Clinical Pathology & Equine Arthropathies

Overview of equine synovial fluid evaluation, for diagnosis and prognostication of common equine arthropathies (including sepsis)

1. Clinical pathology OF Equine Arthropathies Dane M. Tatarniuk, DVM June 25 th , 2014

2. Physiology & Anatomy Joints, Bursas, Tendon Sheaths Plasma filtrate that provides Lubrication Regulatory cytokines Proteolytic enzymes Nutritional reservoir Highly viscous Gross appearance Pale yellow Transparent

3. 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

4. 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

5. 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 10 9 / L

6. 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?

7. 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)

8. 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

9. 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

10. 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

11. 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 ‘ X anthochromia ’

12. 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 10 9 / L 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.

13. 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 10 9 /L 1 Bertone AL. Update on infectious arthritis in horses. Equine Veterinary Education 1999; 11(3):143–52.

14. 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

15. 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

16. 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

17. 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

18. Analysis Hemocytometer

19. Analysis Total Nucleated Cell Count (TNCC) Joint Normal: 1.0 x 10 9 / L or less cells 1 Often as low as 0.2 x 10 9 / L Tendon Sheath Normal: 0.2 to 3.5 x 10 9 / L 2 Van Pelt RW. Interpretation of synovial fluid findings in the horse. J Am Vet Med Assoc 1974;165 :91–5 . 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.

20. 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

21. Analysis Protein (TP) True reference range for horses is 1.8 to 3.0 g /dL 1 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 /dL 2 Van Pelt RW. Interpretation of synovial fluid findings in the horse. J Am Vet Med Assoc 1974;165 :91–5 . 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.

22. 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

23. Analysis Cytological Exam Subjective Nucleated Cell Cellularity Normal or mild / moderate / markedly i ncreased 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

24. 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

25. Analysis Synoviocytes

26. Analysis Neutrophils, macrophage, erythrocytes

27. Analysis Erythrophagocytosis

28. Analysis Degenerate neutrophils, scattered cocci

29. Analysis Septic Inflammation

30. 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 10 9 /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 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 .

31. Non-septic Arthropathies Idiopathic synovitis, osteochondrosis dissecans, osteoarthritis TNCC less than 1.0 x 10 9 /L Normal to mild increase in TP Predominately mononuclear cells In osteoarthritis specifically TNCC can be up to 5.0 x 10 9 /L, but often is normal Depends on whether active synovitis is present

32. Non-septic Arthropathies Traumatic Synovitis Cell counts can rise to up to 10 x 10 9 / L Non-degenerate neutrophils predominate

33. 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 10 9 /L Duration ~1 to 3 days

34. Non-septic Arthropathies Immune-mediated Synovitis secondary to Rhodococcus Equi . i nfection in foals 1,2 Single or multiple joint distension Synovitis resolves once pneumonia responds to antibiotics Negative bacterial culture Mean TNCC: 8.0 x 10 9 / L Predominate neutrophils Immunofluorescence of synovium positive for immune complexes MadisonJB,ScarrattWK.Immune-mediatedpolysynovitisinfourfoals.JAmVetMedAs- soc 1988;192:1581–4 . Paradis M. Cutaneous and musculoskeletal manifestations of Rhodococcus equi infection in foals. Equine Veterinary Education 1997;9(5):266–70.

35. Non-septic Arthropathies Idiopathic Polysynovitis 1 No underlying systemic disease Stiffness & Polysynovitis Cytology Mean TNCC of 7.0 x 10 9 / 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 Pusterla N, Pratt SM, Magdesian KG, et al. Idiopathic immune-mediated polysynovitis in three horses. Vet Rec 2006;159:13–5.

36. Non-septic Arthropathies Eosinophilic Synovitis Rare In (limited) case reports, c oncurrent with peripheral eosinophilia Eosinophils in case reports were <20% of TNCC

37. 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) 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.

38. Septic Arthropathies Arthrocentesis 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.

39. 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 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 . 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.

40. 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 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.

41. 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 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.

42. 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) 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.

43. Septic Arthropathies Cytology P aramount in determining sepsis on initial exam TNCC is usually at least >30 x 10 9 /L And often are much higher Can be as low as 10 x 10 9 /L TNCC over 100 x 10 9 /L are considered pathognomonic TNCC between 5 ( x10 9 / L) to 10 ( x10 9 / L) Grey zone, mild cases, interpret with suspicion

44. Septic Arthropathies Cytology TNCC between 5 ( x10 9 / L) to 10 ( x10 9 / L) Differentials Early stages of sepsis Secondary to corticosteroid injection (anti-inflammatory) Open and draining Low virulence organism Traumatic non-septic arthropathies Chemical synovitis

45. 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 / d L is consistent with sepsis

46. 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 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.

47. 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 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.

48. 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 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.

49. 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 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.

50. 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 10 9 / 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 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.

51. 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 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.

52. 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 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.

53. Septic Arthropathies Neutrophil Viability / Cell Death Proportion V iable 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 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.

54. Septic Arthropathies Neutrophil Viability / Cell Death 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.

55. 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 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.

56. Septic Arthropathies Myeloperoxidase (MP) 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.

57. Septic Arthropathies Myeloperoxidase (MP) Cut-offs: Total MP = 5000ng/ml Active MP = 350ng/ml Adequate sensitivity, specificity, PPV, NPV at these cut-offs

58. 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 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.

59. 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: S eptic 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 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. 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.

60. Septic Arthropathies D-dimer 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.

61. 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 10 9 /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

62. QUESTIONS?