Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Summary

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

Abstract

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Introduction

Fine Needle Aspiration (FNA) is widely used in the diagnosis of cancer and non-neoplastic diseases, both in human and domestic animals. The technique has been standardized over the years and is described in numerous textbooks^1,2.

It largely consists of the percutaneous aspiration of palpable masses, organs or effusions with a thin needle fitted onto an empty syringe, using the negative pressure to withdraw cells or fluid from the mass^1,3. Needles are typically 22 to 25 G (gauge corresponding to the needle inner diameter), and the use of a larger bore needle (large diameter, e.g., 21 G) is helpful to increase the cellularity, although this can produce excessive blood contamination. Needle length will depend on the depth of the mass but 1 or 1½ inch is commonly used for superficial masses. Syringes are typically 5 to 10 mL, with larger syringes achieving higher vacuum, which in turn increases aspirate yield. Non-palpable deep-seated masses can also be aspirated, with longer needles and under image guidance (ultrasonography). The retrieved aspirate can then be smeared, air dried, wet-fixed, stained and observed under a microscope to achieve a diagnosis³ (Figure 1).

This is a simple, inexpensive, painless and minimally invasive technique mainly used in the preoperative setting to achieve a diagnosis on palpable masses and also for organs, like lymph nodes, thyroid, prostate or even the external male reproductive structures¹. In addition to being a diagnostic tool, this technique can be used for the collection of cells for other purposes as well, namely cytogenetics, electron microscopy (Figure 1D), flow cytometric characterization^4,5,6,7, establishment of cell cultures⁸. A common example in clinical practice is sperm retrieval for in vitro fertilization⁹.

Aspiration can be repeated several times in the same mass to obtain multiple smears. In case of a heterogeneous lesion, e.g., a solid area and a cystic space, it is important that cells are aspirated from each region. The material collected by FNA is in general highly cellular, which in most cases allows for the diagnosis of diseases without the need for a tissue biopsy. Special stains, immunofluorescence. immunocytochemistry (Figure 1D), and molecular techniques may also be performed in smears obtained through FNA, e.g., for the identification of infectious agents when not recognizable by morphology alone¹⁰. A brief overview of the general applications and of the equipment and supplies needed for FNA are summarized in Tables 1 and 2, respectively.

The first report on the use of the needle puncture for diagnostic purposes is described in early writings of Arab medicine, but it’s in the early 20th century that modern needle aspiration techniques were implemented¹¹. Notably, perhaps the first report that suggests the use of FNA for the diagnosis of infectious diseases was a study in 1904, where Grieg and Gray reported needle aspirations of lymph nodes from patients with sleeping sickness revealed motile trypanosomes¹². The authors reported the presence of trypanosomes in both early and advanced cases, and at a higher density than that seen in blood smears, where these are often rare events¹².

Current diagnosis of Trypanosomiasis in cattle relies on the direct observation of parasites in the blood, lymph or in immunodiagnostic techniques^13,14,15. We have previously shown that in experimental Trypanosoma infections in the mouse, Trypanosoma brucei (T. brucei) has a remarkable tropism to adipose tissue¹⁶ and also to some of the external male reproductive structures, namely epididymis¹⁷. Parasites accumulate in the stroma of these tissues in large numbers¹⁶.

The protocol depicted below describes a detailed step-by-step technical procedure for FNA in live mice, aimed at the aspiration of trypanosomes present in the external male reproductive structures (testis, epididymis, and epididymal fat), followed by conventional cytology and immunostaining for specific parasite proteins (VSG)^16,17. Aspiration was performed 6 days after the infection and safety procedures that apply are those commonly established for routine handling of experimental animals. Additional measures are required for animals that have immune deficiencies (wearing a steam-sterilized gown, mask, hair bonnet, sterile gloves, and ensuring aseptic technique at all times) to mitigate accidental exposure to opportunistic pathogens.

Protocol

All animal experiments in this protocol were performed according to EU regulations and approved by the Animal Ethics Committee of Instituto de Medicina Molecular (iMM), (AEC_2011_006_LF_TBrucei_IMM). The animal facility of iMM complies with the Portuguese law for the use of laboratory animals (Decree-Law 113/2013) and follows the European Directive 2010/63/EU and the FELASA (Federation of European Laboratory Animal Science Associations) guidelines and recommendations concerning laboratory animal welfare.

1. Aspiration of Parasites from the External Male Reproductive Organs of the Mouse

NOTE: Fine needle aspiration (FNA) was performed in wild-type male C57BL/6J mice, 6 to 10 weeks old, infected with T. brucei through intraperitoneal injection of 200 µL of saline with 2,000 parasites as described previously¹⁶.

1. For FNA of the external male reproductive organs, place the mouse in a laminar flow hood, anesthetize the animal with an intraperitoneal injection of 200 µL of a mixture of 75 mg/kg Ketamine + 1 mg/kg Medetomidine in saline.
   1. Confirm anesthetization with the toe pinch method. When the reflex of the retraction of the leg is absent, position mouse in dorsal recumbency (Figure 2A).
   2. Carefully palpate the testis, assessing size and distance from the overlying skin. Restrain the organ between the index and middle finger or between the index finger and thumb. Stretch the overlying skin tightly across the mass to further immobilize the target. Clean the surface with alcohol wipes (Figure 2A).
   3. Hold the assembled 22 G needle and 5 mL syringe and insert the needle tip into the target, always with the plunger in the rest state (Figure 2B-C).
   4. Apply suction by retracting the syringe plunger to the 4 mL to 5 mL mark 2-3 times. Redirect the needle within the organ either in a straight line or along several different tangents to increase the probability of a representative sample and of targeting smaller structures like the epididymis. Make sure that this procedure is gentle, to minimize the tissue damage (Figure 2C-D).
   5. Release the suction and then withdraw the needle. Do not redraw the needle with the retracted plunger as this will lead to the suction of the aspirate into the barrel of the syringe and impede its recovery (Figure 2E). After the withdrawal of the needle, control any bleeding by applying pressure with a sterilized gauze sponge at the puncture site.
   6. Disconnect the syringe from the needle, fill it with air, reconnect the needle and gently eject the contents of the needle onto a slide. Place the tip of the needle very close or even on the slide to avoid splattering (Figure 2F-I).
   7. Perform at least one additional aspiration per organ/animal to ensure replicate sampling.
   8. Revert anesthesia with a subcutaneous injection of 200 µL of 1 mg/kg Atipamezole in saline and return animals to their home cage for recovery and observe until full recovery. 

2. Smear Preparation from the Aspirated Material

NOTE: Use gloves throughout the procedure and ensure safe disposal of needles and syringes.

1. Two steps pull method
   1. Pick up the slide that has the drop of the aspirate using the nondominant hand, and pinch the frosted end between the thumb and index finger (Figure 3A).
   2. Pick up a second clean slide, the spreader slide, with the dominant hand and bring it across the first slide with the drop of the aspirate. Place the smooth clean edge of the slide on the specimen slide just on the top of the drop at an angle of approximately 30° (Figure 3B).
   3. Glide the slide forward with one light, continuous and steady movement to obtain a thin film (Figure 3C).
   4. Rest the slide and allow for the complete and fast air drying of the material (Figure 3D). Do not heat-fix. Label the frosted edge of the slide with a pencil.
      NOTE: The protocol can be paused at this step and smears can be stored indefinitely until ready to be stained.

3. Staining of the Smears

NOTE: Use gloves throughout the procedure and ensure that steps 3.1.4 and 3.2.9 are performed inside a fume hood.

1. Giemsa staining protocol
   1. Fix air-dried smears by immersing the slides into a Coplin jar containing 100% methanol for 5 min (Figure 3E).
   2. Transfer the slide into a Coplin jar containing 20% Giemsa solution (diluted to 1/5 in distilled water) for 30 min, or 10% Giemsa for 10 min (Figure 3F).
   3. Rinse off in tap water and dry thoroughly using tissue paper to dab.
   4. Hold the slide horizontally and apply one drop of the non-aqueous 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.
2. Immunocytochemistry in FNA smears
   1. Fix air-dried smears in 100% methanol at room temperature for 10 min.
   2. Wash the slide for 5 min in a Coplin jar with 1x phosphate buffer (PBS), repeating this step 3 times using fresh 1x PBS every time.
   3. Remove the slide from Coplin jar, wipe excess buffer without touching the smear and draw a circle around the smear with a water repellent pen (Table of Materials).
   4. Hold the slide horizontally and apply 150 μL of diluted primary antibody solution to each smear and incubate for 1 h at room temperature.
      NOTE: Primary antibody used here is a non-purified rabbit serum anti-T. brucei VSG13 antigen (cross-reactive with many T. brucei VSGs, produced in-house), diluted in 1x PBS at 1:50,000. Perform negative controls by replacing the appropriate primary antibody with preimmune serum (Table of Materials) to allow for the assessment of the non-specific binding of the secondary antibody.
   5. Wash the slide for 5 min in a Coplin jar with 1x PBS, repeating this step 3 times using fresh 1x PBS every time.
   6. Hold the slide horizontally and apply 150 μL of commercially available horseradish peroxidase/DAB visualization system to each smear. Incubate for 30 min at room temperature (Table of Materials).
   7. Wash for 3x for 5 min in 1x PBS.
   8. Counterstain by immersing the slides into a Coplin jar containing Harris hematoxylin. Rinse off in tap water and dry thoroughly using paper to dab.
   9. Hold the slide horizontally and apply one drop of the non-aqueous 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.

Discussion

Fine Needle Aspiration (FNA) is a method widely used to diagnose disease in human and domestic animals. The technique has been standardized over many years^1,2, which makes use of a small-bore needle to aspirate cells or fluid from a palpable mass or organ^1,3. The aspirate is then typically smeared on a glass slide and stained for microscopic observation to achieve a diagnosis, but the technique can also be used to retrieve cells for other purposes^4,5,6,7,8,9.

The procedure is quick (<5 min per mouse, for an experienced researcher) and the risk of complications is minimal, similar to the risk incurred when undergoing simple venous puncture. For this reason, for FNA of palpable masses, anesthesia is only required for sensitive anatomic locations or when a good immobilization of the animal and stabilization of the organ or mass to be aspirated is extremely crucial for optimum results. This is frequent for small laboratory animals, as safe and effective restraint of a small rodent with one hand while ensuring the best access to the area for aspiration with the other hand, is hard to achieve without anesthesia. Short-term anesthesia is in most instances sufficient, nonetheless necessary, for a good FNA in the mouse. A good immobilization maximizes the chances of obtaining an aspirate that is representative of the cellular components of the lesion and also minimizes the chances of externalizing the tip of the needle while negative pressure is applied.

Although the procedure itself is very simple, coordinated application and release of vacuum are the most critical steps. After insertion of the needle, the plunger of the syringe is retracted to achieve a controlled vacuum (suction), and the needle can only be removed from the mass after releasing the negative pressure by letting go of the plunger (Figure 2); otherwise the aspirate is sucked into the barrel of the syringe and is then very hard to recover. Another very important step is the preparation of high-quality smears. There are various methods to make a smear but regardless of the method, smears should be prepared immediately after the material has been placed onto the glass. The biological material should be spread out gently to avoid cell-crush artifacts. Use of a coverslip as spreader can help prevent these artifacts. However, the application of too much force while making the smear will break the coverslip. A good quality smear typically has most of the cell population distributed as monolayer so that they can easily transmit light. Cellular material should not be excessively trapped in the blood clot.

The goal of an FNA is to collect cells from a mass, tissue or organ. The use of larger bore needles is helpful to increase the cellularity, but can be associated with excessive blood contamination, while sampling with smaller needles yields higher quality, though less abundant material. In our case, percutaneous aspiration of the external male reproductive structures in mice experimentally infected with T. brucei, was performed with 22-gauge needles coupled to a 5 mL syringe. Mice were anesthetized, the testis was stabilized with one hand and puncture and aspiration guided and performed with the other hand. We retrieved numerous trypanosomes admixed with germ cells, spermatozoa, epithelial cells and stromal cells, which were smeared and immunostained for the parasites’ surface proteins (VSG) (Figure 4).

There is always the potential sampling error in aspirates yielding negative results because although multiple areas of a given lesion can be sampled multiple times, this is a blind aspiration where we do not visualize the needle tip and target organ or mass. This is extremely relevant in a clinical setting, where a negative FNA of a suspicious lesion does not obviate further investigations, but it’s not so relevant in animal models of disease. Thus, general limitations include mainly false negative results, and less frequently false positive results (e.g., from blood contamination), but the most important limitation in the setting of animal experimentation is the lack of information on tissue architecture¹⁷, like we have with histopathology, e.g. distribution pattern of parasites, of immune cells, and cell-cell interaction features. Nonetheless, advantages are that FNA is a non-terminal procedure allowing for the repeated sampling in the same animal over-time and always allows for better preserved cellular morphology (Figure 5). Alternatives to FNA for harvesting host cells, immune cells or microorganisms from a mouse always rely on euthanizing the mouse to collect the mass or organs of interest.

To our knowledge, there are only a few reports on the use of FNA in small laboratory animals, one from 1949 corresponding to the aspiration of bone marrow with 22 G needle for studying hematopoiesis¹⁸, and all other in combination with flow cytometry to quantify either tumor-associated inflammatory cells or endothelial cells^7,19,20. Our work shows that this technique can be extended to the diagnosis and study of infectious disease models and can combine cytology with techniques like immunocytochemistry or electron microscopy. Two of the major advantages of the method in experimental animals are: (1) this procedure is not terminal, i.e., can be performed in live mice; and (2) due to its mild severity it allows for serial aspirations in the same animal. Hence fewer mice are required for each study, and the correlation between progression of clinical disease and evolution of the cellular and molecular features of a disease and/or microorganism can be easily performed, thus enabling longitudinal studies.

Perhaps the first report on the use of FNA for diagnosing an infectious diseases is a study by Grieg and Gray in 1904 that reports needle aspirations of lymph nodes from patients with sleeping sickness, which revealed motile trypanosomes¹². If our findings in laboratory mice find translation to cattle, i.e., that if Trypanosoma can be easily sampled by FNA from the external male reproductive structures, one can expect that this technique will be useful to veterinarians for diagnosing animal trypanosomiasis in-farm, in livestock.

Materials

Name	Company	Catalog Number	Comments
Atipamezole (ANTISEDAN 10 mL)	Bio 2	7418046	Anesthesia reversal
Cover slips (24 x 60 No.1)	VWR	631-0664	Smear making
DAB	Dako	K3468	Immunocytochemistry
Entellan (500 mL)	VWR	1.07961.0500	Mounting media
Envision Flex antibody diluent	Dako	8006	Immunocytochemistry
EnVision Flex conjugated w/ HRP (anti-rabbit)	Dako	K4010	Immunocytochemistry
Envision Flex Wash Buffer	Dako	K8007	Immunocytochemistry
Giemsa stain	Atom Scientific Ltd	RRSPSS-A	Smear staining
Glass slides (Superfrost Plus)	VWR	631-9483	Smear making
Harris Haematoxylin	Bio-optica	05-06004E	Immunocytochemistry
Hydrogen Peroxidase solution	Sigma	H1009-500ML	Immunocytochemistry
Hypodermic needles Microlance 3 (23G)	Henry Schein	902-8001	Aspiration technique
Ketamin (IMALGENE 1000 - 10 mL)	Bio 2	7410928	Anesthesia
Medetomidine (DOMITOR 10 mL)	Bio 2	7418335	Anesthesia
Methanol	Merck	1.06009.2511	Smear fixative
Pap pen	Merck	Z377821-1EA	Immunocytochemistry
Protein Block Serum free	Dako	X0909	Immunocytochemistry
Syringes (5 mL, 10 mL)	Henry Schein	900-3311, 900-3304	Aspiration technique