Читать книгу Equine Reproductive Procedures - Группа авторов - Страница 51

Оглавление

18 Uterine Culture/Cytology: Low Volume Lavage

John J. Dascanio

School of Veterinary Medicine, Texas Tech University, USA

Introduction

Typically a double‐guarded swab or a brush is used to obtain a sample for uterine culture or cytology. However, a low volume uterine lavage may be more diagnostic in situations where localized uterine infections are present or if an infection is suspected and no microbial growth was obtained after collection of a uterine sample with a guarded swab. The goal is to have the lavage fluid distribute throughout the uterine lumen and therefore “sample” the entire uterus instead of one specific region as would occur with a traditional swab. The negative aspects of performing a low volume uterine lavage are the extra time required to perform the procedure, the possibility of contamination since the tubing is not guarded, the need to have sterilized lavage tubing, and the centrifugation step used to concentrate the cellular material in the effluent.

Equipment and Supplies

Tail wrap, tail rope, non‐irritant soap, roll cotton, stainless steel bucket, disposable liner for bucket, paper towels, obstetrical sleeve, obstetrical lubricant, examination gloves, sterile lubricant, sterile obstetrical sleeve, sterile 80 cm (30 inch) uterine lavage catheter, sterile single‐line tubing, 50 ml centrifuge tubes, 150 or 250 ml sterile 0.9% sodium chloride or phosphate‐buffered saline, catheter tipped 60 ml syringe, 60 ml syringe, microscope slides, Diff‐Quik® stain, microscope, centrifuge, oxytocin, needles and syringes.

Technique

Remove feces from the rectum.

Place a tail wrap and tie the tail out of the way (see Chapter 4).

Clean and dry the perineum of the mare (see Chapter 3).

Don a sterile obstetrical sleeve.

Place the end of an 80 cm uterine lavage catheter with a deflated 75 ml cuff into the palm of the hand.

A 150–250 ml bag of 0.9% sodium chloride can be attached directly to the catheter and the catheter is filled prior to placement. This prevents air influx into the uterus and allows for better recovery of the lavage fluid. Alternatively, sterile, single‐line tubing can be used to connect the bag of saline to the lavage catheter (Figure 18.1).

Place sterile lubricant on the back of the gloved hand, being careful not to get lubricant onto the palm. If the end of the lavage tubing becomes inundated with lubricant, the lubricant may get into the sample thus interfering with interpretation of the cells.

Rub lubricant from the knuckles on to the vulva, straighten the fingers and insert through the vulva, being careful to not rub the clitoris as this structure has a significant natural bacterial flora.Figure 18.1 Low volume lavage supplies, including a uterine catheter, single‐line tubing, and 150 ml bag of sterile saline.

Using a slight rotating motion, pass the hand into the vagina so that the mid‐forearm is to about the level of the vulva. This should enable palpation of the external cervical os.

Gently insert the index finger into the external cervical os. Sometimes the os may be off‐center, located slightly downward, or to the left or right of center.

Pass the index finger through the cervix to the last knuckle (metacarpo‐phalangeal joint). Usually there is a feeling of entering the uterine body lumen when the tip of the finger exits the internal cervical os.

Pass the uterine lavage catheter past the inserted index finger, past the internal cervical os, and into the caudal uterine body. Sometimes, if the cervical canal is under the influence of progesterone and is toned, the index finger may need to be removed prior to passing the lavage catheter. Typically, the catheter would be pointed in a slightly downward direction when being passed through the cervix due to the dependent nature of the suspended uterus within the abdomen. The cuff on the uterine catheter should be inflated with approximately 75 ml of air to prevent reflux of fluid into the vagina. The cuff should be gently snugged against the internal cervical os.Figure 18.2 Infusion of sterile saline into the uterus by gravity flow for low volume lavage.

The 150–250 ml bag of 0.9% sodium chloride or other sterile fluid is elevated to allow the fluid to flow by gravity into the uterus (Figure 18.2).

The fluid is allowed to stay in the uterus for approximately 3 minutes to allow for distribution of fluid throughout the lumen.

In addition, the uterus may be gently massaged per rectum to ensure distribution of the fluid. Care should be taken to not irritate the endometrium with the lavage catheter.

The empty bag is subsequently lowered to allow a return of fluid via gravity flow (Figure 18.3). Gentle lifting of the uterus per rectum may increase return of fluid. If the fluid does not flow readily through the tubing, gentle aspiration with a catheter tipped 60 ml syringe may be used to initiate flow.

A single 20 IU dose of oxytocin may be administered intravenously to stimulate uterine contractions and augment return of the uterine fluid.

Once adequate fluid has been obtained, the clamp on the single‐line tubing is closed or the catheter is disconnected to make sure that there is no contamination of the recovered fluid with bacteria from the vaginal vault or caudal reproductive tract.Figure 18.3 Recovery of fluid from the uterus during a low volume lavage procedure.

The cuff is subsequently deflated and the catheter removed from the reproductive tract.

An alternative method involves passage of an insemination pipette into the uterus and the infusion of 60 ml of 0.9% sodium chloride via a syringe. The uterus is massaged to distribute the fluid and then the fluid is aspirated back into the syringe. Fluid recovery may be more difficult with this technique.

Recovered fluid is placed into one or two 50 ml centrifuge tube(s) and centrifuged at 400× g for 10 minutes. Alternatively, the cellular material may be allowed to settle by gravity for a few hours.

All but 5 ml of supernatant is removed and the pellet is re‐suspended in the 5 ml of lavage fluid.

Sterile culture swabs may be placed into the re‐suspended lavage fluid to obtain samples for cytology and culture. The culture swab is transferred to a transport media and the cytology swab is gently rolled across a microscope slide. The slide is air‐dried prior to staining with a modified Wright’s stain such as Diff‐Quik®.

An additional sample may be obtained from the centrifuge tube for polymerase chain reaction (PCR) analysis for bacterial or fungal DNA.

Interpretation

It is important that sufficient fluid is recovered from the uterus to be of diagnostic use.

Enough fluid should pass through the lavage tubing so that assessment of the uterine environment is performed. Care should be taken to not just pass fluid into and out of the catheter without actually contacting the uterus.

Note any discharge present on the sterile sleeve when the gloved arm is pulled from the vagina. There should not be any purulent or hemorrhagic discharge, only the lubricant and clear mucous should be present.

The clarity and presence of debris and mucus should be noted in the effluent fluid recovered from the uterus. Typically, the recovered fluid is graded as clear, cloudy, or cloudy with mucus.

Microscopic examination is performed at 400× (10× eye piece and 40× objective) magnification or under oil immersion at 1,000× (10× eye piece and 100× objective).

Evaluate slide quality (e.g., the presence of normal uterine epithelial cells) as well as the presence and relative number of inflammatory cells (neutrophils, macrophages, lymphocytes, etc.) and any bacterial or fungal organisms.

The amount of background debris and sediment appearing on stained slides from a low volume lavage is often greater than with swab or brush techniques. The amount of debris may be correlated with the degree of inflammation.

The presence of >5–10 neutrophils per high power field is an indication of inflammation, providing that an adequate cellular sample was obtained.

The greater the ratio of neutrophils : uterine epithelial cells, the greater the degree of inflammation or endometritis.

Neutrophils are the primary white blood cells present with acute inflammation. Macrophages, lymphocytes, and plasma cells may be noted with chronic inflammation. Eosinophils may be noted in cases of fungal endometritis, urine pooling, or aspiration of air into the uterus.

The absence of white blood cells (with normal endometrial cells present) often suggests that there is no inflammation present in the uterine lumen. However, some bacteria such as Escherichia coli and Pseudomonas aeruginosa may not stimulate a large inflammatory response and a “negative cytology” (i.e., the absence of white blood cells) may provide incorrect evidence of a “clean” or non‐infected uterus. Consequently, it is recommended that both culture and cytology samples be collected and evaluated and not just one or the other.

Please see Chapter 17 for further description of cell types found on cytology.