Vet Clin Equine 24 (2008) 437454

Equine Synovial Fluid Analysis

Catherine M. Steel, BVSc, FACVSc
Department of Veterinary Clinic and Hospital, The University of Melbourne,
250 Princes Highway, Werribee, Victoria 3030, Australia

Synovial uid (SF) of joints, tendon sheaths, and bursae normally functions as a biologic lubricant and a biochemical pool through which nutrients
and regulatory cytokines traverse. SF is normally a clear to pale yellow
highly viscous uid composed primarily of plasma ltrate, which is similar
to blood plasma, except that there are fewer proteins. SF is therefore described as an ultraltrate (a dialysate) of plasma, into which several macromolecules are secreted. Filtration occurs through the synovial membrane,
a system that is well reviewed elsewhere [1,2]. Briey, the synovial membrane
is a thin lining composed of tissue macrophage A cells, broblast-like B cells,
and fenestrated capillaries. Underneath the synovial membrane lies the subsynovium, a thicker layer of loose connective tissue that contains an extensive system of lymphatics for clearance of transported molecules. The cells
of the synovium form a discontinuous layer separated by intercellular
gaps several microns in width. The extracellular matrix in these gaps contains collagen and various other molecules, including hyaluronan (HA),
chondroitin sulfate, biglycan and decorin proteoglycans, and bronectin.
It provides the permeable pathway through which exchange of molecules occurs yet also oers sucient outow resistance to retain large molecules
within the SF. As a result, glucose and electrolytes occur in concentrations
similar to those in plasma or serum but proteins cross to a limited extent. SF
should contain few if any erythrocytes and low numbers of nucleated cells
(!1  109/L) of which most are mononuclear cells [3,4]. The SF total protein (TP) concentration is approximately 25% to 35% of the plasma protein
concentration of the same animal. The molecules believed to play a key role,
alone or in combination, in lubrication of the joint surfaces are largely produced by the B cells of the synovium and include proteoglycan 4 [5], HA [6],
and surface-active phospholipids [7].

E-mail address: csteel@unimelb.edu.au

0749-0739/08/$ - see front matter. Crown Copyright 2008 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.cveq.2008.05.004
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Alterations in transsynovial diusion or changes in synoviocyte metabolism may occur in response to a variety of insults or disease conditions, with
all these changes aecting the composition of the SF. Some generalizations
regarding these changes in various joint diseases can be made, and SF analysis can therefore provide valuable information in conjunction with history,
physical examination ndings, ultrasonography, and radiography in the diagnosis of these conditions. The most common and important application
for SF analysis in equine patients is to distinguish between septic and nonseptic synovitis. Although this dierentiation is clear-cut in many cases, in
some patients, it can be a challenge because of the large variation in clinical
symptoms and SF alterations in these conditions and the high rate of falsenegative bacterial culture results in septic synovitis. In other conditions, SF
analysis provides an indication of the degree of synovitis and metabolic derangement within the joint.
Sample collection and handling
Sampling of SF must be performed under strict aseptic conditions. Introduction of a needle through contaminated or infected skin or through subcutaneous tissue may lead to infection in a sterile synovial cavity or
contamination of septic SF with additional organisms. The reader is referred
to other texts for discussion of appropriate techniques [8].
In most cases, sample collection is straightforward; however, SF may be
dicult to obtain in open joint or tendon sheath injuries if SF is draining
through the wound, in small joints, and in infected joints in which brin
clots may prevent aspiration. If attempts at sample collection are unsuccessful, sterile 0.9% saline or lactated Ringers solution can be injected into
the synovial structure and uid can then be aspirated. Clearly, this dilutes
the SF and aects the clinicopathologic parameters; however, because the
urea concentration in SF normally mimics that in serum, the urea concentrations in SF and serum collected simultaneously can be compared to determine the degree of SF dilution [9,10].
After sampling, normal SF does not clot, but it often becomes gelatinous. This phenomenon is referred to as thixotropism, and agitation of the
sample returns it to its uid state. If there is iatrogenic blood contamination
of the SF, hemarthrosis, or an elevated SF TP concentration, the sample
may actually clot. Hence, it is important to immediately transfer a portion
of the sample to an anticoagulant tube. Ethylenediamine tetraacetic acid
(EDTA) is the most appropriate anticoagulant because it is best for cytology
and suitable for other tests, with the exception of the mucin clot test, which
requires a sample that has been placed in heparin. Ideally, the tube should
be lled to the appropriate mark to maintain optimal cell morphology and
to prevent clotting. Some of the sample should be kept sterile and free from
anticoagulant in case it is required for bacterial culture. Preferably, if septic
synovitis is possible, some SF should be inoculated directly into a biphasic

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439

blood culture bottle for aerobic culture and into appropriate enrichment
media, such as cooked meat broth, for subsequent anaerobic culture.
Analysis and test priorities
Complete SF analysis consists of a description of the gross appearance of
the uid, determination of TP and total nucleated cell count (TNCC), and
cytologic examination. If synovial sepsis is suspected, a Gram stain and aerobic and anaerobic cultures are performed. The TNCC, or SF white blood
cell (WBC) count, TP concentration, and percentage of neutrophils on cytologic examination are the most useful parameters to evaluate in making a diagnosis of septic synovitis. The mucin clot test (mucinous precipitate
quality) evaluates the quality and quantity of SF mucin (and hence HA)
and may be included in complete SF analysis; however, in many laboratories
that handle equine samples, it is no longer performed routinely.
When the volume of SF obtained is limited, cytologic examination is the
single most useful test. Therefore, if only a few drops of SF are available, the
color and turbidity should be noted while the sample is in the syringe and
the viscosity noted as the sample is expelled onto a glass slide for the immediate preparation of a direct smear for cytologic examination and subjective
assessment of cellularity. A mean of zero to two WBCs on examination of
10 high-power elds (400 magnication) on a slide from such a wet
drop preparation of SF corresponds with an actual WBC count less than
1.3  109/L [11]. Estimates from smears with higher WBC counts are
less consistently correlated [11].
The importance of using a laboratory that is familiar with SF analysis has
been highlighted by several published quality control studies for total and
dierential WBC counts of SF in the medical literature [12,13]. In one study,
a single SF sample was analyzed by 26 dierent hospital laboratories [13],
whereas in the other study, results from 3 dierent hospital laboratories
were compared with those of a reference laboratory [12]. Both studies reported a high degree of variation in results. For example, total WBC count
on the same uid varied from 2.5 to 12.0  109/L [13]. Considerable interobserver error and intraobserver error in the identication of cells were also
recognized. Similar studies do not seem to have been published in the veterinary literature; nevertheless, the laboratory to analyze the sample should be
selected carefully.
In a joint with arthritis, abnormal matrix turnover results in the release
into the SF of many molecules and fragments of matrix components derived
from such tissues as the articular cartilage. Such substances are referred to
as biomarkers (ie, biochemical measures that are indicative of a biologic
process or the response to an intervention). Numerous SF biomarkers derived from cartilage or from noncartilaginous sources, and as indicators
of anabolic or catabolic processes, have been studied [14,15]. Analysis for
many of these biomarkers is not widely available, and they are therefore

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not part of the routine evaluation of SF. Aside from highlighting the potential use of some markers in dierentiating septic from nonseptic synovitis,
these assays are not discussed in the present review.
Gross appearance
Visual inspection of SF for color and clarity at the time of collection is
extremely useful. As mentioned previously, normal SF is transparent, clear
to pale yellow, and highly viscous. It does not clot because it lacks brinogen and other clotting factors.
When SF is bloody, hemarthrosis must be distinguished from iatrogenic
hemorrhage caused by arthrocentesis. Usually, this distinction is made at
the time of specimen collection. With hemarthrosis, such as may occur in
an acute traumatic situation or with sepsis, the uid is uniformly bloody,
whereas with iatrogenic hemorrhage, blood may not be present throughout
the sample collection. Previous hemorrhage is characterized by dark yellow
or yellow-orange discoloration, especially of the supernatant. Such discoloration is termed xanthochromia. It is attributable to the presence of hemoglobin breakdown pigments and is often associated with chronic traumatic
arthritis.
Turbidity is usually caused by increased cellularity attributable to inammation. Septic SF is usually turbid or occulent, cloudy, and nonviscous. As
a guide, if the degree of turbidity prevents newspaper print to be read
through the sample, the cell count is greater than 30.0  109/L and strongly
suggests infection [16]. Septic SF is pale yellow to orange or red in color.
Bloody uid is not unusual in such cases because of hemorrhage from the
markedly inamed synovium. If clinical signs consistent with synovial infection are present, especially if combined with a risk factor (eg, septicemia in
a foal, a traumatic wound over a synovial structure, recent intrasynovial injection or surgery), prompt initiation of treatment is important. In these
cases, if the gross appearance of the SF is consistent with sepsis, appropriate
treatment, including intravenous broad-spectrum antibiotics and lavage,
should be initiated before the diagnosis is conrmed with laboratory
analysis.
Viscosity
The viscosity of SF is directly related to the quantity and degree of polymerization of HA [3]. With synovitis, loss of viscosity can occur with dilution of SF and enzymatic breakdown of HA. This implies inammation
but not necessarily infection. The lower viscosity of septic SF is attributable
to a lower HA concentration and may also be the result of decreased HA
synthesis [17].
Viscosity is usually evaluated subjectively by observing the length of the
strand formed by a drop of SF as it is expelled from the end of a needle or

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441

syringe. It usually strings 5 to 7 cm before separating. Alternatively,

a drop of SF may be placed on the thumb and then touched with the index
nger, with separation of the thumb and nger normally producing a string
2.5 to 5 cm long before breaking [8]. Decreased stringing occurs with decreased viscosity. Such stringiness may be reported as normal, decreased,
or markedly decreased.
Viscosity can also be evaluated qualitatively during cytologic examination. In slides made by directly smearing the specimen, the cells from normal
SF are arranged in rows (a phenomenon known as windrowing) because
of the viscosity of the uid. As viscosity decreases, the cells become progressively more randomly distributed.
Mucin quality and concentration
The mucin clot test (or mucinous precipitate quality) provides a crude estimate of the HA concentration and degree of polymerization. It is poor or
absent in sepsis. Because it may also be poor in nonseptic synovitis, however, it is of little use in making this important dierentiation [18].
Protein
SF TP concentration can be measured by refractometry or by biochemical
assays. The reference value for normal horses has been documented as 18 
3 g/L [4], and, in general, a value less than 20 g/L is considered normal [19].
Joint trauma and inammatory conditions increase the TP concentration,
as protein leaks from damaged vessels. A TP concentration greater than
40 g/L indicates severe inammation, with noninfectious inammatory conditions generally having TP concentrations less than this level. In cases of
septic synovitis, the TP can vary depending on the duration and severity
of disease. Although SF TP concentration is often greater than 40 g/L in
cases of sepsis and levels greater than 60 g/L are recognized in some chronically infected joints [20], the concentration may be lower earlier in the disease. As discussed elsewhere in this article, TP values less than 25 g/L have
also been reported in SF samples with a positive culture [21], indicating
the importance of considering all SF clinicopathologic parameters, with
the history and clinical ndings, when dierentiating septic from nonseptic
synovitis in some cases.
Total nucleated cell count
A TNCC can be done manually using a microscope and hemocytometer,
or, provided the uid is not too turbid or occulent, an electronic particle
counter (automated analyzer) that dilutes the specimen in isotonic buer
may be used. The volume of SF needed varies among dierent analyzers
but can be small (in the range of 1020 mL). It is important to use

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physiologic saline as the diluent and not a diluent that contains acetic acid,
which would precipitate the HA-protein complex (performing a mucin clot
and interfering with the cell count). As mentioned previously, a TNCC less
than 1.0  109/L is generally considered normal, with many such samples
having less than 0.2  109/L [3,4,19]. Fluid from a tendon sheath is similar
to joint uid with reference values reported as 0.2 to 3.5  109/L [22].
Increases in the SF WBC count occurred within 8 hours of experimental
inoculation of the tarsocrural joint of horses with Staphylococcus aureus,
and values were signicantly increased within 12 to 24 hours [18]. Such an increase in the WBC count may be delayed if a smaller number of bacteria or
less virulent organisms are involved. Therefore, if early infection is suspected
and the TNCC is not markedly increased as expected with sepsis, repeat sampling after 12 to 24 hours is indicated. It must be recognized that repeat sampling may in itself cause some increase in the TNCC of SF, however [23].
Cytologic examination
Cytologic examination is the most important part of the analysis of SF.
Smears of SF that has been placed in EDTA immediately after collection are
prepared for dierential cell counts in a similar way to peripheral blood
smears. If the TNCC count is substantially increased, the smear is made directly from the SF. Otherwise, if a cytocentrifuge is available, a cytospin
preparation is made for microscopic examination. If not, the sample may
be centrifuged and the sediment resuspended in 0.5 mL of supernatant, after
which the smear is made. The smears are air-dried and stained with a Romanowsky stain (eg, Wrights stain, Di-Quik [Di-Quik Solution I, II and
Fixative; Siemens Medical Solutions Diagnostics, Deereld, Illinois]).
Initial examination of the smear should include subjective evaluation of
cellularity (as normal or mildly, moderately, or markedly increased) and
the number of erythrocytes in addition to determination of the presence or
absence of windrowing (as an indication of viscosity). A more thorough microscopic examination is then performed to classify nucleated cells and evaluate their morphology and to note any micro-organisms. Normal SF consists
of large mononuclear cells, which are derived from blood monocytes, tissue
macrophages, and synoviocytes (Fig. 1) in addition to fewer small mononuclear cells or lymphocytes (Fig. 2). Neutrophils usually constitute less than
10% of the nucleated cells, and eosinophils are rare (!1%) [19]. Vacuolation
and phagocytic activity of mononuclear cells (Fig. 3) and any degenerative
changes in neutrophil morphology are noted. Mucin appears as a nely granular pink material throughout the background of smears.
Hemarthrosis and its dierentiation from blood contamination
Erythrocytes are not considered normal constituents of SF; however,
most samples contain some, and their presence is usually attributed to blood

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443

Fig. 1. (A) Synoviocytes (sheet of synovial membrane cells) (May Grunwald Giemsa stain, original magnication 1000). (B) Synoviocyte with granular cytoplasm (right) and neutrophil.
Note granular mucinous background (May Grunwald Giemsa stain, original magnication
1000). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

contamination during sample collection. Microscopic evidence of erythrophagocytosis (Fig. 4) occurs when hemorrhage is more than a few hours
old. Hemorrhage typically incites inammation; thus, the cell count and
number of neutrophils are increased. When hemorrhage is more chronic,
xanthochromia (orange discoloration) may be visible grossly in the supernatant of the uid. In such cases, microscopy of the smear may reveal hemosiderin granules in macrophages and hematoidin crystals free in the

Fig. 2. Note lymphocyte and large nonreactive macrophage at the center of the eld. Increased
numbers of neutrophils indicate inammation rather than normal joint uid. Also free
erythrocytes suggest mild hemorrhage or blood contamination at collection (May Grunwald
Giemsa stain, original magnication 1000). (Courtesy of Peter M. Lording, BVSc, MVetSc,
Melbourne, Australia.)

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Fig. 3. Note leukophagocytic macrophage (bottom center) and reactive macrophage (top center). Other less reactive macrophages and many neutrophils with good cell morphology are
also present. Inammatory arthritis (May Grunwald Giemsa stain, original magnication
1000). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

background of the smear or within leukocytes. Any platelet aggregates must

be noted. Because these usually remain little more than 1 hour after hemorrhage into SF, their presence suggests blood contamination rather than true
hemarthrosis. If blood contamination occurs during sample collection,

Fig. 4. Erythrophagocytic macrophage, indicating hemorrhage before collection (May Grunwald Giemsa stain, original magnication 1000). (Courtesy of Peter M. Lording, BVSc,
MVetSc, Melbourne, Australia.)

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445

however, and a delay of more than several hours occurs between sample collection and analysis, it may be mistaken for pathologic hemorrhage because
platelets degenerate in old samples and erythrophagocytosis can occur in vitro. If results of a peripheral blood leukogram are available, the distribution
of leukocytes in the SF and leukogram can be compared to determine
whether the distribution of cells is consistent with the degree of peripheral
blood contamination suspected.
Nonseptic arthropathies
Idiopathic synovitis and osteochondritis dissecans are generally associated with a TNCC less than 1.0  109/L [8]. In osteoarthritis, the TNCC
is within reference limits to mildly increased, depending on the degree of active synovitis, with values usually less than 5.0  109/L. In these conditions,
the TP concentration is within reference limits or mildly increased and the
cells are predominantly mononuclear.
Traumatic synovitis may be associated with cell counts less than 10.0 
109/L [8] with neutrophils predominating. Injection of sterile 0.9% saline
(1 mL) into the tarsocrural joint of horses and repeated arthrocentesis
for sample collection produced a signicant increase in the TNCC (!14.0 
109/L), the proportion of neutrophils (!71%), and TP (!43 g/L), which
generally returned to normal within 3 to 7 days of arthrocentesis
[18,24,25]. This highlights the potential for signicant alterations in SF clinicopathologic parameters with what may be considered a minor traumatic
or chemical insult.
Chemical synovitis, often referred to a joint are, can also occur in response to injection of corticosteroids, local anesthetics, HA, and polysulfated
glycosaminoglycans [2628]. In addition to the response reported after 0.9%
saline injection [24], clinicopathologic values in the SF of the equine carpal
joint increase to greater than reference ranges and can approach values suggestive of sepsis after serial arthrocentesis and injection of local anesthetic
[29], gentamicin sulfate [25,30], ceftiofur sodium [31], or amikacin [32]. Similar increases in TNCC, percentage (%) of neutrophils, and TP concentration
occur after injection of amikacin or lactated Ringers solution into the equine
digital exor tendon sheath [23], with TNCC greater than 30.0  109/L and
80% or greater neutrophils observed in some cases. Prior sampling and treatment may therefore complicate interpretation of SF analysis.
Early onset of signs (around 24 hours), synovial TNCC less than 30.0 
109/L, and resolution within 1 to 3 days have been suggested to be helpful in
dierentiating chemical synovitis from septic arthritis [26]. Clearly, overlap
with the ndings expected with septic synovitis may still occur, and repeated
lameness examination and follow-up sampling are therefore appropriate.
Although marked lameness and synovial eusion can occur with a nonseptic
are, acute synovitis after arthrocentesis is best treated as if septic if doubt
exists.

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Nonseptic inammation may be immune mediated, and several noninfectious nonerosive syndromes have been described in horses, with and without
evidence of infection elsewhere in the body. Synovitis is not uncommon in
foals with Rhodococcus equi infection [3335] and usually resolves as the
pneumonia resolves with antimicrobial therapy. These foals have single or
multiple joint distention but negative bacterial culture of SF, no or only
mild lameness or stiness, and a SF TNCC less than 8.0  109/L, [33,35]
with predominance of neutrophils [33] or mononuclear cells [36]. Immunouorescence of the synovia of aected joints was positive for immune complexes in some cases [33,36]. Although positive rheumatoid factor activity
was identied in the SF of one case [34], in other cases, there were no rheumatoid factor or antinuclear antibody titers and lupus erythematosus cell
preparations were negative. Similar ndings have also been described in
a foal with bacterial endocarditis caused by Escherichia coli [33] and
a foal infected with equine herpes virus-4 [37].
A lupus erythematosuslike syndrome was diagnosed in two horses based
on the presence of a positive lupus erythematosus cell preparation and titers
of antinuclear antibody [38,39].
A third syndrome, referred to as idiopathic polysynovitis because systemic underlying disease was absent, has been reported in four adult horses
[40,41] and was characterized by stiness or lameness, fever, polysynovitis
with a TNCC less than 7.0  109/L, TP !36 g/L, neutrophil or mononuclear cell predominance (12%81% neutrophils), failure to identify a microbial cause on culture of SF, no antinuclear antibody titer, negative lupus
erythematosus cell preparations, and response to immunosuppressive therapy. Histologically, there may be evidence of chronic polysynovitis with inltration of lymphocytes and plasma cells [41].
Eosinophilic synovitis is rare but has been reported in several horses
[42,43] and after the intra-articular injection of streptococcal antigen in
horses hyperimmunized with Streptococcus equi M protein vaccine [44]. In
one case of tarsocrural synovitis, SF analysis revealed an increased TNCC
of 12.8  109/L, with 76% eosinophils [43]. There was histologic evidence
of an intense inltrate of eosinophils, lymphocytes, and a few mast cells
in the synovium, suggestive of an immune-mediated response. In this case,
peripheral eosinophilia (0.6  109/L eosinophils, with a reference range of
00.1  109/L) was also present. In the other reported clinical case [42], carpal synovitis occurred after intra-articular injection of methylprednisolone
acetate and was thought to be a sterile are. In the latter case, and in two
horses that developed eosinophilic synovitis after the intra-articular injection of bacterial antigen [44], the SF eosinophil counts were less than 20%.
Septic synovitis
The gold standard for the diagnosis of septic synovitis is a positive bacterial culture; however, cultures from infected joints are negative in almost

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447

50% of clinical cases when enrichment media are not routinely used [21] and
in 27% when they are [45]. Furthermore, only approximately 25% of cases
demonstrate bacteria on Gram stain [45]. Factors that may contribute to
this include the presence of antibiotics and sequestration of bacteria in the
synovial membrane. Culture success is improved if SF is inoculated immediately into a biphasic blood culture bottle that contains ideal media for
the growth of most aerobic microorganisms, allows for a larger volume of
SF to be used, and dilutes and contain inhibitors of antimicrobial drugs.
In addition to conrming infection, isolation of the causative organism(s)
and determination of their sensitivity allow antimicrobial therapy to be better directed. Despite the fairly low success rate in demonstrating bacteria,
a Gram stain is considered useful. If bacteria are seen, it can assist with identication of the organism and the initial selection of antimicrobial agent(s)
well before culture results are available [16].
Because of the limitations of bacterial isolation, cytologic examination of
SF is the single most useful means of diagnosis (Figs. 5 and 6). With synovial sepsis, the TNCC of SF is usually greater than 10.0  109/L and often
much higher [4548]. Septic synovitis is therefore characterized by the highest WBC counts, although, as discussed elsewhere in this article, counts
!10.0  109/L have been have been reported in culture-positive uid in
some circumstances [21]. Although not validated by presentation of results
from culture-positive joints, this observation has led some to recommend
that cell counts greater than 5.0  109/L should be viewed with suspicion
and that counts greater than 10.0  109/L should be taken to indicate synovial sepsis in foals [49], although sympathetic synovitis secondary to septic physitis should also be considered in foals with a moderate increase in the
TNCC and less than 90% neutrophils [50]. Others consider cell counts
greater than 30.0  109/L suggestive of infection and counts greater than
100.0  109/L virtually pathognomonic [4,19,51]. Most cells are neutrophils,
yet degenerative changes are often not present, possibly because bacterial
toxins do not occur in SF to the same extent that they do in other septic
bodily uids. In summary, as a general rule, SF with a TNCC greater
than 30.0  109/L, greater than 80% neutrophils, or TP greater than
40 g/L should be considered infected, particularly if these correlate with clinical signs and predisposing circumstances [52].
Problems encountered in the dierentiation of septic
from nonseptic synovitis in some cases
The overlap among clinicopathologic values in septic synovitis with a low
TNCC (as may occur in early sepsis), sepsis resulting from corticosteroid injection into a synovial structure, an open draining synovial structure or infection with an organism of low virulence, and nonseptic inammation that can
occur secondary to trauma or therapeutic intervention (eg, synoviocentesis
alone, injection of medications) can make dierential diagnosis of these

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Fig. 5. Septic inammation. Note the predominantly neutrophilic inammatory response and
single cell to the right of center with phagocytosed cocci. Despite sepsis, cell morphology is reasonably good, suggesting that bacteria are weak toxin producers (May Grunwald Giemsa stain, original magnication 1000). (Courtesy of Peter M. Lording, BVSc, MVetSc, Melbourne, Australia.)

conditions dicult or impossible. Madison and colleagues [21] reported

a TNCC less than 10.0  109/L in 5 (14%) of 35 culture-positive joints. Of
these, 3 had an open draining synovial stulae and 1 had been injected with
a corticosteroid [21]. In a series of open digital exor tendon sheath injuries
in horses, there was a wide range of values with a TNCC from 1.3 to 92.6 
109/L, 33% to 98% neutrophils, and TP of 30 to 62 g/L [53]. Wright and colleagues [48] also reported a wide range of values in horses with synovial

Fig. 6. Septic inammation (Streptococcus zooepidemicus). Note organisms at the center of the
eld and poor cell morphology, indicating cellular degeneration secondary to bacterial toxins
(May Grunwald Giemsa stain, original magnication 1000). (Courtesy of Peter M. Lording,
BVSc, MVetSc, Melbourne, Australia.)

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449

contamination or infection, most of which were open wounds or self-sealing

punctures, with a TNCC less than 5.0  109/L to greater than 100.0  109/L,
TP from 22 to 82 g/L, and with 25% samples having 80% or less neutrophils.
Corticosteroid injection can delay the onset of clinical signs and the increase in SF TP [18,54] and TNCC [10] in horses with septic synovitis, particularly during the initial few days. Neutrophils greater than 90% and pH
less than 6.9 may be the most reliable parameters for diagnosis of septic arthritis after corticosteroid injection [54].
Other assays that may be of use in dierentiating
septic from nonseptic synovitis
The pH of SF (7.30  0.06) in healthy joints is similar to that of serum
[18] and can decrease with infection [18,54]. The normal SF lactate concentration of less than 3.9 mmol/L increased to greater than 4.9 mmol/L in 66%
of equine tarsocrural joints inoculated with S aureus yet remained less than
4.4 mmol/L in control joints that were injected with saline [18]. In the latter
study, serum and SF glucose dierence (SSGD) was also evaluated as a diagnostic aid. The SF glucose concentration is usually the same or marginally
lower than that of serum, with SSGD reference values of 0.85  0.59 mmol/L
(mean  SD), whereas an SSGD of greater than 2.2 mmol/L was seen in
83% of SF samples with experimentally induced septic arthritis [18].
SF pH, lactate, and glucose can easily be measured using a portable glucometer and other spectrophotometric units; however, although they may
provide supportive evidence of sepsis, they are not uniformly reliable and
should not be used as a primary diagnostic parameter [18].
Serum amyloid A (SAA) concentration in SF is signicantly higher in
horses with septic synovitis than in normal horses and those with lowinammation joint conditions [55]. It seems to be synthesized locally in
the inamed joint [56]. Furthermore, SF SAA does not change in response
to repeated arthrocentesis [55], and although further validation is required,
it has potential as a useful marker of synovial sepsis.
The activity of the latent form (the proenzyme) and bioactive form of matrix metalloproteinase (MMP)-9, measured by gelatin zymography, is increased in SF of horses with septic arthritis [57,58], with the active form
being directly proportional to the SF TNCC [58]. Because most WBCs in septic SF are neutrophils, these are thought to be the main source of proMMP-9
[58,59]. Indeed, analysis of the concentration of the proactive and active
MMP-2 and MMP-9 in SF may be of value in dierentiating septic from nonseptic SF, because in one study, septic SF had signicantly higher concentrations of proactive and active MMP-9 compared with nonseptic SF [57].
Furthermore, the concentration of proMMP-9 and the ratio of proMMP9 to proMMP-2 were predictive of survival [58], and therefore may be of
use in determining prognosis in addition to assisting in diagnosis should these
assays become more widely available.

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The potential for myeloperoxidase activity [60] and assay for proteolytic
enzymes [61], cytokines (including tumor necrosis factor-a), and eicosanoids
[62] in SF to aid in the diagnosis of septic arthritis in horses for which routine SF analysis is inconclusive needs to be validated in large case studies.
Such studies should include SF samples with positive bacterial culture yet
low TNCC and samples from acute traumatic synovitis with high TNCC before their clinical usefulness can be fully determined.
Gram staining
In cases of potential sepsis, an additional smear should be prepared for
a Gram stain. Although the sensitivity of this test is quite low for the diagnosis of septic synovitis and the absence of bacteria in Gram-stained specimens does not rule out infection, if microorganisms are present, knowledge
of their appearance may be of use in antibiotic selection. In one study, microorganisms were evident on smears in 58% of joints from which a positive
culture was obtained and in 21% of SF samples from which no bacteria
were cultured [21].
Bacterial culture
Using conventional agar plate methods alone to detect bacteria in SF (in
which SF is inoculated directly onto Columbia agar with 5% sheep blood
[blood agar; Oxoid, Columbia, Maryland] and incubated at 37 C with 5%
carbon dioxide for several days) has a high rate of false-negative results
and should be augmented by blood culture media (BCM) techniques. After
aseptic collection of an SF sample, the maximum volume of SF available,
within the recommendations for the bottle used, should immediately be inoculated into a biphasic blood culture bottle. Some SF should also be transported to the laboratory in suitable enrichment broth for anaerobic culture.
BCM enrichment methods reportedly result in detection rates of 55% to
78% [21,45,63]. There is further evidence in the human literature that commercial BCM systems (eg, BACTEC plus Aerobic/F medium; BACTEC,
BD, Franklin Lakes, New Jersey) increase the chances of culturing aerobic
pathogens from SF, particularly in patients receiving antibiotic treatment.
Larger volumes of inoculum and medium used when incubating in BCM
compared with conventional plate methods allow detection of extremely
low numbers of viable bacteria and the dilution of SF growth inhibitors
and antibiotics [64]. A disadvantage of the BCM, however, is the time
needed from inoculation of an SF specimen to the detection of infection,
with up to 7 to 10 days required for some organisms.
Polymerase chain reaction techniques
Polymerase chain reaction (PCR) oers great benet because it provides
rapid sensitive testing that can detect selected bacterial species in the presence of antibiotics. Performing PCR in addition to incubation of SF in

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451

BCM results in a signicant increase in sensitivity compared with BCM enrichment alone [63]. PCR diagnosis does not provide antibiotic sensitivity
testing, however, and knowledge of local antimicrobial susceptibility patterns is therefore necessary to guide the choice of an appropriate antimicrobial. Furthermore, false-positive results attributable to contamination
during sampling and analysis and, theoretically, from the presence of noninfective microbial contaminants that may enter the SF within phagocytic
cells from elsewhere within the host (bacterial translocation) are a concern
with this methodology but seem to occur uncommonly [63]. Detection of
bacterial DNA in SF samples of horses with presumed synovial infection
is therefore considered indicative of active or recent synovial infection
[63]. False-negative results have also been reported and may be attributed,
at least in part, to the considerably smaller sample volume used for PCR
(200 mL) compared with BCM (13 mL) [63].
Summary
The most important application for SF analysis in the horse is in the diagnosis of synovial sepsis. Misdiagnosis of synovial sepsis is costly, and SF
analysis makes correct diagnosis more likely, although far from certain. It is
well recognized that there is a gray zone of overlap in clinical signs and SF
assay ndings between cases of acute traumatic synovitis with a high TNCC
and those with septic synovitis with a low TNCC [8]. The precision of diagnosis may be increased with PCR analysis for detection of bacterial DNA in
SF and with assays for various enzymes and cytokines. These tests are currently not widely available, however, and routine SF analysis (determination
of the total and dierential cell counts and TP) remains of prime importance
in diagnosis.
With continued research and the evaluation of newer assays on a larger
scale, more information should be available on whether these tests can differentiate septic arthritis from other forms of acute arthritis, in addition to
monitoring the response to treatment and aiding in determination of the
prognosis.
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