Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine needle aspiration cytology is an inexpensive, simple, quick and minimally invasive technique that we use to collect trypanosomes from palpable lesions in organs in live mice. Given that fine needle aspiration is a non-terminal procedure, it allows for the repeated sampling in the same animal over time. If these findings can be translated to cattle, this method will be very useful for veterinarians to diagnose trypanosomiasis in cattle in a farm.

Demonstrating the smear staining procedure will be Ana Margarida Santos, an assistant technologist from my laboratory. To begin, confirm anesthetization with the toe pinch method. When the reflex of the retraction of the leg is absent, position the mouse in dorsal recumbency.

Carefully palpate the testes to determine the size and distance from the overlying skin and then restrain it between the index and middle finger or between the index finger and thumb. To further immobilize the target, stretch the overlying skin tightly and clean the surface with alcohol wipes. Next, insert the needle tip of a 23 gauge needle connected to a five milliliter syringe into the target making sure the plunger is in the rest state.

To apply suction, retract the syringe plunger to the three to four millimeter mark two to three times. To increase the probability of obtaining a representative sample and of targeting smaller structures like the epididymis, redirect the needle within the organ either in a straight line or along several different tangents. Release the suction and then withdraw the needle.

Needle can only be removed after the negative pressure is released by letting go of the plunger. Otherwise, the aspirate gets sucked into the syringe and is irrecoverable. Apply pressure with a sterile gauze sponge at the puncture site to control any bleeding.

Disconnect the syringe from the needle and fill it with air. Reconnect the needle, place the tip of the needle very close to or even on the slide and gently eject the contents of the needle onto the slide. To ensure replicate sampling, perform at least one additional aspiration per organ and animal.

After reverting anesthesia with a subcutaneous injection of 200 microliters of one milligram per kilogram Atipamezole in saline, return animals to their home cage for recovery. Using the nondominant hand, pick up the slide that has the drop of aspirate pinching the frosted end between the thumb and index finger. Using the dominant hand, pick up a clean spreader slide and place it smooth clean edge across the second slide just on the top of the drop at an angle of approximately 30 degrees.

To obtain a thin film, glide the slide forward with one light continuous and steady movement. Rest the slide and allow for the complete and fast air drying of the material. Label the frosted edge of the slide with a pencil.

For the Giemsa staining protocol, fix air dried smears by immersing the slides into a Coplin jar containing 100%methanol for five minutes. Then transfer the slides into a Coplin jar containing 20%Giemsa solution and distilled water for 30 minutes. Rinse the slides in tap water and dab with tissue paper to dry thoroughly.

While holding the slide horizontally, apply several drops of nonaqueous mounting medium onto the smear. Place the edge of a cover glass onto the slide, lower it and press gently to remove any air bubbles. For immunocytochemistry, fix air dried smears in 100%methanol at room temperature for 10 minutes.

Wash the slide for five minutes in a Coplin jar with 1X PBS repeating this step three times using fresh 1X PBS every time. After removing the slides from the Coplin jar, wipe excess buffer without touching the smear and draw a line around the smear with a water repellent pen. While holding the slide horizontally, apply 150 microliters of diluted primary antibody solution to each smear and incubate for one hour at room temperature.

After incubation, wash the slides for five minutes in a Coplin jar with 1X PBS repeating this step three times using fresh 1X PBS every time. While holding the slide horizontally, apply 150 microliters of commercially available horse radish peroxidase DAB visualization system to each smear and then incubate for 30 minutes at room temperature. Wash the slides again three times for five minutes in PBS.

Then counterstain by immersing the slides into a Coplin jar containing Harris hematoxylin for 10 seconds. Rinse off in tap water and dab with paper to dry thoroughly. While holding the slide horizontally, apply several drops of nonaqueous mounting medium onto the smear.

Place the edge of a cover glass onto the slide, lower it and press gently to remove any air bubbles. A good quality smear was characterized by a monolayer of cells with good density and preserved morphological features allowing for the identification of their tissue of origin and relative proportion between one another. Furthermore, parasites were efficiently immunostained, identifiable and countable.

Poor or negative fine needle aspiration results may be due to too little material and thus under representative of the mass or organ. It can also be due to too much material on a single slide making overly thick smears and impairing cytological evaluation. So called crushed artifact happens due to too much force being applied when making the smear leaving to disrupted cells resulting in a lot of naked nuclei and DNA streaks.

When not enough force is applied during smear preparation, cells do not disaggregate resulting in a stratified layer that impedes evaluation of the morphological features of the cells. Comparison of fine needle aspiration cytopathology with histopathology, cytopathology had the advantage of better preserved cellular morphology and better assessment of relative proportions and counting of cells. Immunocytochemistry is simpler, faster, and easier to optimize than immunohistochemistry.

For fine needle aspiration, always ensure a good immobilization of the animal, stabilization of the organs or mass to be aspirated, and a coordinated vacuum application and release. Then for high quality smears, immediately spread after the material has been placed onto the glass and be gentle on the strength applied to make the smear to avoid cell crushed artifacts. In addition to routine microscopy and immunocytochemistry, this material can also be used for electron microscopy, biochemical analysis, flow cytometry, molecular biology, or even for in vitro assays.

This technique allowed us to demonstrate that fine needle aspiration can be used to diagnose and study disease in experimental animals. We were able to diagnose tropism of Trypanosoma parasites to the external male reproductive organs and also to characterize this disease over the course of the infection.