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)

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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.

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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