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Cell counting in equine synovial uid samples

woensdag 15 mei 13

Lameness in horses
Orthopedic problems with lameness is the most common problem in equine veterinary practice.
The lameness examination aims to:
rst localize the origin of pain, then obtain more information about that source of pain, and nally to determine the best possible treatment.

Boule 2013-02-07 woensdag 15 mei 13

Lameness in horses
Diagnostic methods in investigation of lameness in horses
Trotting-up and joint exion tests Nerve blocks or joint blocks Imaging techniques (radiographs, ultrasound, scintigraphy, MRI) Synovial uid (joint uid) analysis

Collection of synovial uid from a joint in a horse using sterile technique

Boule 2013-02-07 woensdag 15 mei 13

Synovial uid analysis

Total nucleated cell counts in equine synovial uid are important tools in evaluating joint inammation.
Information for:
Diagnosis Prognosis Treatment

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 inammation

Boule 2013-02-07 woensdag 15 mei 13

Normal joint synovial uid Color Turbidity Viscosity

Low; no RBC; very few nucleated cells: Horses: <500/L (<0.5x109/L) Cattle: <1000/L (<1.0x109/L) Dogs/cats: <1500/L(<1.5x109/ L) <6-12% 90-100% 18-48 g/L (generally <25 g/L) No toxic cells or microorganisms Transparent and colorless / yellow None Very viscous 2-5 cm string

Normal joint uid:

High viscosity Few cells

Cell count

Neutrophils Mononuclear cells* Total protein Other features

Inamed joint uid:

Decreased viscosity Increased cellularity (neutrophils)

Boule 2013-02-07 woensdag 15 mei 13

Manual nucleated cell count

Labor-intensive Time-consuming Unsuitable for rapid analysis Poor reproducibility Variation among and within laboratories

Hemocytometer + microscope

There is need for an automated nucleated cell counting method for synovial uid that would be faster, easier, and more precise.
Boule 2013-02-07 woensdag 15 mei 13

Automated nucleated cell count of equine synovial uid

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 uid sometimes causes analytical errors due to irregular ow
Impedance-based hematology analyzer

Boule 2013-02-07 woensdag 15 mei 13


Boule 2013-02-07 woensdag 15 mei 13

Reducing viscosity in synovial uid samples

Treatment of sample with the enzyme hyaluronidase before analysis
Decreases viscosity Avoid instrument error ags The enzyme breaks up the large molecule hyaluronan (hyaluronic acid) by hydrolysis

Boule 2013-02-07 woensdag 15 mei 13

Optimal use of hyaluronidase


Test for the optimal use of hyaluronidase enzyme on viscous equine synovial uid samples
Equine synovial samples with (normal) low cell counts (WBC <1x109/L, n=3), in EDTA-tubes These samples gave instrument error ags due to (normal) viscosity Treatment with Hyaluronidase (Roche Diagnostics Scand. AB, Sweden) was tested to abolish viscosity-related instrument error Tested nal concentrations of enzyme in synovial uid: 1 to 0.000025 mg/ml diluted in the hematology analyzers diluent (Mediton II-Vet, Boule Medical AB, Sweden)

Boule 2013-02-07 woensdag 15 mei 13

Optimal use of hyaluronidase

Tested conditions:
Incubation temperatures: Room temperature (RT) and 37C 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 -18C and thawed after 7 days Hematology analyzer: Medonic CA620-VET (a.k.a; Heska CBCdiff; Boule Medical AB, Sweden)

Boule 2013-02-07 woensdag 15 mei 13

Optimal use of hyaluronidase

! Enzyme solution was stable and produced reproducible cell counts without error ags
Hyaluronidase enzyme was potent: dilution to 0.001 mg/ml gave good results. However, for practical purposes, 0.01 mg/ml (10 times higher nal concentration) gives a more convenient volume of enzyme solution, still with neglible dilution of samples of small volume. Incubation in RT and 37C 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.

Boule 2013-02-07 woensdag 15 mei 13

Practical protocol & advice

Add 0.01 mL of hyaluronidase 0.5 mg/mL (diluted in Mediton IIvet) to 0.5 mL of equine synovial uid from an EDTA-tube. Incubate for at least 2 minutes before automated analysis. Apply the protocol for equine synovial uid 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 uid samples).

Boule 2013-02-07 woensdag 15 mei 13

This PowerPoint presentation was prepared by Gittan Grndahl, DVM, PhD National Veterinary Institute Uppsala, Sweden

Gittan Grndahl, DVM, PhD

Boule 2013-02-07 woensdag 15 mei 13