clinical pathology & equine arthropathies

CLINICAL PATHOLOGY OF EQUINE ARTHROPATHIES

Dane M. Tatarniuk, DVM

June 25th, 2014

Physiology & Anatomy• Joints, Bursas, Tendon Sheaths• Plasma filtrate that provides

• Lubrication• Regulatory cytokines• Proteolytic enzymes• Nutritional reservoir

• Highly viscous• Gross appearance

• Pale yellow• Transparent

Physiology & Anatomy• Intimal Layer

• Macrophage type A synoviocytes• Phagocytosis

• Fibroblast type B synoviocytes• Hyaluronic Acid, Aggrecan, etc.

• Sub-intimal Layer• Loose connective tissue (type II

collagen)• Lymphatics

• Clear intrasynovial molecules

• Vasculature• Nerves

Physiology & Anatomy• Intimal Layer

• Extracellular matrix gaps in intimal layer• Gaps are several microns in length

• No basement membrane

• Synovial Fluid• Plasma ultra-filtrates from vessels within subintimal layer, • Passes through intimal layer• Becomes basis for SF in joint cavity

• Due to intimal layer• Exchange of small molecules between circulation and joint• Precludes large molecules from passing into SF

Physiology & Anatomy• Specifically:

• Glucose, electrolytes similar in SF vs. serum/plasma• Proteins become limited in cross over

• ~25% to 33% the concentration within plasma• Predominately proteins present produced by type B synviocytes

• Proteoglycans, Hyaluronic acid, Phospholipids

• Rare or no erythrocytes• Low number of nucleated cells normally

• < 1.0 x 109/L

Synovial Fluid Assessment• Why?

• Can help establish important difference between septic and non-septic synovitis• Can have similar clinical presentation• Immediate results

• Culture of synovial fluid often unrewarding• False negatives• Delayed results

• Provides subjective assessment of degree of inflammation• Mild, moderate, severe

• Can trend change in joint response to therapy• Trending back to normal?

Synoviocentesis• Collection

• Strict aseptic technique• Variety of locations to insert needle depending on joint

• Attempt to pick location distant from wounds / traumatic areas

• Risk• Introduce a new infection to a previously sterile synovial cavity• Contaminate an already infected synovial cavity with a new organism

• Counter-indicated• Subcutaneous tissue infection (cellulitis)

Synoviocentesis• Can be difficult if

• Synovial structure is actively draining• Fibrin clots present that interfere with aspiration

• If difficulty collecting fluid• Can distend joint with 0.9% saline first• Then aspirate synovial fluid – saline sample• Measure urea concentration in synovial fluid and serum

• Urea in SF should be near identical to that of serum

• Compare to the two results to determine degree of dilution present

Analysis• Sample Handling

• ‘Thixotropism’• Does not clot, but becomes gelatinous• Should return to normal fluid state when agitated

• Can clot if,• Iatrogenic blood contamination• Hemoarthrosis• Elevated total protein concentration in SF

• EDTA (ethylenediamine tetraacetic acid) • Preferred• Suitable for cytology

• Culture• Sterile heparin container• Directly inoculated into blood culture bottle or Amies media/swab

Analysis• Characterize / Quantify:

• Gross appearance of fluid• Color, viscosity, turbidity

• Total Nucleated Cell Count• Total Protein• Cytological Examination

• If suspicious of sepsis,• Gram Stain• Culture

• Aerobic• Anaerobic

Analysis• Visual inspection

• Should be transparent• Clear to pale yellow• Very viscous• Should not clot, since it lacks fibrinogen• Septic SF is can be pale yellow, orange or

red in color

• Iatrogenic Hemorrhage vs. Hemoarthrosis• Iatrogenic

• May not be diffuse

• Hemoarthrosis• Associated with trauma• Diffuse through entire sample• Dark yellow or yellow-brown discoloration

• ‘Xanthochromia’

Analysis• If sample limited (ie, few drops)

• Subjectively assess color, turbidity, and viscosity• Make a smear,• Estimate,

• If you note ‘0 to 2’ WBC at 400x magnification (10 high-power fields), this corresponds roughly to 1.3 x 109/L1

1. Clayburne G, Baker DG, Schumacher HR Jr. Estimated synovial fluid leukocyte numbers on wet drop preparations as a potential substitute for actual leukocyte counts. J Rheumatol 1992;19(1):60–2.

Analysis• Turbidity

• Increased cellularity due to increased inflammation• Sepsis

• Flocculent, cloudy, non-viscous

• Rule:• If you can’t read (size 12) printed words through the sample, then the

total cell count is greater than 30.0 x 109/L 1

1. Bertone AL. Update on infectious arthritis in horses. Equine Veterinary Education 1999; 11(3):143–52.

Analysis• Viscosity

• Relates to the quantity and degree of hyaluronic acid polymerization

• Viscosity decreases with inflammation• Dilution effect• Enzymatic breakdown of HA• Decreased HA synthesis

• Subjective• One drop of SF on a index finger• Compress thumb and index finger• Should normally ‘string’ 2 to 5cm prior to separating

• Cytology Smear• Normal viscosity, cells ‘windrow’• Decreased viscosity, cells more randomly

distributed

Analysis• Mucin Clot Test

• Evaluates mucinous precipitate quality• Provides crude estimate of quantity

• Indirect assessment of hyaluronic acid content• Clinically,

• Minimal to non-existent in sepsis• Poor in non-septic synovitis• Does not help differentiate between sepsis or joint flair

• Requires SF in heparin vial, not EDTA• Add 2.5% acetic acid to equal volume SF• Mucin precipitates, forms white clot

• Not routinely performed

Analysis• Total Nucleated Cell Count (TNCC)

• Manual• Requires microscope and hemocytometer

• Automatic• Electronic particle counter (analyzer)• Dilute the sample with saline

• Acetic acid precipitates hyaluronic acid & protein = mucin clot

• Can utilize samples as small volume as 10 – 20 μL• Need to use hyaluronidase to avoid clotting the system

Analysis• Hemocytometer

• Grid pattern etched on slide• Area of square and depth known• Count cells within given area

• Measure number of cells per volume of SF

• Only measure cells on top or left of square, not on bottom or right

• If need to dilute, use saline

Analysis• Hemocytometer

Analysis• Total Nucleated Cell Count (TNCC)

• Joint• Normal: 1.0 x 109/L or less cells 1

• Often as low as 0.2 x 109/L

• Tendon Sheath• Normal: 0.2 to 3.5 x 109/L 2

1. Van Pelt RW. Interpretation of synovial fluid findings in the horse. J Am Vet Med Assoc 1974;165:91–5.2. Malark JA, Nixon AJ, Skinner KL,et al. Characteristics of digital flexor tendon sheath fluid from clinically normal horses. Am J Vet Res

1991;52(8):1292–4.

Analysis• Total Nucleated Cell Count (TNCC)

• Delayed effect• Can take up to 8 to 24 hours for increase influx of white blood cells

following bacterial inoculation of joint• Influenced by number of bacteria present• Influenced by virulence of bacteria present

• If SF is sampled shortly after the suspect introduction of contamination, WBC may yet be in normal range• Can re-sample 12 to 24 hours later

Analysis• Protein (TP)

• True reference range for horses is 1.8 to 3.0 g/dL1

• Generally considered, normal is < 2.0 g/dL• Trauma & inflammation increase permeability of synovial

membrane• Generally, > 4.0 g/dL suggests severe inflammation• Variable, whether acute vs. chronic• Sepsis,

• Protein often between 4.0 g/dL to 6.0 g/dL• Lower protein in early sepsis

• Reports of positive culture with protein less than 2.5 g/dL2

1. Van Pelt RW. Interpretation of synovial fluid findings in the horse. J Am Vet Med Assoc 1974;165:91–5.2. Madison J, Sommer M, Spencer P. Relations among synovial membrane histopathologic findings, synovial fluid cytologic findings, and bacterial

culture results in horses with sus- pected infectious arthritis: 64 cases (1979–1987). J Am Vet Med Assoc 1991;198:1655–61.

Analysis• Cytological Exam

• Differential cell counts performed similar to blood smears• Preparation:

• Make smear directly from SF• Works well if TNCC is elevated

• Centrifuge SF and re-suspend sediment in 0.5cc of supernatant• Air dry slide• Stain with Wright’s (Diff-Quik) stain

Analysis• Cytological Exam

• Subjective Nucleated Cell Cellularity• Normal or mild / moderate / markedly increased

• Presence of absence of windrowing• Viscosity

• Presence of erythrocytes• Classify mononuclear cells

• Normal• Mostly large mononuclear cells (macrophages, monocytes, synoviocytes)• Neutrophils <10%• Eosinophils rare

• Abnormal• Increased neutrophils• Vacuolation, degeneration• +/- extra or intracellular bacteria

Analysis• Interpreting erythrocytes

• Likely is from iatrogenic hemorrhage during collection• Erythrophagocytosis

• Implies hemorrhage is at least a few hours old• Can occur in-vitro (evaluate sample immediately)

• Chronic• Xanthochromia, orange tinged SF

• Hemorrhage incites inflammation• So neutrophils and NTCC will also increase

• Platelets• Usually only are visible for 1-2 hours • Suggest iatrogenic blood contamination over hemoarthrosis

• Can compare systemic leukocyte distribution to see if similar to SF

Analysis

Synoviocytes

Analysis

Neutrophils, macrophage, erythrocytes

Analysis

Erythrophagocytosis

Analysis

Degenerate neutrophils, scattered cocci

Analysis

Septic Inflammation

Non-septic Arthropathies• What is the effect of arthrocentesis alone?

• Study design 1

• Injection of 1cc of saline into tibiotarsal joint• Multiple injections

• Significant increase in TNCC• Up to 14.0 x 109/L• ~70% neutrophils• Mean TP of 4.3 g/dL• Cytology returned to normal within 3 – 7 days

• Conclusion: • Needle introduction itself causes joint inflammation• Difficult to interpret origin of mild inflammatory conditions in SF

1. Tumalo R, Bramlage L, Gabel A. Sequential clinical and synovial fluid changes associated with acute infectious arthritis in the horse. Equine Vet J 1989;21:325–31.

Non-septic Arthropathies• Idiopathic synovitis, osteochondrosis dissecans,

osteoarthritis• TNCC less than 1.0 x 109/L• Normal to mild increase in TP• Predominately mononuclear cells• In osteoarthritis specifically

• TNCC can be up to 5.0 x 109/L, but often is normal• Depends on whether active synovitis is present

Non-septic Arthropathies• Traumatic Synovitis

• Cell counts can rise to up to 10 x 109/L• Non-degenerate neutrophils predominate

Non-septic Arthropathies• Chemical Synovitis

• “Joint Flair”• Corticosteroids, hyaluronic acid, PSGAGs, antibiotics, IRAP• Typically early onset of signs

• Within 24 hours

• TNCC less than 30 x 109/L• Duration ~1 to 3 days

Non-septic Arthropathies • Immune-mediated

• Synovitis secondary to Rhodococcus Equi. infection in foals 1,2

• Single or multiple joint distension• Synovitis resolves once pneumonia responds to antibiotics

• Negative bacterial culture• Mean TNCC: 8.0 x 109/L• Predominate neutrophils

• Immunofluorescence of synovium positive for immune complexes

1. MadisonJB,ScarrattWK.Immune-mediatedpolysynovitisinfourfoals.JAmVetMedAs- soc 1988;192:1581–4.2. Paradis M. Cutaneous and musculoskeletal manifestations of Rhodococcus equi infection in foals. Equine Veterinary Education 1997;9(5):266–

70.

Non-septic Arthropathies• Idiopathic Polysynovitis 1

• No underlying systemic disease• Stiffness & Polysynovitis• Cytology

• Mean TNCC of 7.0 x 109/L• TP < 3.6 g/dL• Neutrophil predominance

• Negative SF culture, negative for lupus, no antinuclear antibody titer, and responds to immunosuppressive therapy

• Infiltration with lymphocytes and plasma cells histologically

1. Pusterla N, Pratt SM, Magdesian KG, et al. Idiopathic immune-mediated polysynovitis in three horses. Vet Rec 2006;159:13–5.

Non-septic Arthropathies• Eosinophilic Synovitis

• Rare• In (limited) case reports, concurrent with peripheral eosinophilia• Eosinophils in case reports were <20% of TNCC

Septic Arthropathies• Etiology

• Hematogenous • Foals

• Increased physeal vasculature

• Trauma• Wounds• Fractures

• Iatrogenic• Arthrocentesis

• Risk of septic arthritis following intra-articular medication is low with good technique• 1 case per 1279 injections 1 (n = 16624)

1. Steel CM, Pannirselvam RR, Anderson GA. Risk of septic arthritis after intra-articular medication: a study of 16, 624 injections in TB racehorses. Aust Vet J., (2013), 91 (7): 268 – 73.

Septic Arthropathies• Arthrocentesis

1. Steel CM, Pannirselvam RR, Anderson GA. Risk of septic arthritis after intra-articular medication: a study of 16, 624 injections in TB racehorses. Aust Vet J., (2013), 91 (7): 268 – 73.

Septic Arthropathies• ‘Culture’ is Gold Standard

• In a perfect world, positive culture confirms sepsis• However,

• Cultures are positive in 27% of cases with no enrichment media 1

• Cultures are positive in 50% of cases with enrichment media used 1

• Only 25% of cases display bacteria on gram-stain 2

• Factors limiting culture growth• Previous initiation of systemic antimicrobials• Bacteria tend to sequester in synovium and/or fibrin

1. Madison J, Sommer M, Spencer P. Relations among synovial membrane histopathologic findings, synovial fluid cytologic findings, and bacterial culture results in horses with sus- pected infectious arthritis: 64 cases (1979–1987). J Am Vet Med Assoc 1991;198:1655–61.

2. Schneider R, Bramlage L, Moore R, et al. A retrospective study of 192 horses affected with septic arthritis/tenosynovitis. Equine Vet J 1992;24:436–46.

Septic Arthropathies• Culture is delayed knowledge

• Consider gram stain• If bacteria are seen, can determine if gram +/-• May help better direct initial antimicrobial therapy

• Likely still place on broad spectrum antibiotics

• Positive culture• Bacteria seen in 58% of gram stains 1

• Negative culture• Bacteria seen in 21% of gram stains 1

1. Madison J, Sommer M, Spencer P. Relations among synovial membrane histopathologic findings, synovial fluid cytologic findings, and bacterial culture results in horses with sus- pected infectious arthritis: 64 cases (1979–1987). J Am Vet Med Assoc 1991;198:1655–61.

Septic Arthropathies• Culture can be improved via:

• Directly transfer of SF into biphasic blood culture bottle• Media for most aerobic organisms

• Larger size to facilitate larger SF volume• Maximal amount of SF possible per bottle size is ideal

• Contains inhibitors of antimicrobials• Provide media for anaerobic organisms

• Using blood culture media• Results in detection rates of 55% to 78% 1

• Culture can be delayed• Growth from infected SF can take up to 7 – 10 days

1. Schneider R, Bramlage L, Moore R, et al. A retrospective study of 192 horses affected with septic arthritis/tenosynovitis. Equine Vet J 1992;24:436–46.

Septic Arthropathies• Culture results as a prognostic indicator

• Discharge from hospital survival• 89.9%, 185 of 206 horses

• Overall survival & long-term return to function• Positive culture = 50% return (24/48)• Negative culture = 70.5% return (74/105)

1. Taylor A, Mair T, Smith L, Perkins J. “Bacterial culture of septic synovial structures of horses: does a positive bacterial culture influence prognosis? Equine Vet J. (2010) 42, 213 – 218.

Septic Arthropathies• Cytology

• Paramount in determining sepsis on initial exam

• TNCC is usually at least >30 x 109/L• And often are much higher • Can be as low as 10 x 109/L

• TNCC over 100 x 109/L are considered pathognomonic

• TNCC between 5 (x109/L) to 10 (x109/L)• Grey zone, mild cases, interpret with suspicion

Septic Arthropathies• Cytology

• TNCC between 5 (x109/L) to 10 (x109/L)

• Differentials• Early stages of sepsis• Secondary to corticosteroid injection (anti-inflammatory)• Open and draining• Low virulence organism• Traumatic non-septic arthropathies• Chemical synovitis

Septic Arthropathies• Cytology

• TNCC will be composed predominately of neutrophils• Neutrophils over 80%

• Degenerate vs. non-degenerate• Degenerate is intuitive• However predominately non-degenerate is fairly common

• Bacterial toxins are not released as much as in other body fluids

• Total Protein• Over 4.0 g/dL is consistent with sepsis

Septic Arthropathies• Sepsis post-corticosteroid administration

• Characteristics:• Delayed onset of clinical signs, TNCC, and total protein 1

• Anti-inflammatory effect

• High neutrophil percentage 1

• >90%

• pH of SF less than 6.9 1

1. Tumalo R,Bramlage L,Gabel A.The influence of corticosteroids on sequential clinical and synovial fluid parameters in joints with acute infectious arthritis in the horse. Equine Vet J 1989;21(5):332–7.

Septic Arthropathies• pH of synovial fluid

• Can use subjective pH strips

• Normal is similar to serum• 7.3 +/- 0.1

• pH decreases with infection 1

• < 6.9

1. Tumalo R,Bramlage L,Gabel A.The influence of corticosteroids on sequential clinical and synovial fluid parameters in joints with acute infectious arthritis in the horse. Equine Vet J 1989;21(5):332–7.

Septic Arthropathies• Lactate

• Can be performed with stall-side lacto-meter

• Normal is less than 3.9 mmol/L

• Tarsocrural joints experimentally inoculated with S. aureus 1

• Lactate was > 4.9 mmol/L in 66% of joints• All saline controls were < 4.4 mmol/L

1. Tumalo R,Bramlage L,Gabel A.The influence of corticosteroids on sequential clinical and synovial fluid parameters in joints with acute infectious arthritis in the horse. Equine Vet J 1989;21(5):332–7.

Septic Arthropathies• Glucose

• Use stall-side glucometer• Serum & synovial fluid difference (SSFD)

• Normal• Serum & SF are approximately the same• SF can be slightly lower than serum• Normal SSFD = 0.85 (+/- 0.59 mmol/L)

• Tarsocrural joints experimentally inoculated with S. aureus 1

• SSFD was > 2.2 mmol/L in 83% of samples

1. Tumalo R,Bramlage L,Gabel A.The influence of corticosteroids on sequential clinical and synovial fluid parameters in joints with acute infectious arthritis in the horse. Equine Vet J 1989;21(5):332–7.

Septic Arthropathies• Serum Amyloid A (SAA)

• Acute phase inflammatory protein• Synthesized in liver

• Significantly higher in horses with septic joints compared to healthy joints or low inflammatory arthropathies 1

• Healthy joints, osteoarthritis, non-septic synovitis (WBC < 0.5 x 109/L)

• Does not change in response to repeat arthrocentesis• Unlike protein

• Increases 4 – 8hr following experimental induction of arthritis• Easily measured

• Results within 30 minutes

1. Jacobsen S, Thomsen MH, Nanni S. Concentrations of serum amyloid A in serum and synovial fluid from healthy horses and horses with joint disease. Am J Vet Res 2006;67(10): 1738–42.

Septic Arthropathies• Serum Amyloid A (SAA)

• Group 1 – Healthy• Group 2 – Non-septic arthropathies

• 7 horses

• Group 3 – Septic arthropathies• 8 horses

• Group 4 - Osteoarthritis

1. Jacobsen S, Thomsen MH, Nanni S. Concentrations of serum amyloid A in serum and synovial fluid from healthy horses and horses with joint disease. Am J Vet Res 2006;67(10): 1738–42.

Septic Arthropathies• Matrix Metalloproteinases

• Proteolytic enzymes up-regulated with inflammation• Actively degrade cartilage• Assay’s not widely available for clinical-setting

• MMP-9 • Gelatinase• Up-regulated with joint sepsis• Active form of MMP-9 is increased proportional to TNCC• MMP-9 is predominately produced by neutrophils• Concentration of MMP-9 and ratio of MMP-9 : MMP-2 1

• Predictive of survival of septic arthritis

1. Kidd JA, Barr AR, Talton JF. Use of matrix metalloproteinases 2 and 9 and white blood cell counts in monitoring the treatment and predicting the survival of horses with septic arthritis. Vet Rec 2007;161:329–34.

Septic Arthropathies• Neutrophil Viability / Cell Death

• Proportion • Viable versus dead neutrophils• Apoptotic to necrotic neutrophils

• Apoptosis: Plasma membrane blebbing, chromatin condensation, nuclear fragmentation, apoptotic body formation

• Necrosis: Rapid plasmic swelling, plasma membrane rupture, organelle breakdown

• Neutrophils apart of both septic and non-septic inflammation• During infection, neutrophils live longer

• Accumulate in the SF

• Resolution of infection via bacterial phagocytosis, intracellular killing, and apoptosis of neutrophils

1. Wauters J, Pille F, Martens A, et. al. Viability and cell death of synovial fluid neutrophils as diagnostic biomarkers in equine infectious joint disease: a pilot study. Res Vet Sci. (2012) 92: 132 – 137.

Septic Arthropathies• Neutrophil Viability / Cell Death

1. Wauters J, Pille F, Martens A, et. al. Viability and cell death of synovial fluid neutrophils as diagnostic biomarkers in equine infectious joint disease: a pilot study. Res Vet Sci. (2012) 92: 132 – 137.

Septic Arthropathies• Myeloperoxidase (MP)

• Enzyme released by neutrophils• Stored in granules, released in presence of bacteria• Active and inactive state

• Involved in the production of reactive oxygen species

• Evaluation as a biomarker 1

• Both total MP and active state of MP• Significantly higher MP in (culture-positive) septic SF

• Compared to health, OCD and non-septic synovitis cases

1. Wauters J, Pille F, Martens A, et. al. Equine myeloperoxidase: A novel biomarker in synovial fluid for the diagnosis of infection. EVJ 2013. 45: 278 – 283.

Septic Arthropathies• Myeloperoxidase (MP)

1. Wauters J, Pille F, Martens A, et. al. Equine myeloperoxidase: A novel biomarker in synovial fluid for the diagnosis of infection. EVJ 2013. 45: 278 – 283.

Septic Arthropathies• Myeloperoxidase (MP)

• Cut-offs:• Total MP = 5000ng/ml• Active MP = 350ng/ml

• Adequate sensitivity, specificity, PPV, NPV at these cut-offs

Septic Arthropathies• Polymerase Chain Reaction (PCR)

• Rapid, accurate• Can detect bacteria in presence of antibiotics• No antibiotic sensitivity information• False positives

• Contamination during handling / lab analysis• Non-infective microbial contaminants

• PCR and ‘culture using BCM’ together has significant increase in sensitivity compared to ‘culture using BCM’ alone 1

1. Pille F, Martens A, Schouls LM, et al. Broad range 16S rRNA gene PCR compared to bac- terial culture to confirm presumed synovial infection in horses. Vet J 2007;173:73–8.

Septic Arthropathies• D-Dimer

• Fibrinolytic system activated in inflamed joints• Protective, homeostatic response

• D-dimer measures fibrinolytic activity• Higher in foals with septic joints compared to foals without joint

disease 1

• Groups:• Septic foals with septic joints• Septic foals without septic joints• Non-septic foals with septic joints• Healthy foals

• Also higher with osteochondrosis, but not osteoarthritis 2

1. Ribera, T., Monreal, L., Armengou, J. “Synovial fluid d-dimer concentration in foals with septic joint disease.” J Vet Intern Med (2011) 25: 1113 – 1117.

2. Ribera, T. Monreal, L. Delgado, M. “Synovial fluid d-dimer concentration in horses with osteochondritis dissecans and osteoarthritis.” Vet Comp Orthop Traumatol. (2013) 26: 54 – 60.

Septic Arthropathies• D-dimer 1

1. Ribera, T., Monreal, L., Armengou, J. “Synovial fluid d-dimer concentration in foals with septic joint disease.” J Vet Intern Med (2011)25: 1113 – 1117.

Take Home Points

• Can be difficult to…• Separate marked non-septic arthropathies from mild-septic arthropathies

• Attain positive culture

• Parameters that are highly indicative of infection:• Total Nucleated Cell Count: > 30 x 109 /L

• Cell Differential: > 80% neutrophils

• Total Protein: > 4.0 g/dL

• Presence of bacteria on gram stain

• Novel biomarker research is being developed, but limited

availability for clinical setting

QUESTIONS?