Ethylenediaminetetraacetic acid (EDTA) anticoagulant affects the refractometric measurement of total protein concentration in equine synovial fluid

The objectives of this study were to (i) determine the effect of ethylenediaminetetraacetic acid (EDTA) concentration on the refractometric measurement of the total protein (TP) concentration of equine synovial fluid and (ii) assess the effect of time and temperature on the refractometric measurement of TP concentration. Synovial fluid was retrieved aseptically from horses presenting to Liphook Equine Hospital either (a) immediately after euthanasia or (b) from those requiring arthrocentesis as part of their clinical investigation. Samples were aliquoted into commercially available (i) plain tubes (0.5 mL; control) and (ii) EDTA tubes (at different volumes to obtain serial dilutions of EDTA:synovial fluid). TP concentration was measured by two independent observers using a calibrated hand-held refractometer. Samples from a separate group of horses were stored for 24 h at 4°C and 20°C before analysis to assess the effects of time and temperature. Results were compared using repeated measures ANOVA. TP concentration was significantly higher at all EDTA concentrations compared to control samples, with the greatest effect seen with higher concentrations (P<0.0001 for samples with ≥3.17 mg EDTA/mL). Storage time or temperature did not significantly affect TP concentration. Refractometric measurement of small volumes of synovial fluid in commercially available EDTA tubes leads to artificially elevated TP concentrations. Delays in sample analysis and alterations in storage conditions did not significantly affect TP concentration within the parameters studied. The magnitude of overestimation of synovial total protein concentration could result in misclassification of suspect synovial sepsis cases where EDTA tubes are used.