Cell counting in equine synovial fluid samples woensdag 15 mei 13
Cell counting in equine synovial fluid samples
woensdag 15 mei 13
Boule 2013-02-07
Lameness in horses
Orthopedic problems with lameness is the most common problem in equine veterinary practice.
The lameness examination aims to: first localize the origin of pain, then obtain more information about that source of pain, and finally to determine the best possible treatment.
woensdag 15 mei 13
Boule 2013-02-07
Lameness in horses
Diagnostic methods in investigation of lameness in horses Trotting-up and joint flexion tests Nerve blocks or joint blocks Imaging techniques (radiographs, ultrasound, scintigraphy, MRI) Synovial fluid (joint fluid) analysis
Collection of synovial fluid from a joint in a horse using sterile technique
woensdag 15 mei 13
Boule 2013-02-07
Synovial fluid analysis
Septic arthritis (caused by bacterial infection):
Nucleated cell count (NCC) Bacterial culture Cytologic smears
Nonseptic conditions (not caused by bacterial infection):
Changes in neutrophil counts can indicate the magnitude of synovial membrane inflammation
Information for: Diagnosis Prognosis Treatment
Total nucleated cell counts in equine synovial fluid are important tools in evaluating joint inflammation.
woensdag 15 mei 13
Boule 2013-02-07
Normal joint synovial fluidColor Transparent and colorless /
yellow
Turbidity None
Viscosity Very viscous
Viscosity 2-5 cm string Viscosity
Cell count
Low; no RBC; very few nucleated cells:
Cell count Horses: <500/µL (<0.5x109/L)Cell count Cattle: <1000/µL (<1.0x109/L)Cell countDogs/cats: <1500/µL(<1.5x109/
L)Neutrophils <6-12%
Mononuclear cells* 90-100%
Total protein 18-48 g/L (generally <25 g/L)
Other features No toxic cells or microorganisms
Normal joint fluid: High viscosity Few cells
Inflamed jointfluid:Decreased viscosityIncreased cellularity (neutrophils)
woensdag 15 mei 13
Boule 2013-02-07
Labor-intensive Time-consuming Unsuitable for rapid analysis Poor reproducibility Variation among and within
laboratories
Manual nucleated cell count
There is need for an automated nucleated cell counting method for synovial fluid that would be faster, easier, and more precise.
Hemocytometer + microscope
woensdag 15 mei 13
Boule 2013-02-07
Automated nucleated cell count of equine synovial fluid
High correlation and low pseudomedian difference compared to manual cell count from a smear
Better reproducibility (CV 1.5-2.7%) than the manual method (CV 6.1-15.7%)
Viscosity of synovial fluid sometimes causes analytical errors due to irregular flow
Impedance-basedhematology analyzer
woensdag 15 mei 13
Boule 2013-02-07
Article
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woensdag 15 mei 13
Boule 2013-02-07
The enzyme breaks up the large molecule hyaluronan (hyaluronic acid) by hydrolysis
Reducing viscosity in synovial fluid samples
Treatment of sample with the enzyme hyaluronidase before analysis Decreases viscosity Avoid instrument error flags
woensdag 15 mei 13
Boule 2013-02-07
Optimal use of hyaluronidase ! Test for the optimal use of hyaluronidase enzyme on viscous
equine synovial fluid samples Equine synovial samples with (normal) low cell counts (WBC <1x109/L, n=3), in
EDTA-tubes These samples gave instrument error flags due to (normal) viscosity Treatment with Hyaluronidase (Roche Diagnostics Scand. AB, Sweden) was tested
to abolish viscosity-related instrument error Tested final concentrations of enzyme in synovial fluid: 1 to 0.000025 mg/ml diluted
in the hematology analyzer’s diluent (Mediton II-Vet, Boule Medical AB, Sweden)
woensdag 15 mei 13
Boule 2013-02-07
Optimal use of hyaluronidase Tested conditions: Incubation temperatures: Room temperature (RT) and 37°C Incubation times:
2, 3, 5, 6, 10, 11, 30 and 31 minutes at RT with 0.01 mg/ml enzyme Effect of storage of enzyme solution tested after 0,7 and 9 days in RT;
and if frozen in -18°C and thawed after 7 days Hematology analyzer: Medonic CA620-VET (a.k.a; Heska CBCdiff;
Boule Medical AB, Sweden)
woensdag 15 mei 13
Boule 2013-02-07
! Enzyme solution was stable and produced reproducible cell counts without error flags Hyaluronidase enzyme was potent: dilution to 0.001 mg/ml gave good results.
However, for practical purposes, 0.01 mg/ml (10 times higher final concentration) gives a more convenient volume of enzyme solution, still with neglible dilution of samples of small volume.
Incubation in RT and 37°C both worked. Incubation times between 2 to 31 minutes produced similar results. The enzyme solution was stable after at 7 and 9 days storage
in a refrigerator, and after freezing for 7 days and thawing.
Optimal use of hyaluronidase
woensdag 15 mei 13
Boule 2013-02-07
Apply the protocol for equine synovial fluid samples as a routine, or when viscosity-related error occurs.
The stock solution of enzyme can be frozen in vials and used for at least 9 days after thawing.
The cost of the hyaluronidase is minimal (around 70 USD for 100 g, which is enough for 100,000 synovial fluid samples).
Practical protocol & advice
Add 0.01 mL of hyaluronidase 0.5 mg/mL (diluted in Mediton II-vet) to 0.5 mL of equine synovial fluid from an EDTA-tube.
Incubate for at least 2 minutes before automated analysis.
woensdag 15 mei 13
Boule 2013-02-07 14
Gittan Gröndahl, DVM, PhD
This PowerPoint presentation was prepared by Gittan Gröndahl, DVM, PhDNational Veterinary InstituteUppsala, Sweden
woensdag 15 mei 13