, examined, and reported accordingly.
Preparation of Thick and Thin Blood Films
Some parasites (microfilariae and trypanosomes) can be detected in fresh blood by their characteristic shape and motility, but specific identification of the organisms requires a permanent stain. Two types of blood films are recommended. Thick films allow a larger amount of blood to be examined, which increases the possibility of detecting light infections (2). However, species identification by thick film, particularly for malaria parasites, can usually be made only by experienced workers. The morphologic characteristics of blood parasites are best seen in thin films, in which the red blood cell (RBC) morphology is preserved and the size relationship between infected and uninfected red cells can be determined after staining. This characteristic is often valuable in determining the species of Plasmodium present from the thin blood film.
The accurate examination of thick and thin blood films and identification of parasites depend on the use of absolutely clean, grease-free slides for preparation of all blood films. Old (unscratched) slides should be cleaned first with detergent and then 70% ethyl alcohol; new slides should also be cleaned with alcohol before use. When a new box of slides is opened, the slides are coated with a substance that allows them to be pulled apart; these slides should be cleaned before use for preparation of blood films. Do not use cotton; gauze is recommended with 70% alcohol. The advantages and disadvantages of the thin and thick blood films can be seen in Table 7.1.
Blood should be collected immediately on admission or when the patient is first seen in the emergency room and/or clinic; if the initial blood films are negative, collect daily specimens for 2 or 3 additional days (ideally between paroxysms if present; however, there is often no periodicity seen). After a finger stick, the blood should flow freely; blood that has to be “milked” from the finger will be diluted with tissue fluids, decreasing the number of parasites per field. If the specimen is sent directly to the laboratory, thus eliminating laboratory-patient contact, the following approach can be used. Unless you are positive that you will receive well-prepared slides, request a tube of fresh blood (EDTA anticoagulant is preferred/lavender top) and prepare the smears. In general, the use of finger stick blood has declined, particularly in areas of the world where automated hematology instruments have become much more widely used. 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 will still be acceptable. Potential problems with anticoagulants can be seen in Table 7.2. Although blood films can be prepared from the small amount of blood left in the needle after the venipuncture collection using anticoagulant, it is not recommended for several reasons. The blood tends to clot fairly quickly in the needle, and there are safety recommendations that limit the handling of needles. Therefore, the finger stick and EDTA venipuncture are recommended for collection of specimens for blood film preparation.
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. However, with travelers who are immunologically naive (have never come in contact with malaria before), there may not be any fever periodicity at all, and the symptoms may be very general and nonspecific for a malaria infection. There should also be some indication on the report that is sent back to the physician that one negative specimen does not rule out the possibility of a parasitic infection.
Note Although most laboratories use commercially available blood collection tubes, the following approach can be used when necessary. 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 tube should be filled with blood to provide the proper blood/anticoagulant ratio.
Fresh Blood
To prepare the thick film, place 2 or 3 small drops of fresh blood (no anticoagulant) on an alcohol-cleaned slide. With the corner of another slide and using a circular motion, mix the drops and spread them over an area ∼2 cm in diameter. Continue stirring for 30 s to prevent the formation of fibrin strands that may obscure the parasites after staining.
Anticoagulant
If blood containing an anticoagulant is used, 2 or 3 drops may be spread over an area about 2 cm in diameter; it is not necessary to continue stirring for 30 s, since fibrin strands do not form. If the blood is too thick or any grease remains on the slide, the blood may flake off during staining. It is far better to make the preparation too thin, rather than too thick.
Allow the thick film to air dry (room temperature) in a dust-free