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

Abstract

Summary 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.

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

“…Although protein is the predominant solute, other substances including electrolytes, glucose, urea and lipids also contribute to the angle of refraction and therefore the refractometric determination. EDTA is most frequently used in the form of salts (ie, K 2 EDTA, K 3 EDTA) to store equine SF samples to prevent sample coagulation and preserve cell morphology 4 5. The use of EDTA salts as an anticoagulant preservative caused an overestimation of the refractometric TP determinations on different body fluid samples on different species 4 5 19.…”

“…EDTA is most frequently used in the form of salts (ie, K 2 EDTA, K 3 EDTA) to store equine SF samples to prevent sample coagulation and preserve cell morphology 4 5. The use of EDTA salts as an anticoagulant preservative caused an overestimation of the refractometric TP determinations on different body fluid samples on different species 4 5 19. The overestimation effect seems to be concentration‐related and more frequent when commercial solutions of EDTA salts are used 5.…”

“…Overestimation does not occur when recommended concentrations of EDTA salts are used,20 but is associated with situations when the collection tubes are underfilled 19. It has been suggested that the overestimation may be related to the addition of EDTA salts that affect the total solids content and consequently the refractory index4 5 reported significantly increased TP refractometric determinations for SF samples collected in K 2 EDTA at concentrations from 3.17 mg/ml and above. In this study, a steady increase in the refractometric determination was observed as the K 2 EDTA concentration increased, but the difference was only significant for samples containing 17.6 mg/ml of K 2 EDTA (figure 1).…”

“…Blood contamination on the samples was considered minor by the operator and only cases from which a minimal volume of 3.5 ml of SF could be easily obtained were included. Centrifugation of blood samples before refractometric determination can be performed before refractometric determination of TP on serum or plasma,4 although high correlation in refractometric TP values was observed between centrifuged and non‐centrifuged serum samples in calves 28. Other body fluid samples such as SF or peritoneal fluid are not routinely centrifuged before refractometric TP determination in clinical practice.…”

“…However, in some instances, the amount of SF available is lower than the recommended volume for the collection tube, which causes the concentration of K 2 EDTA in the mixture to be higher than ideal. Previous studies have shown that TP determinations may be overestimated when the concentration of K 2 EDTA is higher than recommended in equine SF4 and peritoneal fluid 5. Lithium heparin (LH) has anticoagulant properties, is available in collection tubes and its concentration does not affect the determination of TP in equine peritoneal fluid 5…”