Concentration of dipotassium ethylenediaminetetraacetic acid but not lithium heparin affects total protein determination in equine synovial fluid

Abstract

BackgroundRefractometric determination of total protein (TP) in synovial fluid (SF) is commonly used for diagnosis and monitoring of synovial sepsis in horses. Previous studies have shown that elevated concentrations of certain anticoagulants may overestimate refractometric determination of TP concentration.ObjectivesThe aim of the study was to evaluate the effect of different concentrations of dipotassium EDTA (K2EDTA) and lithium heparin (LH) on TP determination by using a hand-held refractometer in equine synovial fluid.Study designCross-section observational study.MethodsThirty samples of synovial fluid obtained from 22 horses with different synovial conditions were collected. Synovial fluid samples were separated into different aliquots and placed in commercially available collection tubes containing K2EDTA or LH at four different concentrations (1.76, 3.52, 7.04 and 17.6 mg/ml for K2EDTA; 16, 32, 64 and 160 IU/ml for LH) . Refractometric TP determination was performed on untreated and K2EDTA and LH aliquots with a hand-held refractometer and by spectophotometric Biuret method as the gold standard.ResultsRefractometric TP determination was overestimated in SF samples containing 10 times the recommended K2EDTA concentrations. Lower concentrations of K2EDTA and LH concentrations did not affect refractometric TP determinations.Main limitationsLimited number of samples mostly obtained from large synovial structures.ConclusionTo avoid incorrect TP determination, the use of LH containing collection tubes may be an appropriate alternative when the SF volume available is not enough to fill the K2EDTA collection tube.

Effect of dipotassium‐ethylenediaminetetraacetic acid or lithium‐heparin treatments and storage times on selected clinicopathologic analytes in equine synovial fluid

“…This suggests that either heparin or EDTA salts may be used for anticoagulation to determine accurate TP concentrations. Rihuete et al 7 found, similar to our study, that the TP concentrations in equine SF were similar across the EDTA, heparin, and control groups, provided that the EDTA was used within the recommended concentrations. They showed that TP concentrations are only overestimated when EDTA is used in excess (more than four times the recommended 23 concentration of 1.2-2.0 mg/mL).…”

“…[4][5][6] Elevation of equine SF total protein (TP) to levels above 2 and 4 g/dL are likely compatible with inflammation and infection, respectively, while pH levels below 6.9 suggest the presence of joint infections. [6][7][8] Sample processing methods, storage times, and conditions affect the outcome of clinicopathologic and biochemical analyses. The American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance Guideline 9 emphasizes that the sample preparation method is an important source of pre-analytical errors, which not only encumbers reproducibility but also elevates the coefficient of variation of final results in metabolomic investigations.…”

“…The American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance Guideline 9 emphasizes that the sample preparation method is an important source of pre-analytical errors, which not only encumbers reproducibility but also elevates the coefficient of variation of final results in metabolomic investigations. While setting storage times, temperatures, or anticoagulant treatments as independent variables, several studies have reported significant variations in selected analytes of equine mesothelial and SFs, 7,10 and murine, 11 bovine, and ovine plasma. 12 Normal equine SF does not clot in vitro due to the absence of fibrinogen in the fluid; however, clinicians intending to analyze SF for diagnosis or prognosis of joint disease routinely collect SF samples in anticoagulated tubes because SF can clot under inflammatory conditions or when blood contamination of the SF sample occurs during collection.…”

“…LDH activity in equine SF has been used to detect sepsis with potential advantages of speed, ease of measurement, and lower cost relative to bacterial cultures 4–6 . Elevation of equine SF total protein (TP) to levels above 2 and 4 g/dL are likely compatible with inflammation and infection, respectively, while pH levels below 6.9 suggest the presence of joint infections 6–8 . Sample processing methods, storage times, and conditions affect the outcome of clinicopathologic and biochemical analyses.…”

“…The American Society for Veterinary Clinical Pathology (ASVCP) Quality Assurance Guideline 9 emphasizes that the sample preparation method is an important source of pre‐analytical errors, which not only encumbers reproducibility but also elevates the coefficient of variation of final results in metabolomic investigations. While setting storage times, temperatures, or anticoagulant treatments as independent variables, several studies have reported significant variations in selected analytes of equine mesothelial and SFs, 7,10 and murine, 11 bovine, and ovine plasma 12 …”