cytology in food animal practice

Vet Clin Food Anim 23 (2007) 443–479

Cytology in Food Animal Practice

Andrea A. Bohn, DVM, PhDa,*,Robert J. Callan, DVM, MS, PhDb

aDepartment of Microbiology, Immunology, and Pathology, College of Veterinary

Medicine and Biomedical Sciences, Colorado State University, Fort Collins,

CO 80523–1619, USAbDepartment of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences,

Colorado State University, Fort Collins, CO 80523, USA

Cytology is an often overlooked diagnostic modality in food animal med-icine, even though it can be relatively easy to perform and can provide im-portant diagnostic information in a timely manner. The reasons for this arevaried but often revolve around unfamiliarity with the indications, proce-dures, methods of evaluation, or criteria for interpretation. Cytologic eval-uation is useful in detecting abnormalities in body cavity fluids and tissueaspirates. Detecting a septic or nonseptic inflammatory process and differen-tiating between an inflammatory versus neoplastic process may be two of themore commonly used and clinically significant indications for cytologic eval-uation. The information gained from cytologic evaluation may help to directfurther diagnostic testing, determine the best treatment option, or determinethe prognosis for an animal.

The premise of cytologic evaluation in clinical practice is that diseased tis-sues respond to injury and inflammation in a predictable manner and thatthis is represented by the fluid and cytologic characteristics of the tissue.Therefore, one should have a basic understanding of what is normal andwhat types of cellular responses can be seen within a particular tissue beforeinterpreting a sample. Interpretation of many cytologic specimens isstraightforward and can be made in practice; there also are many veterinarydiagnostic laboratories to which samples can be sent that employ specialistswith extensive training in cytopathology. Regardless of who evaluates thesample, the best way to obtain useful diagnostic information from an aspi-rate is to make sure that a quality representative sample is obtained and thatit is handled appropriately. This article discusses some of the more common

* Corresponding author.

E-mail address: [email protected] (A.A. Bohn).

0749-0720/07/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.cvfa.2007.07.007 vetfood.theclinics.com

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tissues evaluated by cytologic evaluation, with discussion on collection tech-niques, sample handling, and sample evaluation and interpretation.

General recommendations for cytologic evaluation

Submission information

If sending to an outside laboratory, do not hesitate to contact the labo-ratory for information on obtaining a sample or proper handling methods.To make the best interpretation of a sample, the cytologist needs to knowthe animal’s signalment as well as pertinent history and clinical findings. Al-ways include details about the site of collection, physical characteristics ofa sampled lesion, and specifics of collection events if they may affect inter-pretation (eg, blood contamination of a fluid, quantity/flow rate of a fluid).

Air-dry

Allow cytology slides to fully dry while exposed to room air. No otherfixation is necessary before staining the slides and may actually ruin the sam-ple. Exposing the slides to formalin fumes can also be detrimental to thequality of the sample. If sending samples to an outside laboratory, it isbest to ship cytology slides separate from biopsy samples or at least toseal them separately in an airtight bag or container. Slides should be storedat room temperature in a protective container that protects from moisture,scratches, and breaking.

Staining

Romanowsky-type stains are typically used (eg, Wright’s, Giemsa, Diff-Quick). Stain solutions should be changed regularly and whenever obviouscontamination occurs.

Even if sending your sample to an outside laboratory, it is a good idea tostain one slide (choose the worst first rather than the best) to evaluatewhether you have obtained a good sample. Make sure that cells are presentand that they are intact and spread adequately. Cytopathologists prefer touse their own stain; thus, if there is a possibility that a sample may be sub-mitted to an outside laboratory, it is recommended to leave some slidesunstained.

Fluids

Collect fluid in an ethylenediaminetetraacetic acid (EDTA) blood tubefor cytologic evaluation. EDTA keeps the sample from clotting, helps topreserve the cells, and is bacteriostatic. Do not underfill the tube, becauseEDTA can affect protein determination if using a refractometer (fill tubeat least one quarter full, or shake out excess EDTA from the tube). Because

445CYTOLOGY IN FOOD ANIMAL PRACTICE

EDTA is bacteriostatic, a separate sample needs to be collected using a se-rum tube, other sterile vial, or culture swab if the sample may be cultured. Itis best to store and ship fluid samples in a cold environment, being carefulnot to freeze the sample.

Make fresh slides at the time of sampling if there might be any delay(O1 hour) in processing the sample. If fluid is not processed quickly, cellscan deteriorate over time. If the sample gets exposed to extreme heat orcold, it may become nondiagnostic. Fresh slides are useful in helping to de-termine if there is contamination from blood or bacteria (eg, inadvertent en-terocentesis during paracentesis, oropharyngeal contamination of an airwaywashing) or if these substances are present as the result of a pathologic pro-cess. Cells phagocytize contaminants while in the collection tube; thus, con-tamination can appear to be a pathologic process if sample processing isdelayed. With bacterial contamination of a fluid sample, fresh slides shouldonly show extracellular organisms and neutrophils should appear nondegen-erate. If slides are made from an aged contaminated fluid, bacteria may beseen intracellularly and the cells may appear degenerate, which are indica-tions of an infectious process. If sending to an outside laboratory, alwayssend some freshly made slides with the tube of fluid.

Detecting bacteria

It is easiest to find bacteria in cytologic samples stained with Romanow-sky stains, but these stains do not differentiate between gram-negative andgram-positive bacteria. It is therefore useful also to have slides availablefor Gram staining.

Samples for aerobic bacterial culture may be placed in a sterile tube ormay be kept in a capped syringe. The preferred method for submitting fluidsamples for anaerobic culture is to apply the sample to a sterile swab andplace in Port-A-Cul (Becton, Dickinson and Company, Franklin Lakes,New Jersey) transport medium. Alternatively, samples can be placed ina capped syringe with all the air removed. In most cases, it is best to refrig-erate samples until submission. Timely submission of culture samples is crit-ical for obtaining accurate bacterial culture results.

Body cavity effusions

There is normally a small amount of transudative fluid (plasma dialysate)present in the peritoneal, pleural, and pericardial spaces. If the amount offluid increases within any of these cavities, it is considered an effusion. Col-lection and analysis of an effusion can help to determine what is causing thefluid accumulation. There are three general classifications of effusions basedon cell numbers and protein concentration. A transudate (or ‘‘pure’’ transu-date) is characterized as a fluid with a low protein concentration and cellcount. Normal body cavity fluid falls into this category, and it is important

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for the evaluator to know if excess fluid was present in the body cavity forthe interpretation of a transudate. Modified transudates typically have in-creased protein concentration but normal or only mildly elevated cellcounts. Exudates are effusions with an increased protein concentrationand cell count. It is important to remember that classification guidelinesare not absolute and that fluid analysis must include cytologic examination.There can be some overlap in the numbers of cells or protein content be-tween the different classifications, depending on disease processes, cell integ-rity, and timing of sample collection. Some specific types of effusions (eg,chylous, neoplastic) can have variable cell numbers and may fall into themodified transudate or exudate classification.

Classification of a fluid and an understanding of underlying mechanismsthat can result in that fluid can help to establish a list of differentials. Theamount of fluid in a body cavity depends on a balance between the amountof fluid that transudes out of arterial capillaries and the amount reabsorbedby venous capillaries and lymphatics. The amount of fluid that transudesout of bloodvessels depends onabalance betweenoncotic pressure andhydro-static pressure within vessels and tissues as well as on the integrity of vascularwalls. Oncotic pressure, predominantly related to albumin concentration, actsto hold fluid within the vasculature, whereas hydrostatic pressure acts to pushfluid out. Therefore, alterations in lymphatic drainage, vascular integrity, on-cotic pressure, or hydrostatic pressure can result in fluid accumulation withinbody cavities. A few specific types of effusions (eg, uroperitoneum, some neo-plasias) may have alternative underlying mechanisms that result in fluidaccumulation.

A change in oncotic pressure is most commonly attributable to a decreasein blood albumin concentration. If the decrease in oncotic pressure is severeenough, a watery fluid with few cells and little protein can leak from bloodvessels and accumulate in body cavities. Therefore, if a transudative effusionis present, serum albumin concentrations should be evaluated. Transudatescan also occur secondary to changes in hydrostatic pressure, although mod-ified transudates are more commonly associated with this mechanism.

Transudates, modified transudates, and chylous effusions occur second-ary to changes in the hydrostatic pressure of vessels or to reduced lymphaticdrainage. Hydrostatic pressure and lymphatic drainage can be affected bycardiovascular disease as well as by functional or physical obstruction ofvessels. Functional obstruction can occur with liver diseases that cause por-tal hypertension. Physical obstruction has many potential causes. Any masslesion that impinges on vasculature can result in an effusion. Hepatic ab-scesses or caudal vena caval syndrome can cause portal hypertension andabdominal effusion. Neoplasia (neoplastic cells do not need to exfoliate;a modified transudate is often all that is present) and cystic or walled-off in-flammatory processes can also result in a nonexudative effusion. Organ dis-placements can affect vasculature and result in modified transudates orchylous effusions, including lung torsion and gastrointestinal displacements.


Exudates, with increased cell number and protein content, are typicallyattributable to inflammatory processes. Injured or inflamed tissues respondwith a common cascade of inflammatory mediators, including a diverse col-lection of cytokines and prostaglandins. These mediators direct the recruit-ment of inflammatory cells into the affected region as well as initiatevascular changes resulting in altered blood flow and vascular leakage. Thepercentage of neutrophils is increased in an inflammatory exudate. It isimportant to evaluate cell morphology in these samples, because neoplasticeffusions can have similar cell counts and protein contents. It is also impor-tant to examine the sample for the presence of degenerative changes inneutrophils and infectious organisms. Degenerative neutrophils and intra-cellular and extracellular bacteria are indicative of a septic process. If micro-organisms are not seen, culture may be necessary to confirm a nonsepticexudate.

Collection of body cavity effusions

Abdominocentesis
Collection of abdominal fluid for analysis is a useful procedure in assess-
ing abdominal disease in ruminants. It aids in the diagnosis and differenti-ation of ascites, peritonitis, intestinal crisis, enteritis, uroperitoneum, andabdominal neoplasia (eg, lymphosarcoma). There are two common sitesfor collecting abdominal fluid: the cranial abdomen and the caudal flank.Cranial abdominal abdominocentesis is performed at a site approximately10 cm cranial to the umbilicus and 10 cm to the right of midline (Fig. 1).The area is clipped and prepared with surgical scrub. Lidocaine is locallyinfiltrated beneath the skin and into the deeper muscles. A stab incision is

Fig. 1. Cranial abdominal site for abdominocentesis in cattle is located at the most dependent

portion of the abdomen approximately 10 cm cranial to the umbilicus and 10 cm to the right of

midline. Samples are generally collected with a teat canula, although they can also be collected

with a needle.

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made through the skin and into the muscle layers, feeling the tougher fascialplanes as you penetrate them. Typically, a 3-in metal teat canula is then in-serted through the incision and into the abdomen. In many cases, fluid is notobtained with a teat canula in normal animals. Sometimes, use of an 8- to10-in bitch catheter allows access to distant pockets of fluid for collection.Alternatively, a sleeved trocar can be inserted into the abdomen. After re-moving the trocar, a flexible feeding tube can be placed through the sleeveand into the abdomen. Fluid flows out through the feeding tube or comesout through the sleeve using the feeding tube as a wick. Samples are col-lected directly into purple-top and red-top tubes.

Abomasocentesis or rumenocentesis sometimes occurs inadvertently. Incattle, these generally do not cause significant complications for the animalbut can make interpretation of the sample more problematic. Abomasalcontents can readily be distinguished by measuring the pH of the fluid. Ab-omasal fluid has a pH less than 4, whereas abdominal fluid has a pH near 7.Rumen fluid can be identified by observing protozoa in the sample. Amnio-centesis or allantocentesis can occur in late-term pregnancy but is consideredto have a low risk for pregnancy complications.

The alternative site for abdominocentesis is the caudal flank (Fig. 2). Theprocedure can be performed on the left or right side, but the right side isgenerally used. The caudal flank is clipped just above the udder and cranialventral to the stifle. Generally, no local anesthesia is used. The sample is col-lected by inserting 18-gauge 1.5-in needles through the skin and body wallinto the abdomen. Often, several needles allow fluid to be collected morereadily. Samples are collected directly into purple-top and red-top tubes.Enterocentesis of the small intestine can occur with this technique andcan make interpretation of the fluid more difficult. Amniocentesis or allan-tocentesis can also occur from this site during the second and third trimes-ters of gestation.

Fig. 2. (A) Caudal flank site is located dorsal to the udder and cranial ventral to the stifle, as

depicted by arrows. The sample is collected using 18-gauge 1.5-in needles inserted perpendicular

to the body wall. (B) Multiple needles may improve success in collection of the sample.


Thoracocentesis
Thoracocentesis should be performed any time breath sounds are atten-
uated in the ventral thorax or when pleural effusion is demonstrated by tho-racic radiographs or ultrasound. Thoracocentesis is a diagnostic andtherapeutic procedure and is most commonly used in cases of septic pleuritisattributable to pleuropneumonia or to traumatic reticulopleuritis. Thoraco-centesis is performed in a manner similar to abdominocentesis. A suitablearea over the ventral sixth or seventh intercostal space is clipped and pre-pared with surgical scrub. Staying caudal to the sixth rib minimizes therisk for contacting the heart when performing the procedure. The site forthoracocentesis is best determined by ultrasound. Otherwise, choose a sitein the sixth or seventh intercostal space, dorsal to the olecranon and ventralto the line of dullness observed on thoracic auscultation and percussion(Fig. 3). The area is infiltrated with lidocaine, and a stab incision is madethrough the skin on the cranial aspect of the seventh or eighth rib. A 3-inteat canula is inserted through the incision and into the thorax. A pain re-sponse is often elicited on piercing the pleura. Generally, fluid flows freelyfrom the canula and can be collected into purple-top and red-top tubes.An extension set can be attached to the canula, and the fluid can be drainedby gravity flow or by attaching a three-way stopcock and a 60-mL syringe.Drainage of the fluid often improves the animal’s condition and allows timeto institute other appropriate treatment.

Pericardiocentesis

Pericardiocentesis is used to collect pericardial fluid in conditions inwhich pericardial effusion is suspected, such as traumatic reticulopericarditisor cardiac lymphosarcoma. Clinical signs that are suggestive of pericardial

Fig. 3. Thoracocentesis may be performed on the left or right thorax at the ventral third of the

sixth or seventh intercostal space. The sample can be collected with a teat canula, an 18-gauge

1.5- to 2-in needle, or a spinal needle.

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effusion include tachycardia, distended jugular veins, brisket or ventral ab-dominal edema, muffled heart sound, ‘‘washing machine–like’’ cardiac mur-mur, or evidence of thoracic pain. Radiographs or cardiac ultrasound canconfirm the presence of pericardial effusion.

Pericardiocentesis is performed at the left fifth intercostal space, 2 to 20cm dorsal to the olecranon (Fig. 4). The area is clipped and prepared withsurgical scrub. Lidocaine is infiltrated into the area to the level of the pleura.A stab incision is made with a no. 15 scalpel blade through the skin and deepinto the muscle just cranial to the sixth rib. A 3-in teat canula or a 16- or 18-gauge 3.5- to 5-in spinal needle is carefully inserted through the incision andinto the pericardial space. Occasionally, the teat canula may not be able toenter the pericardial space, because the pericardial membrane is often thick-ened and tough. In these cases, a spinal needle should be used. Care must betaken when using a spinal needle, however, because it is easier to contact theventricle inadvertently and initiate an arrhythmia or to enter the left ventri-cle and cause hemopericardium. Once in the pericardial space, fluid shouldfreely flow from the canula or needle. The sample may be drained by gravityflow, or an extension set and syringe may be connected to aspirate the fluid.Potential complications include acute cardiac arrhythmia or hemopericar-dium, both of which can result in sudden death. Relief of pericardial effu-sion often dramatically improves the cardiac function of the patient,however, and allows time for additional treatment.

The two most common pericardial diseases are pericarditis associatedwith traumatic reticulopericarditis and cardiac lymphosarcoma. In bothcases, these patients are often severely compromised because of cardiac tam-ponade and the resulting cardiac failure. Removal of pericardial effusion of-ten dramatically improves stroke volume and cardiac function. Althoughthe long-term prognosis for these animals is poor, they can occasionally

Fig. 4. Pericardiocentesis is most often performed at the ventral third of the left fifth intercostal

space (arrow). The sample can be collected similar to thoracocentesis.


be maintained for weeks to months to salvage a valuable late-termpregnancy.

Evaluation of body cavity effusions

Gross observations and the determination of protein content and nucle-ated cell count are essential components of fluid analysis.

Gross observations
A fluid sample should first be evaluated for gross findings: the quantity of
fluid obtained and how easily it was collected, the color and transparency ofthe fluid, and the presence of a strong or unusual odor. Normal fluid shouldbe colorless to yellow and clear to slightly hazy. Normal bovine peritonealfluid can clot; clotting of the sample does not correlate well with total pro-tein or fibrinogen and should not be used as a criterion for assessing thefluid.

Cellularity
Nucleated cell counts can be performed using an automatic cell counter
or manually using a hemocytometer. Unopette systems (Becton, Dickinsonand Company) can be used for cell counts. If neither of these methods isavailable, cell counts can be estimated during cytologic evaluation of a directfluid preparation. A definitive upper limit of normal cell numbers in bovineabdominal fluid has not been established. Many clinicians consider less than5000 cells/mL as normal [1,2]. Others consider less than 10,000 cells/mL asnormal [3,4]. A few studies have reported cell counts greater than 10,000cells/mL in apparently healthy cattle [5–7]. It must be considered that occultlesions were likely present in these animals. One study reports that a nucle-ated cell count greater than 6000 cells/mL and protein concentration greaterthan 3 g/dL were almost always associated with peritonitis; evaluation offluid from healthy cows was not included as a control in this study [8]. An-other study reported significant overlap between cows with peritonitis andapparently healthy cows at greater than 6000 cells/mL and greater than3 g/dL of protein [7]. In general, a cell count greater than 10,000 cells/mLlikely indicates inflammation [1]. Cattle with peritonitis often do not havecell counts in peritoneal fluid as high as might be seen in other species.Also, because cattle are relatively proficient in walling off foci of inflamma-tion, it is possible that an obtained sample does not represent the primaryinflammatory site. Cellularity of abdominal fluid from apparently healthypregnant cows was lower than fluid from nonpregnant cows [7]. Cellularityof abdominal fluid from 8-week-old calves was higher than from adult cowsin the second trimester of pregnancy [9]. Interpretation of cell numbersneeds to be performed in conjunction with cytologic evaluation of the sam-ple and other laboratory data (eg, hemogram) as well as signalment, history,and clinical presentation of the animal.

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Cytologic evaluation
Fluid analysis should always include cytologic evaluation of the sample.
Direct films of the fluid can be made in a manner similar to making a bloodfilm. In addition to the direct smear, concentrated preparations are useful,especially if the nucleated cell count is not elevated. To make a sedimentpreparation, the sample can be centrifuged at a similar speed and time asa urine or blood sample. Most of the supernatant is removed from thespun sample, and the cell button is gently mixed with the remaining fluid.A drop of the sample is placed on a clean glass slide and is spread as thoughmaking a blood film, or a second slide can be placed flat on top of the dropbefore gently pulling the slides apart horizontally. If a centrifuge is not avail-able, cells can be concentrated on a slide by proceeding as if making a directpreparation; however, instead of sliding the spreader slide off the end of thebottom slide, it is stopped short of the end and lifted vertically. Nucleatedcells tend to be dragged with the leading edge of a smear; therefore, thelast part of the smear often has higher cellularity.

Slides should be evaluated for the types of cells present, the morphologyof those cells, and the presence of microorganisms or other objects not nor-mally seen in fluid samples (Fig. 5). The normal cell distribution within bodycavity fluids can vary; however, typically, there are roughly equal numbersof neutrophils and large mononuclear cells [4–7,9,10]. Lymphocytes com-prise less than 20% (usually !10%) of cells present. A few mast cells orplasma cells may be seen. A high proportion of eosinophils (O70%) canbe seen in the peritoneal fluid of apparently healthy cattle [4,5,7]. Eosino-phils are less commonly observed in thoracic fluid. Fluid accumulationfrom any cause can alter normal cell distribution; therefore, the percentageand absolute number of a particular cell type need to be taken into accountduring fluid analysis.

Fig. 5. Cells commonly seen during cytologic evaluation of abdominal cavity fluids include neu-

trophils (N), lymphocytes (L), large mononuclear (monocytoid) cells (M), and eosinophils (E)

as indicated by arrows (Wright-Giemsa, �100 objective).


Exudates are typically characterized by high numbers of neutrophils(Fig. 6). Increased numbers of macrophages are also often seen. In somecases of septic peritonitis, neutrophils deteriorate rapidly, such that a lowcell count is maintained. Cytologic evaluation of these samples reveals de-generative neutrophils and intracellular and extracellular bacteria (Fig. 7).After routine exploratory celiotomy, the abdominal fluid routinely shows in-flammatory exudates [5]. This makes it difficult to evaluate whether or notpostoperative sepsis has occurred. Generally, the postoperative abdominalfluid contains nondegenerative neutrophils unless there is bacterial contam-ination. The observation of intracellular and extracellular bacteria also indi-cates postoperative sepsis.

Shifts in proportions of neutrophils can occur in transudates, modifiedtransudates, and chylous effusions, but cell numbers should remain rela-tively low and the cells should appear nondegenerate. Chylous effusionsare characterized by an increased proportion of lymphocytes [11]. Lympho-cytes often comprise most of the cells present; however, over time, moderateincreases in proportions of neutrophils and macrophages can occur in a chy-lous effusion. High numbers of lymphocytes may also be seen with lympho-sarcoma (Fig. 8). Morphologic evaluation of cells is important in helping todifferentiate neoplasia from other processes.

Mesothelial cells are frequently present in body cavity fluid samples andcan be a diagnostic challenge, especially when reactive (Fig. 9). Reactive me-sothelial cells can acquire characteristics of malignant cells, but their num-bers and proportions should stay relatively low. Mesothelioma is rare buthas been reported in cattle [12]. Squamous cell carcinoma has also been de-tected in peritoneal fluid [8]. If poorly differentiated, carcinomas can be dif-ficult to differentiate from mesothelioma.

Fig. 6. Suppurative inflammatory response with a predominance of nondegenerate neutrophils

as indicated by arrows (N). A lymphocyte (L), large mononuclear cell (M), and small cluster of

reactive mesothelial cells (Meso) are also present as indicated by arrows (Wright-Giemsa, �100objective).

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If erythrocytes are present, differentiation between blood contaminationand previous hemorrhage may be necessary. Blood contamination may havebeen obvious at the time of fluid collection; however, if there is uncertaintyas to when the hemorrhage occurred, cytologic evaluation can help. Afterhemorrhage into a body cavity, platelets disappear fairly quickly and mac-rophages begin to ingest erythrocytes. Therefore, the presence of erythro-phagocytosis or hemosiderin-laden macrophages indicates previoushemorrhage. The presence of platelets without evidence of erythrophagocy-tosis indicates probable blood contamination, although recent hemorrhage(within a few hours) is also possible.

With accidental enterocentesis, a mixed population of bacteria is present(Fig. 10). Cells are not normally present unless there is mixing with

Fig. 7. Septic suppurative inflammation with degenerative neutrophils, extracellular bacteria,

and intracellular bacteria (Wright-Giemsa, �100 objective).

Fig. 8. Neoplastic effusion containing high numbers of large round cells with abnormal mor-

phology consistent with lymphosarcoma. Note the size of the large lymphoblasts compared

with the small mature lymphocytes (L) and neutrophils (N) as indicated by arrows (Wright-

Giemsa, �100 objective).


peritoneal fluid. If slides are made quickly, peritoneal fluid cells should ap-pear nondegenerate and should not contain bacteria if the bacteria are con-taminants. If bacteria are present because of gastrointestinal leakage orrupture, cells should appear degenerate and contain intracellular bacteria(see Fig. 7).

Fig. 9. Cluster of mesothelial cells in an inflammatory effusion. Mesothelial cells are commonly

seen in low numbers in body cavity effusions and should not be mistaken for neoplastic cells

(Wright-Giemsa, �100 objective).

Chemical analysis
Protein content of body cavity fluid is typically measured using a refrac-
tometer. Ideally, the measurement should be done on the supernatant of thefluid after centrifugation of the sample. Some authors consider normal pro-tein content in peritoneal fluid as less than 3 g/dL [1,4], although higher peri-toneal fluid protein contents have been reported from apparently healthy

Fig. 10. Accidental enterocentesis containing mixed bacteria and amorphous debris. Rare squa-

mous cells (arrows) are present, but there are no nucleated cells (Wright-Giemsa, �100objective).

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adult cattle (up to 4 g/dL [9], 4.6 g/dL [7] and 4.2 � 0.9 g/dL [5]) and calves(up to 3.3 g/dL, [10] 3.8 g/dL, [9] or 6.4 g/dL [6]). Protein concentration wasslightly lower in the abdominal fluid of pregnant cows compared withnonpregnant cows [7]. The abdominal fluid protein concentration in 1- to3-day-old calves was higher than at 2 to 4 weeks [6,10]. The protein contentof normal pleural fluid should be less than 2 g/dL [2] or less than 2.5 g/dL [1].

Creatinine measurement can be performed in abdominal fluid to diagnoseuroperitoneum. Serum creatinine needs to be measured concurrently, anda fluid creatinine–to–serum creatinine ratio greater than 2:1 indicates leak-age of urine into the abdominal cavity. This procedure is more commonlyperformed in small ruminants with urinary tract obstruction.

Airway washes

A tracheal wash aspirate is the single best way to determine the patho-logic character of respiratory disease and to determine a specific cause. Be-cause tracheal wash specimens are diluted to differing degrees, total cellcounts and protein measurements are not routinely performed as part ofthe analysis. Cytologic evaluation is used to detect the presence of possibleinflammatory processes.

Collection of airway washes

The transtracheal wash technique is a simple procedure that can be per-formed in the field with basic head restraint. To perform this procedure, theanimal is restrained manually (calves) or in a head-catch (adults). The ven-tral neck is clipped in the middle trachea region and prepared with a surgicalscrub. While palpating the trachea, a lidocaine bleb is placed beneath theskin and in the subcutaneous tissue over the trachea. A stab incision ismade with a no. 15 scalpel blade. A 10-gauge 2-in needle is placed throughthe tissue into the trachea. The tip of the needle is used to identify a spacebetween tracheal cartilage rings. Once in the lumen of the trachea, polypro-pylene tubing is inserted through the needle and into the trachea down toa level approximately 10 cm beyond the thoracic inlet. Commercialthrough-the-needle intravenous catheter kits can also be used to performa transtracheal wash. Twenty to thirty milliliters of sterile saline or lactatedRinger’s solution is infused into the trachea and then aspirated back into thesyringe. You typically recover only 5 to 10 mL of the solution. This can berepeated an additional two times if necessary without adverse effect. A por-tion of the sample can be transferred to an EDTA tube, and the remainingsample is kept in the syringe or transferred to a sterile tube. Tracheal washspecimens can also be collected by endoscopy by way of the biopsy port.

Bronchoalveolar lavage (BAL) is performed by passing a flexible 7- to15-mm tube intranasally and into the trachea. Extending the head andneck and passing the tube through the larynx at the time of inhalation


can aid in successful nasal-tracheal intubation. A reflex cough is often elicitedwhen the tube is in the trachea, and the tube can be felt to rattle in the tracheawhen the trachea is shaken. The tube is then passed down the trachea to thelungs, where it is lodged into a bronchus. The tube is held tightly in the bron-chus, or if the tube has an inflatable balloon, the balloon is inflated to occludethe bronchus. Lactated Ringer’s solution or normal saline is used to lavagethe terminal airways. For calves, approximately 30 mL of fluid is sufficient.For adult cattle up to 180 mL of fluid is used for the lavage. Once the fluidis instilled into the airway, it is immediately aspirated for sample collection.The recovered fluid may be pooled and submitted for evaluation.

Evaluation of airway washes

Slides should be made soon after collection of the sample. Especially withtracheal washes, cells are often in poor morphologic condition to start withbecause they have been in the airways for awhile. Cells continue to deterio-rate in the saline or other collection fluid. Also, there is often some degree ofcontamination from the oropharyngeal region in airway wash samples,which can result in bacterial overgrowth or misinterpretation as an infec-tious process if the cells are given time to phagocytize the organisms. Directpreparation of particulates floating in the fluid should be made. These par-ticulates typically are composed of mucus that has trapped cells. The mucushelps to preserve cell morphology. Slides can also be made from the fluidcomponent of the sample, ideally after centrifugation.

Normal tracheal wash samples should be of low cellularity. The predomi-nant inflammatory cell in a healthy animal is a macrophage. Airway epithelialcells are also typically present. These cells are cuboidal to columnar cells withsmall nuclei, often with intact eosinophilic cilia; they can become hyperplasticin the face of inflammation or irritation. A small amount of mucus (purple tomagenta-colored proteinaceousmaterial) can be seen in normal fluid, increas-ing with inflammation or decreased mucociliary clearance.

The average ratio between macrophages and neutrophils in BAL fluidfrom healthy calves is approximately 9:1 [13,14]. This ratio was nearly re-versed in healthy calves only 1 week of age [15]. Environmental conditions,feeding practices, and whether the sample is a tracheal wash versus a BALsample are likely to have some influence on the number of neutrophils andlymphocytes present in an airway wash sample. Lymphocytes comprise ap-proximately 10% of leukocytes in healthy adult cattle [16]. In general, neu-trophils should be less than 20% to 30% of the total white blood cells inairways of healthy animals. An elevated percentage of neutrophils is indic-ative of an inflammatory process (Fig. 11). This would suggest a septic ornonseptic (eg, toxic, immune-mediated, irritant, parasitic, hypersensitivity)process. Degenerative neutrophils are generally associated with septicprocesses. Increased numbers of macrophages are seen with chronicityand various disease processes.

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In the early stage of the bovine respiratory disease complex, multinucle-ated syncytial cells are frequently seen [17]. Macrophages containing eryth-rocytes or hemosiderin are indicative of previous hemorrhage into therespiratory tract, as can be seen associated with some pneumonic processes,neoplasia, or pulmonary thromboembolism. The presence of eosinophils(normally !1% of cells) suggests parasitic pneumonia (Dictyocaulusviviparous) or an immune-mediated process, such as allergic pneumonitis.Occasionally, lungworm ova or larvae can be seen, which are best detectedat low magnification because of their large size and few numbers.

Bacterial cultures should routinely be submitted for any inflammatorywash. Virus isolation may be performed on tracheal wash specimens, whichis a good method for the detection of bovine respiratory syncytial virus(BRSV). Plant material, squamous epithelial cells, pollen, rumen protozoa,and fungal elements are often observed in tracheal wash aspirates of cattle.These are often natural contaminants or contaminants that occur because ofaspiration of pharyngeal contents during the procedure. The presence ofsquamous epithelial cells typically indicates oropharyngeal contamination,which is commonly associated with accompanying extracellular bacteria(Fig. 12). It is important not to mistake contaminant bacteria for sepsis.Contaminating bacteria should be extracellular and are typically of mixedtype. Finding bacteria engulfed by neutrophils in a freshly made samplewould be indicative of a pathogenic infection (see Fig. 11). BAL samplesare not considered suitable for bacterial culture because of inherent contam-ination with nasal-pharyngeal bacterial flora.

Sheep seem to have a similar cell distribution in BAL fluid as cattle, al-though they may have more eosinophils. A study of 20 sheep revealed an

Fig. 11. Sedimentation preparation of transtracheal wash fluid, interpreted as septic suppura-

tive inflammation. The predominant cells present are neutrophils. Bacteria can be seen extracel-

lularly (small arrows) and intracellularly (fat arrows). Finding bacteria within neutrophils in

a freshly made smear indicates that the bacteria are pathogenic and not contaminants

(Wright-Giemsa, �100 objective).


average of 74% macrophages, 12% lymphocytes, 5% neutrophils, and 8%eosinophils [18].

Fig. 12. Transtracheal wash fluid sample. The cluster of squamous epithelial cells with associ-

ated bacteria is indicative of oropharyngeal contamination of the sample. Care must be taken

not to overinterpret the presence of bacteria when squamous cells are present and bacteria are

seen only extracellularly (Wright-Giemsa, �100 objective).

Cerebrospinal fluid analysis

Cerebrospinal fluid (CSF) analysis is used to evaluate neurologic condi-tions in cattle and small ruminants, including septic, toxic, parasitic, neo-plastic, and traumatic causes of neurologic disease. One of the morevaluable uses of CSF evaluation is to differentiate between inflammatoryversus metabolic disease processes [19–21]. CSF results often do not providea definitive diagnosis and must be interpreted with clinical information andother diagnostic tests.

Collection of cerebrospinal fluid

Lumbosacral CSF taps are more commonly performed because they canbe done on conscious animals with no or minimal chemical restraint. Theprocedure is most easily performed with the animal standing or in sternalrecumbency. It can be done in lateral recumbency as well, but assessing nee-dle positioning tends to be more difficult. The lumbosacral space is locatedon midline, just caudal to a line extending between the cranial aspects of thetuber coxae (Fig. 13). You can palpate a noticeable depression at the lum-bosacral space. An 18- or 20-gauge 3.5-in needle is used in calves and smallruminants, and an 18-gauge 5- or 7-in needle is used in adult cattle. The areais clipped and prepared with surgical scrub. The local site is anesthetizedwith lidocaine, and a stab incision is made with a no. 15 scalpel blade inthe middle or caudal third of the lumbosacral space. The spinal needle is

460 BOHN & CALLAN

positioned parallel to the longitudinal plane and tilted slightly caudally fromthe transverse plane. One hand holds the hub of the needle to steady the po-sition, and the other hand is used to insert the needle gently in 1-cm incre-ments near the body. This technique helps to minimize curving andmisdirection of the needle. As the needle progresses through the tissues,you should feel the tough supraspinal ligament and then the interspinal lig-ament before you reach the dura mater sheath. The animal typically flincheswhen the dura mater sheath is touched or pierced. At this time, the stylet isremoved and the hub of the needle is watched for free-flowing spinal fluid.Twisting or slight repositioning of the needle may be required to obtainfluid. It may be necessary to aspirate fluid with a syringe, because the sub-arachnoid pressure may not be sufficient to push fluid up the total length ofthe needle in animals that are standing or in sternal recumbency. Fluid maybe collected directly into sample tubes or into a syringe and then placed ina purple-top tube for cytologic examination and in red-top tubes for chem-ical analysis, immunologic testing, and culture. It is helpful to collect twored-top tubes so that one can be used for chemical analysis and immuno-logic testing while keeping one sterile for possible culture.

Fig. 13. Lumbosacral space is the location of choice for collecting CSF from food animals. A

noticeable depression can be palpated on midline, just caudal to a line extending between the

cranial aspects of the tuber coxae (arrow).

Sample handling of cerebrospinal fluid

Because of the low protein and cell counts of CSF fluid, these samples arebest analyzed at a medical laboratory. They should be refrigerated and sub-mitted within 1 hour of collection. If a veterinary laboratory is not nearby,the samples may be submitted to a human clinical pathology laboratory ata nearby hospital. Because of the low protein content of the sample, a delayin CSF analysis may result in rupture of the cells present, making accurateanalysis difficult. Studies of human CSF have shown rapid deterioration ofcells (within a few hours) in CSF held at room temperature [22,23]. If


a sample cannot be analyzed within 30 minutes, it should be refrigerated orplaced on ice until use. If there is to be a prolonged (O1 hour) delay in CSFanalysis, a portion of the sample can be mixed with serum to preserve cellintegrity. Adding autologous serum at a concentration of 11% (30 ml [se-rum] to 250 ml [CSF]) to CSF and keeping the samples at 4�C adequatelypreserved the morphology of canine CSF cells for 48 hours [24]. Withoutadded serum, most cells were unidentifiable, but total cell count was not af-fected when the sample was maintained at 4�C for 48 hours. If serum isadded to CSF for cell preservation, remember to save a quantity of unadul-terated CSF for submission to do a cell count, measure protein content, andperform other chemical analysis.

Cell counts can be done in practice with a standard duel-chambered he-mocytometer. Because of the low cellularity, no dilution of the sample isneeded. The sample is gently mixed, and both chambers are carefully loadedwith CSF. By lowering the condenser of the microscope, white and redblood cells can, with experience, be distinguished and individually countedin the same sample. White blood cells appear granular, have a slightly irreg-ular cytoplasmic border, and contain an indistinct nucleus, whereas redblood cells tend to be smaller, smoother, and possibly crenated and internalstructures, including a nucleus, are not present. The number of cells countedwithin the nine large squares of the hemocytometer is multiplied by 1.1 togive the number of cells per microliter in the sample. Ideally, two sides ofthe hemocytometer should be counted and the results averaged. Specialstaining techniques can be used to help differentiate white blood cellsfrom red blood cells [25–29].

For cytologic evaluation, direct preparations are not useful if the count isless than 500 cells/ml. Sedimentation with routine centrifugation techniquesis also typically unrewarding. Adding protein to the sample may help tomaintain cell integrity, settle the cells, and increase cellular adherence tothe slide. The primary method used to concentrate CSF cells at referral lab-oratories is cytocentrifugation, after adding a drop of 22% bovine albuminto the aliquoted sample before spinning. The cost of a cytocentrifuge pre-cludes purchasing this piece of equipment for most veterinary practices,but other techniques can be used. The most practical ‘‘in-house’’ methodof concentrating the sample in practice is gravitational sedimentation. Var-ious methods to perform this sedimentation have been described [27,28,30].A simple method of gravitational sedimentation for CSF fluid is shown inFig. 14. The needle end of a tuberculin syringe is cut off. Filter paper con-taining a hole the size of the syringe barrel opening is wrapped arounda glass slide. The plunger end of the syringe is placed over the hole in thefilter paper, and the lips of the syringe are clamped to the slide with binderclips, sandwiching the filter paper between. After CSF (1 mL) is loaded intothe syringe barrel, cells settle onto the glass slide as the filter paper absorbsthe fluid. After all the fluid is absorbed, the filter paper is carefully removedfrom the slide and the slide is allowed to air-dry.

462 BOHN & CALLAN

Evaluation of cerebrospinal fluid

Fig. 14. Preparation of a slide for cytologic evaluation of CSF using gravitational sedimenta-

tion. A tuberculin syringe barrel with the needle hub removed is clamped to a microscope slide

(small black arrows) that has been wrapped with filter paper containing a hole cut the diameter

of the syringe to allow fluid to contact the slide. One milliliter of CSF fluid is added to the

syringe (white arrow) and allowed to be absorbed by the filter paper (black arrowheads) while

the cells are sedimented onto the microscope slide.

Analysis of CSF should minimally include gross observations, total pro-tein concentration, total nucleated cell and erythrocyte counts, and cytologicexamination. Further testing can be performed to measure chemical constit-uents or protein fractions, to detect specific antibodies or specific organismsusing molecular techniques, or to culture infectious agents.

Normal CSF is colorless and clear. The sample can be held against a whitebackground to aid in detection of subtle color changes and against a paperwith black printing to aid in detection of turbidity of the sample. A reddishcolored sample may indicate more recent hemorrhage, reflecting hemor-rhage into the subarachnoid space or iatrogenic hemorrhage at the timeof sample collection, whereas a yellowish sample (xanthochromia) may in-dicate previous hemorrhage in the subarachnoid space or icterus. A cloudysample may indicate an increase in cell number or protein content. Centri-fugation of the sample with subsequent re-evaluation of the supernatantcan help to determine if gross abnormalities are attributable to intact cellsor to soluble compounds. Even a visually clear sample, however, may


contain increases in nucleated cells and protein. Therefore, a subjectivelyclear sample cannot automatically be regarded as normal.

Protein concentration is generally too low to be able to read using arefractometer. Most practices do not have the equipment necessary to per-form accurate protein quantification, but urine protein dipsticks can be usedas a screening tool while waiting for a more accurate result. Urine dipstickreadings of 2þ (corresponding to a protein concentration of 100 mg/dL)or greater consistently detected CSF samples with significant increases inprotein concentration [28,31]. Urine dipsticks are less reliable in differentiat-ing mildly increased and normal protein concentrations. For accurate totalprotein quantification, CSF should be sent to a reference laboratory.

Protein concentration of the CSF can be elevated for a variety of reasons,and although this is an indication of central nervous system (CNS) disease,it generally does not differentiate disease processes. The lack of an increasein CSF protein can also be useful clinical information. The site of collectioncan influence protein content; sheep with compressive spinal lesions hadhigher protein concentrations in lumbosacral CSF than cisternal CSF [32].Protein concentration can also be influenced by blood contamination. Ingeneral, it is thought that approximately 1 mg/dL of protein may be intro-duced with every 1000 erythrocytes/ml. No effect was seen on protein con-centrations in cattle with less than 2000 erythrocytes/ml of CSF [33].

The range of CSF protein concentration reported from 152 3-week-old to9-year-old cows is 2 to 46 mg/dL [34]. A range of 20 to 33 mg/dL is given formore than 250 normal CSF samples from cattle [35]. Protein concentrationsup to 66 mg/dL were reported in 16 clinically normal adult cows [33]. Inthis study, beef cows had higher protein concentrations than dairy cows(58–66 mg/dL versus 23–50 mg/dL); however, sample sizes were small,and this apparent difference has not been confirmed. CSF protein in 10goats was approximately 16 to 19 mg/dL [36]. Various sources of normalsheep CSF data were combined to report a range of 8 to 70 mg/dL of pro-tein, with average values of 22.5 to 42 mg/dL (from an unknown number ofanimals) [35].

Cell counts for normal ruminant CSF are variably reported, and, unfortu-nately, well-developed reference intervals have not been substantiated. Mostcattle have a count of less than 10 cells/mL, although slightly higher numbershave been seen in clinically normal animals [34].Data frommore than 250 nor-mal bovine CSF samples included a range of counts from 0 to 3 cells/mL; thissource also referenced another study reporting a range of 0 to 6 cells/mL [35].Ranges of normal CSF cellularity reported in sheep include a range of 0 to5 cells/mL, 1 to 12 cells/mL, and up to 20 cells/mL; the number of sheep usedfor these ranges was not given [35]. An increase in the number of cells inCSF is called pleocytosis. Some amount of blood contamination is a frequentoccurrence and may elevate the white blood cell count. Correction factors fordetermining the contribution of white blood cells from peripheral blood havebeen shown to be unreliable [37].

464 BOHN & CALLAN

Regardless of the total nucleated cell count, a cytologic examinationshould be performed for differential cell counts and examination of the sam-ple for the presence of atypical cells. Common cell types seen include redblood cells, lymphocytes, monocytes, and neutrophils. Lymphocytes arethe predominant cell in normal fluid. Few neutrophils should be seen, al-though blood contamination can introduce neutrophils into the sample,even if not influencing the cell count [38].

CSF creatine kinase measurements can also be performed and should be!20 IU/L. Elevated CSF creatine kinase is suspected to indicate axonaldamage, but the significance remains controversial. CK levels can also be in-creased with blood contamination.

The interpretation of CSF must be made relative to the signalment, his-tory, and clinical signs of the animal. Many viral, toxic, and metabolic CNSdiseases may show no abnormalities in the CSF. Alternatively, at more ad-vanced stages, they may show increased protein or increased cell counts.Typically, if the cell count is elevated, there is mononuclear pleocytosis. Alymphocytic pleocytosis is usually seen with viral infection. With acute sep-tic conditions, there is typically an increase in neutrophils. A neutrophilicpleocytosis is observed with bacterial meningitis, Haemophilus somnus(thrombotic meningoencephalitis [TME]), and CNS or vertebral body ab-scesses. The pleocytosis may become mononuclear with chronicity. Neutro-phils can also be seen in nonseptic inflammation associated with trauma.Listeriosis is often associated with a mononuclear pleocytosis in whichmost cells are macrophages [21], although a combination of macrophagesand lymphocytes or, in some cases, a specific increase in lymphocytes can beseen. A neutrophilic pleocytosis can be seen in sheep with listeriosis [39]. Eo-sinophils are not normally seen in CSF. The presence of eosinophils has beenassociatedwith aberrant parasitemigration, such aParalophostrongylus tenuis(the meningeal worm of white-tailed deer) in small ruminants and camelids[2,39]. Eosinophils may also be associated with other inflammatoryconditions.

Solid tissue cytology

Sample collection

Tissue aspirates are helpful in diagnosing and differentiating inflamma-tory, septic, and neoplastic conditions in tissues. As with any sample, it isimportant to obtain a representative sample of the pathologic process.This is best done by sampling several areas of the lesion. It is also importantto avoid ulcerative surfaces and necrotic areas. If a mass has fluid pockets,do not just collect the fluid but try to aspirate cells from the more solid areasof the mass as well.

Tissue aspirates can be performed using two different techniques. In thefirst technique, a needle is attached to a 10-mL syringe and inserted into the


tissue of interest. Gentle aspiration is applied with the syringe as the needleis repositioned into different areas. Needles ranging in size from 25 to 18gauge are typically used. Smaller gauge needles tend to result in less tissuebleeding and peripheral blood contamination of the sample; however, largerneedles may sometimes facilitate obtaining appropriate cytologic samples.Once the aspirate is obtained, the needle is separated from the syringeand the syringe is filled with air before reattaching it to the needle and ex-pressing the sample onto a glass slide. A second slide is laid on top of thesample, and the two slides are gently pulled apart horizontally to spreadthe sample. The slide is then allowed to air-dry for staining.

The second method for obtaining tissue aspirates is to use a larger sizeneedle (22–18 gauge) and repeatedly insert it into the tissue site (multiplequick jabs) without using a syringe to aspirate. This method works wellfor solid tissue tumors and organs and tends to cause less blood contamina-tion. The needle is then attached to a syringe filled with air, and the contentsof the needle are expressed onto a glass slide. A standard smear is made, air-dried, and stained.

Impression smears can be useful for rapid diagnosis of tissue masses. Afreshly cut surface of the tissue mass is first blotted onto a paper toweland then blotted directly onto the slide and allowed to air-dry. Some tissuesdo not exfoliate well, and it may be necessary to scrape the surface gentlywith a scalpel blade. The tissue is then blotted onto a slide, and the cellulardebris on the scalpel blade may be applied to another slide. Squash prepa-rations are also useful in evaluating biopsy tissues. A small piece of tissue isapplied to a slide, and a second slide is placed over the specimen at right an-gles. The two slides are pressed together to compress the tissue, and the topslide is moved off the end of the bottom slide.

The primary goal of in-house tissue aspirates is often to distinguish be-tween inflammatory and neoplastic processes. Inflammatory aspirates con-tain numerous inflammatory cells. These are typically neutrophilic;however, a preponderance of eosinophils is sometimes observed in nonspe-cific cellulitis in cattle. An inflammatory aspirate should be examined closelyfor the presence of intracellular and extracellular bacteria. If bacteria arepresent, it is advisable to perform a Gram stain and to submit an aspiratefor bacterial culture. Tissue aspirates are especially helpful in quickly diag-nosing or confirming clostridial cellulitis. The clostridial organisms tend tobe large, blocky, gram-positive rods that are present in fairly large numbers.It should be cautioned that the Clostridia sp obtained from many in vivosamples may not have the classic tennis racket or paperclip morphology as-sociated with spore formation. Mixed inflammation may be associated withfungal infections or foreign body reactions.

Tissue aspirates can also help to confirm a diagnosis of neoplasia in cattleand small ruminants. The differentiation of normal cells and neoplastic cells isoften more difficult than the identification of an inflammatory process. Thetwo most common tumors seen in ruminants are lymphosarcoma and

466 BOHN & CALLAN

squamous cell carcinoma. Aspirates of enlarged lymph nodes may be helpfulin confirming a diagnosis of lymphosarcoma. Fine-needle aspirates have beenshown to perform as well as or possibly better than core needle biopsy samplesto diagnose enzootic bovine lymphosarcoma in cattle [40]. Sensitivity was ap-proximately 50%, but reported specificity was 100% for aspirates. It is alsoprudent to confirm a positive bovine leukemia virus titer in cattle before estab-lishing a diagnosis of lymphosarcoma. Lymph node biopsy has been the pre-ferred method for diagnosing lymphosarcoma from enlarged lymph nodes;excisional or wedge biopsy is recommended so that adequate tissue is presentfor evaluation. Identifying pleomorphic lymphocytes from direct impressionsmears or aspirates of non-lymph node masses is highly diagnostic oflymphosarcoma.

It is difficult to get good needle aspirates of squamous cell carcinomas.Instead, a small biopsy sample may be obtained for an impression smear,or a scraping of the cut surface of the tumor may be used for cytology.These samples often show pleomorphic epithelial cells with an eosinophilickeratin background. Nuclear changes described in a study of ocular squa-mous cell carcinoma include condensation of nuclear chromatin, prominentnucleoli, anisonucleosis, bizarre mitoses, multinucleation, and increased nu-clear-to-cytoplasmic ratios [41]. Criteria of malignancy are common and aidin the differentiation from benign lesions; however, for well-differentiatedsquamous cell carcinomas, histopathologic examination is often requiredfor definitive diagnosis.

Liver

Liver aspirates can be used for diagnosing hepatic lipidosis [42]. Thegrade of hepatic lipidosis is based on the accumulation of fat vacuolesand the degree of nuclear distortion (Fig. 15) [42]. Excessive fatty vacuolesand the presence of many shrunken nuclei are consistent with severe hepaticlipidosis and carry a poor prognosis. Occasionally, neutrophils are also ob-served in the aspirates. They are suggestive of hepatic abscessation, perito-nitis, enteritis, or hepatitis. Needle aspiration has been used to confirm liverabscesses identified by ultrasound in several cows [43].

Sample collection of liver

An area is clipped at the eleventh intercostal space just above the middlethird of the rib and prepared with surgical scrub (Fig. 16). A 22-gauge 6-inneedle is passed through an 18-gauge guide needle just off the cranial edge ofthe twelfth rib and directed slightly cranially. Resistance is felt as the needleenters the liver. Aspiration is applied with a 10-mL syringe, and the needle ismoved back and forth within the liver. Suction is released before removingthe needle. The sample is then applied to a glass slide, smeared, and stained.Normal hepatocytes have a diameter approximately five to six times that ofa red blood cell and a nucleus about twice as large as a red blood cell.


Bone marrow aspiration

Indications

Bone marrow evaluation provides important diagnostic information onthe hematopoietic status of an animal. It is typically performed to evaluate

Fig. 15. Cytologic evaluation of liver samples showing various degrees of hepatic lipidosis. (A)

Normal hepatocytes with mild adipose accumulation (Diff-Quick, �100 objective). (B) Moder-

ate hepatic lipidosis (Diff-Quick, �100 objective). (C) Severe hepatic lipidosis with marked lipid

accumulation and small hepatocyte nuclei (Diff-Quick, �100 objective).

Fig. 16. Site for liver aspirate (x). The needle is inserted just cranial to the twelfth rib and above

the middle third of the rib, directed slightly cranially.

468 BOHN & CALLAN

hematopoiesis and to detect evidence of neoplastic or infectious disease [44].Unexplained nonregenerative anemia, neutropenia, thrombocytopenia, orpancytopenia or the presence of atypical cells, unexplained immature cells,or abnormal blood cell morphology on a peripheral blood film is an indica-tion for bone marrow aspiration. Other indications for bone marrow aspi-ration include the presence of lytic or proliferative bone lesions or ifa neoplastic or infectious process is clinically suspected but cannot be foundelsewhere. This suspicion may arise from detecting an unexplained hypercal-cemia or monoclonal gammopathy, which is often associated with neopla-sia; identifying a fever of unknown origin; or recognizing the probabilityof an infectious agent that may have bone marrow involvement.

In general, cytopenias should be persistent and confirmed before bonemarrow evaluation is performed. Rechecking a low cell count by drawinga new blood sample, especially if initial results do not fit with the clinicalpresentation of the animal, is recommended. Repeated complete bloodcell counts (CBCs) can also be used to assess persistence of an abnormality.It can take up to 5 days for the bone marrow to respond to acute anemiaand for regeneration to be evident in the peripheral blood.

Cytologic evaluation of a bone marrow aspirate is more commonly per-formed than core biopsy, because results can be attained quicker and themorphology of the cells is superior, allowing a more accurate assessmentof cell types. The advantage of core biopsy is that architecture can be eval-uated and it can provide a better assessment of bone marrow cellularity. As-sessing cellularity is especially important for hypocellular samples and forthe confirmation of myelofibrosis, generalized bone marrow suppression,or necrosis. When collecting a bone marrow sample for cytologic evaluation,some clinicians also collect a core biopsy sample to store in formalin in theevent that histologic evaluation is later recommended.

If a bone marrow sample is being submitted for evaluation, it is recom-mended to always submit a concurrent peripheral blood sample fora CBC, because interpretation of the bone marrow depends on CBC resultsand changes can occur quickly in the blood.

Location of collection

Hematopoietically active bone marrow is most consistently found in theflat bones (sternum, ribs, pelvis, and vertebrae) and proximal ends of longbones (humerus and femur). The ribs are one of the preferred sites forbone marrow aspirates in cattle. The dorsal ends of the eighth through elev-enth ribs can be accessed for sequential aspirates (Fig. 17) [45]. The vertebralend of the rib more consistently contains marrow than the sternal end of thebone (Fig. 18) [46]. Palpation can be used to find the rib that has the leastamount of fascia covering it. In cattle, place the needle approximately 3in ventral to the ninth or tenth costovertebral junction [47]. The needleshould be perpendicularly inserted at the middle of the rib, midway between


the anterior and posterior borders. In calves, sheep, and goats, the marrowcavity of the rib is small and more difficult to hit [48].

The ventral sternum is a preferred site in small ruminants and cattle (seeFig. 17). The advantages of the sternum are that it is covered by only a thinlayer of bone, it has areas not covered by thick muscles, and samples can be

Fig. 17. Recommended sites for bone marrow aspirates in cattle. Samples are collected from the

dorsal aspect of the eighth through eleventh ribs (numbers) or the ventral sternum (white arrow).

Fig. 18. Cross section of the tenth rib from a cow. A Rosenthal bone marrow needle is inserted

approximately 3 in below the costovertebral junction. Bone marrow is more consistently present

in the vertebral end of the rib.

470 BOHN & CALLAN

reliably obtained from the site. The disadvantages of the sternum are that itis near vital organs and the operator is in an awkward position when work-ing on a standing animal. Sternal bone marrow can be sampled from adultcattle standing in stocks or a squeeze chute. It may be necessary to use tech-niques to prevent kicking, such as jacking the tail, or sedation. Unruly adultcattle and large calves can be cast and held in lateral recumbency. The upperfront leg of the cast animal is held extended along the neck, exposing thesternum, after the other legs are secured with a rope [49]. Calves that weighless than 300 lb can be placed in dorsal recumbency and secured by assis-tants or with ropes [48]. The needle should be placed on the midline and in-serted perpendicular to the third or fourth sternebra. The appropriatesternebra is located by palpating the third or fourth rib and following itto the sternum.

To sample the sternum of small ruminants, the animals are placed in dor-sal recumbency with legs secured, or they can be restrained in a sitting po-sition with an assistant standing behind, supporting the animal and holdinga foreleg in each hand [48]. The location of the appropriate site is midline,between the front legs. In sheep, the second through fourth sternebrae canbe used (Fig. 19). A prominence may be felt between the first and secondsternebrae. The appropriate sternebrae can also be located by palpatingribs and following them to their articulations [50]. In sheep, the needle ad-vances approximately 0.5 cm into the bone (2.5–3.8 cm from the skin) beforeentering the marrow cavity [50].

When sampling the ventral sternum, the needle should be placed on themidline, as near to the center of the bone as possible, and advanced perpen-dicular to the bone. A sudden reduction of resistance may be felt when en-tering the marrow cavity, but because the sternal cortex is so thin, a changein resistance may not be felt. Care needs to be taken not to enter the thoraciccavity, and because a change in resistance may not be felt, once the tip of theneedle is firmly seated in the bone, an aspiration attempt should be made.

The iliac crest can also be used in sheep. The needle should be placedmidway between the two tuberosities of the tuber coxae. The angle of the

Fig. 19. Cross section of the sternum from a sheep. The needle is entering the second sternebra.

The second through fourth sternebrae are recommended as optimal sites for bone marrow col-

lection in the sheep.


needle should be pointing at the coxofemoral joint of the opposite side. Themain benefit of using this method is that there is no risk for inadvertentlyentering the thoracic cavity.

Preparation

Bone marrow aspiration should be performed as a sterile procedure. Af-ter the animal has been restrained and the site of approach identified, hairshould be clipped and the area surgically scrubbed (Fig. 20). Local anes-thetic is injected at the site where the needle is to be inserted, from theskin to the periosteum. A small stab incision is typically made with a scalpelto aid the needle’s approach to the bone. It is important to be organized andhave all supplies ready so that the procedure flows smoothly and the sampledoes not clot before it can be processed (Fig. 21).

To prevent the sample from clotting, the syringe that is to be used for as-pirating the sample can be primed with anticoagulant. One to two drops ofa 3% to 15% EDTA solution should be adequate. The EDTA solution canbe aspirated from a purple-topped blood tube. If the syringe is not primed,as soon as the sample is seen in the syringe, the syringe immediately needs tobe disconnected from the needle and the sample quickly processed before ithas a chance to clot.

To collect the sample, a bone marrow or spinal needle with a styletshould be used. Commonly used needles include Rosenthal, Illinois ster-nal-iliac, and Jamshidi bone marrow needles. Some needles come witha guard that can be adjusted to prevent deeper penetration than desired.The needle used should be 16 gauge or larger and at least 1.5 in long. Largerneedles may be needed, depending on site aspirated and age and size of theanimal.

After an animal has been properly prepared, the bone marrow needle isinserted through the skin, perpendicular to the bone. Once the needle

Fig. 20. Preparation for bone marrow aspirate of a cow. The area over the vertebral end of the

eleventh rib has been clipped and is surgically scrubbed.

472 BOHN & CALLAN

contacts bone, it can be advanced using manual pressure and a clockwise/counterclockwise twisting motion (Fig. 22). If going through dense bone,a wood mallet can also be used to advance the needle.

Once it is suspected that the needle has entered the marrow cavity by de-tection of a sudden reduction of resistance or by needle depth, the styletshould be removed and an aspiration attempted. In dense bone, pliersmay be helpful in removing the stylet [48]. After the stylet is removed,a 10- to 20-mL sterile syringe is securely attached to the needle and theplunger of the syringe is pulled back quickly and sharply, creating negativepressure to dislodge bone marrow particles. Suction can be repeated two tothree times. As soon as blood is seen in the hub of the syringe, suctionshould be discontinued, the needle and syringe removed from the animal,and the sample processed promptly. Additional suction is only likely to re-sult in hemodilution of the sample. The first drop of blood is the most cel-lular, and further aspiration results in lower cellularity of the sample [51].

If the initial aspiration does not obtain any material, remove the syringe,replace the stylet, and carefully advance the needle a little further. If no

Fig. 21. Supplies are organized and ready for the bone marrow aspiration procedure.

Fig. 22. Bone marrow aspirate of a cow. After a local anesthetic and stab incision, the Jam-

shidi-type bone marrow needle is forcibly pushed forward through cortical bone as it is rotated

in a clockwise/counterclockwise motion. The animal was placed in a head-gate and sedated, and

a tail-jack was applied.


sample is still obtained after additional advancement of the needle and re-peated suction, slowly withdraw the needle while applying suction to the sy-ringe. It may be necessary to move the needle to a different site to achievesuccessful aspiration.

Once a sample is obtained, processing needs to proceed rapidly to preventclotting or drying out of the sample. The sample may be expressed (1) di-rectly onto several clean glass slides, (2) into an EDTA blood tube, or (3)into a Petri dish or watch glass. The Petri dish or watch glass must containanticoagulant if no anticoagulant was used in the syringe. The anticoagulantcommonly used is EDTA, but other anticoagulants, such as sodium citrate,are also effective. Samples placed in an anticoagulant should be gentlymixed immediately.

When making slides, if the sample contains a lot of excess blood, the slidecan be briefly tilted on an angle so that the fluid portion can run off onto anabsorbent surface, leaving behind adherent particles for spreading. If thesample was placed into an EDTA tube or Petri dish with anticoagulant,spicules (small grayish particles) can be transferred out of the bloody sampleusing a pipette and placed on a glass slide.

To spread the sample on a slide, place a second glass slide flat on top ofthe sample, allowing the sample to form a thin layer between the slides(Fig. 23A). Without additional pressure other than the weight of the slide,gently pull the slides apart from one another horizontally and without ver-tical separation, which can create suction and rupture cells (see Fig. 23B).This typically results in a nicely spread sample with intact particles andnice monocellular layers for evaluation. Several slides should be made.

Some bone marrow slides should be stained with a Romanowsky-typestain (eg, Wright’s, Giemsa, Diff-Quick) for cytologic examination, and

Fig. 23. (A) To spread bone marrow particles on a slide, a second slide is placed flat on top of

the sample, causing the sample to spread out between the slides. (B) Slides are then carefully

pulled apart in a horizontal plane (arrows) and allowed to air-dry. This technique is useful

for many types of cytologic specimens.

474 BOHN & CALLAN

some should be left unstained in the event that special staining proceduresare wanted. It is highly recommended to stain at least one slide rightaway for microscopic examination to determine sample quality and tomake sure that bone marrow elements are present while the animal is stillavailable and prepared in the event that the first aspiration is nondiagnosticand additional attempts are needed. If a core biopsy sample is obtained, thetissue can be gently rolled onto a slide to make an impression smear for cy-tologic evaluation. The biopsy tissue should then be placed in 10% neutralbuffered formalin for preservation. A larger gauge needle (10–13 gauge) isgenerally recommended for core biopsy. The Jamshidi needle, designed sothat the distal tip tapers to help retain the sample, is the most popular needlefor biopsy. The needle and stylet are initially inserted as for aspiration. Oncethe needle enters the bone marrow, the stylet is removed and the needle isadvanced further with the same twisting motion. Ideally, the needle is in-serted at least an additional 1 to 2 cm, but this may be limited by location.After advancing the needle to obtain the core, it is rotated and rocked forc-ibly to help break the core at its base for successful removal. Once the needleis removed from the animal, a probe is inserted into its lumen at the distaltip, pushing the sample out the hub end.

Often, bone marrow aspirates are placed into an EDTA blood tube forsubmission to an outside laboratory. In this case, it is still recommendedto make slides of the sample to submit along with the fluid sample. If leftin the fluid phase, cells can deteriorate during transit, and it is always helpfulto the cytologist to have a freshly prepared sample to examine (of bone mar-row and peripheral blood). In addition, a slide can be stained and evaluatedfor diagnostic quality before shipping the sample.

If repeated aspirates all result in samples of low cellularity, core biopsymay be indicated. Cytologically, it is usually impossible to differentiate be-tween a sample that is low in cellularity because of a pathologic process orbecause of poor sampling; therefore, biopsy may be needed for the evalua-tion of cellularity.

Evaluation

Bone marrow evaluation requires expertise (advanced training and expe-rience); in most cases, it is anticipated that samples are going to be sent toa trained cytologist for evaluation. To obtain the most information froma bone marrow aspirate, it is important to have a good-quality sample. Re-cent CBC results and freshly made blood films are also essential for full in-terpretation of a bone marrow sample. Relative changes between differentcell lines are assessed in the evaluation of hematopoiesis, because absolutecell counts are unreliable in bone marrow aspirates [51]. Knowledge of theperipheral blood picture is necessary to assess whether changes (or lack ofchanges) in the bone marrow are consistent with normal hematopoiesis.Freshly made blood films are important to have when comparing the


morphology of cells in the bone marrow with those in the peripheral blood.Results from cytologic evaluation of bone marrow should ultimately be cor-related with history, clinical presentation, and other laboratory data.

Cytologic examination
In general, the evaluation of bone marrow includes assessment of the fol-
lowing parameters. On low magnification, the cellularity and quality of thesample as a whole are assessed as well as the cellular density of bone marrowparticles. Iron stores are evaluated within the particles. Megakaryocytenumbers are also best assessed at low magnification. On high magnification,the sample is evaluated for the presence of myeloid and erythroid cell lines.The cell lines are evaluated for orderly maturation, and cell morphology isassessed for evidence of dysplastic changes. The relative proportion of my-eloid to erythroid cells is determined by subjective assessment or by countingcells to derive a myeloid to erythroid (M/E) ratio. The sample is also eval-uated for the presence of other cell types or infectious organisms. Resultsfrom the bone marrow evaluation are then interpreted in accordance withperipheral blood abnormalities.

Cellularity of bone marrow spicules is subjectively determined by assess-ing how much area of a particle is composed of cells versus fat. Cellularity ofnormal marrow can vary according to age of an animal, with higher cellu-larity in younger animals and lower cellularity in older animals. When as-sessing maturation of bone marrow cell lines, the number of cells in eachearly maturation stage increases in somewhat of a pyramid pattern untilpolychromatophilic rubricytes and metamyelocytes; at that point, thereare relatively stable numbers of the subsequent maturation phases. In gen-eral, for each blast, there are 16 mature granulocytes or erythrocytes. Mi-totic figures are normally present in low numbers.

The M/E ratio is used to help determine if the erythrocyte or myeloid cellline is hypoplastic or hyperplastic. It is most accurately determined bycounting a minimum of 500 cells from several different areas of the sampleto make it as representative of the whole sample as possible. Nucleated cellsof all maturation stages are included in the count and categorized as myeloidor erythroid. The ratio is calculated by simply dividing the total number ofmyeloid cells by the total number of erythroid cells. In some sources, the M/E ratio in cattle is listed as less than 1.0, although ranges from 0.27 to 2.59have been reported [45,46,52–54]. The range of M/E ratios in 10 3- to6-year-old pregnant sheep was reported as 0.77 to 1.68 [50]. Blood contam-ination can affect the M/E ratio, especially if leukocytosis is present. AnM/Eratio greater than the reference interval often indicates that granulocyticproduction exceeds erythroid production, but it could also indicate a de-crease in erythropoiesis. Likewise, an M/E ratio less than the reference inter-val often indicates that erythropoiesis exceeds granulopoiesis, but it couldalso indicate suppression of the latter. Determining which interpretation isappropriate requires knowledge of concurrent CBC results. An increase in

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the M/E ratio would be appropriate in an animal with neutrophilia, but ifthe neutrophil count is normal and anemia is present, it would indicate ery-throid hypoplasia. In the face of anemia, a decrease in the M/E ratio wouldindicate erythroid hyperplasia and regeneration, but a decrease in a neutro-penic animal that is not anemic would indicate myeloid hypoplasia. In somedisease states, the myeloid and erythroid series may both be affected, whichcould potentially exacerbate or mask alterations.

Evidence of dysplastic changes in hematopoietic cells includes asynchro-nous maturation between the cytoplasm and nucleus, large cell forms, andabnormal nuclei (eg, ring-shaped nuclei and so forth). Cell types otherthan hematopoietic cells should be evaluated as to their presence and pro-portion of the total cell population. Low numbers of macrophages(!1%), plasma cells (%2%), and small lymphocytes (%10%) are commonlyseen in bone marrow samples.

Interpretation
When investigating cytopenia, one is typically trying to determine if it is
more likely attributable to lack of production or to consumption or destruc-tion of the cell(s) in question. If atypical cells are seen in the blood, the bonemarrow is evaluated for the diagnosis of leukemia, myelodysplastic syn-dromes, and infiltrative disease.

In general, the bone marrow becomes more cellular in a hyperplastic/re-generative response. A shift to the left may be seen, in which all maturationstages are represented but there is a higher proportion of the less maturecells (the proliferating pool) than normal. Blast cells should still compriseless than 20% of all cells present.

Apparent maturation arrest is a term used whenmaturation seems to termi-nate at a particular stage; the earlier maturation stages are present, but laterstages are poorly represented. This can be seen with destruction of cells, aswith immune-mediated disease. Destruction of cells at a particular matura-tion stage results in decreased numbers of the affected cell as well as all laterstages. If a disease process results in a large demand for or rapid mobilizationof cells out of the bone marrow, depletion of the more mature cell stages canappear as a maturation arrest. In these cases, a left shift also is often associ-ated with the increased demand. Suppression of hematopoiesis can result inabnormalities in maturation progression but is often reflected as a decreasein all stages of maturation. If the number of cells within a cell line is toolow, progression of maturation can be difficult to evaluate. Acute leukemiais typically reflected as a severe apparent maturation arrest or left shift.

Atypical cell populations in the bone marrow can represent a myeloprolif-erative disorder, an infiltrative neoplastic process, inflammation, or anti-genic stimulation. Atypical cell populations can be attributable tosignificant increases in cell numbers of types normally seen in the marrow.Increased numbers of blast cells may indicate acute leukemia, althoughcytologic results need to be correlated with other clinical data, because


granulopoiesis characterized by a marked left shift in bone marrow precur-sor cells with rapid mobilization of more mature cells from the bone marrowcan resemble a myeloproliferative disorder. In some instances, sequentialbone marrow aspirates can be useful in helping to determine disease patho-genesis. Abnormalities in maturation progression may also be reflected bythe presence of dysplastic changes.

Another cause for increased numbers of cells normally residing in thebone marrow is inflammation. Suppurative inflammation can be seen, typi-cally associated with septicemia. Histiocytic/granulomatous inflammation ischaracterized by increased numbers of macrophages. Suppurative and his-tiocytic inflammation can be associated with infectious or noninfectious dis-ease processes. If inflammation is present, the sample should be thoroughlyinspected for organisms and culture may be indicated.

Increased numbers of lymphocytes and plasma cells can be seen with an-tigenic stimulation or with neoplasia. In general, it is rare to see the propor-tion of lymphocytes or plasma cells exceed 20% with antigenic stimulation.The morphology of lymphocytes and plasma cells should be well differenti-ated if associated with nonneoplastic processes. Cells associated with neo-plasia can also appear well differentiated; therefore, cell morphology alonecannot be used to establish a diagnosis. Clinical presentation and ancillarytesting (eg, serum protein electrophoresis, flow cytometry) can be helpful ininterpreting lymphocytoses and plasmacytoses.

The presence of cells not typically seen in the bone marrow or cells exhib-iting a high degree of pleomorphism or other criteria of malignancy shouldraise suspicion of a neoplastic process. Neoplasia can arise from within thebone marrow or can metastasize to the bone marrow from other sites.

Degenerative cells or amorphous proteinaceous debris suggests bonemarrow necrosis. This can occur secondary to infarction, neoplasia, or in-flammation. Degenerative cells are often unidentifiable.

After cytologic evaluation of bone marrow and correlation with CBC re-sults, a determination can be made as to whether any of the cell lines are hy-poplastic or hyperplastic and if there is evidence of maturation arrest,dysplasia, neoplasia, or inflammation. This information can then be corre-lated with the clinical information to determine appropriate differentialsand likely disease pathogenesis.

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