Table 17.

Practical guide for detection of circulatingTrypanosoma cruzi trypomastigotes by light microscopya

Obtain whole, anticoagulated blood by venipuncture or fingerstick. Process and examine the blood while it is fresh. Use sterile technique if specimens will also be cultured or inoculated into animals.
Prepare both whole blood and buffy coat for examination.
 If the blood was obtained by venipuncture, remove ∼1 ml of whole blood from the tube, before centrifugation, and place it in a small vial so that whole blood can be examined as described below. Centrifuge the rest of the blood to separate the erythrocyte, leukocyte (buffy coat), and plasma layers. Pass a pipette through the plasma to the buffy coat layer. Carefully remove the buffy coat and place it in a small vial for examination as described below.
 If the blood was obtained by fingerstick, fill at least two microhematocrit tubes with blood. Leave one tube uncentrifuged, so that whole blood can be examined. Centrifuge the other tube to separate the various layers of cells. Break the tube just above the buffy coat layer, remove the buffy coat, and place it in a small vial for examination as described below.
 Prepare multiple slides for examination. To facilitate semiquantitative analysis (see below), if 12-mm-diameter circular coverslips are used, dot 1.5-μl aliquots of blood and separate aliquots of buffy coat onto slides and place a coverslip over each dot; if 22- by 22-mm square coverslips are used, use 6.4-μl aliquots.
Examine slides of both whole blood and buffy coat under high power by light microscopy, preferably phase-contrast, looking for motile trypomastigotes (length, ∼15–25 μm), which often are first manifest by the resultant movement of the other cells on the slide. Stain positive slides with Giemsa.
 Specimens of whole blood can be examined more quickly than specimens of buffy coat because erythrocytes are homogeneous in size and color whereas the leukocytes and debris in the buffy coat are translucent and heterogeneous in size. On the other hand, trypomastigotes are present in higher concentrations in buffy coat than in whole blood. Therefore, both whole blood and buffy coat should be examined.
 If these recommendations about the sizes of aliquots and coverslips are followed, examining 200 high-power fields (magnification, ×400) of whole blood is the equivalent of examining 0.48 μl of blood, and finding on average 1 parasite per high-power field indicates that the specimen contains ∼400,000 parasites/ml.
Residual buffy coat and whole blood can be used in hemoculture, PCR (97,99), and animal inoculation.
  • a L. V. Kirchhoff was instrumental in the development of this table. Appropriate precautions should be used when handling specimens; see the text and Table 1.