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AMBULATORY PRACTICE How to Obtain Useful Information From a Grossly Blood-Contaminated Synovial Fluid Sample James L. Carmalt, MA, VetMB, MVetSc, FRCVS, Diplomate ABVP (Eq), ACVS*; Imma Roquet, DVM, MVetSc; and Steve Hendrick, DVM, DVSc Authors’ address: Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, 52 Campus Drive, University of Saskatchewan, Saskatoon, SK S7N 5B4 Canada; e-mail: [email protected]. *Corresponding and presenting author. © 2013 AAEP. 1. Introduction Synovial fluid examination in the horse is a routine procedure in equine practice. One of the most valuable uses of synovial fluid (SF) analysis is to determine the presence or absence of infection within a synovial structure.1–3 Normal equine SF is clear to straw-colored and highly viscous. It is an ultrafiltrate of plasma, therefore similar concentrations of glucose and electrolytes are expected.3 The total protein (TP) concentration and white blood cell count (WBC), however, are much lower, and the prevailing cell type is mononuclear. SF should contain very few to no erythrocytes. The reported normal values of SF WBC range from ⬍300 to 1000 cells per microliter, containing mainly mononuclear cells with ⬍10% neutrophils on cytologic examination.3–7 Normal TP concentrations have been variably described as ⬍25% to 35% of the systemic plasma TP and ⬍0.8 to 2.5 g/dL.3,4 Values of WBC and TP above 10 ⫻ 109/L and 50 g/L, respectively, with ⬎80% neutrophils, are highly suggestive of septic arthritis; however, lower levels do not necessarily rule this out.3 The definitive diagnosis of a septic synovial structure of bacterial NOTES 62 2013 Ⲑ Vol. 59 Ⲑ AAEP PROCEEDINGS origin is a positive culture of the SF and/or synovial membrane.5 Many instances occur in equine practice, in which clients are faced with the difficult decision of whether or not to initiate aggressive treatment because of economic constraints. Unfortunately, it is not uncommon for an equine practitioner to be unable to provide a definitive diagnosis of septic arthritis for multiple reasons, including an iatrogenically blood contaminated sample. This report shows how to obtain diagnostically useful synovial fluid TP and WBC values from a blood-contaminated sample, given a concurrent complete blood count. 2. Materials and Methods To be able to compute useful WBC counts and TP values from a blood contaminated synovial sample, one must obtain both a synovial fluid sample and a peripheral venous blood sample (usually by means of the jugular vein). The hematocrit (HCT) and WBC counts of both the synovial fluid and blood sample are obtained, as well as the TP concentration of the synovial fluid in the laboratory. AMBULATORY PRACTICE Table 1. Formula for Calculation of Uncontaminated Total Protein From a Blood-Contaminated Synovial Fluid Sample Laboratory Values Horse Name/Number 9 3.49 2.90 9.92 7.00 11.10 30.50 Synovial WBC, *10 /L Synovial TP, g/L Blood WBC, *109/L Blood TP, g/L Synovial hematocrit, % Blood hematocrit, % Calculated values Uncontaminated TP, g/L Uncontaminated WBC, *109/L 1.25 0.78 A ratio of HCT must be calculated to determine the degree of contamination for the synovial fluid sample. Ratio HCT ⫽ HCT SF /HCT Blood (1) The difference between the values obtained from the contaminated synovial fluid sample and those that would have been obtained in a completely blood-free sample is then calculated as: TP DIFF ⫽ ⫺0.063⫺0.0542 (HCT SF ) ⫹ 6.34(RatioHCT) (2) WBC DIFF ⫽ ⫺0.01916 ⫹ 0.01373 WBC Blood ⫹ 0.08689 HCT SF ⫹ 0.01479(WBC Blood * HCT SF ) (3) As TPDIFF ⫽ TP (contaminated SF sample)⫺TP (theoretical blood-free sample), simple substitution is used to calculate the blood-free value for this variable. Similarly, WBCDIFF ⫽ WBC (contaminated SF sample)⫺WBC (theoretical blood-free sample) can be used to calculate the blood-free synovial fluid WBC count. This process seems cumbersome; however, in the authors’ practice, a Microsoft Excel spreadsheet has been constructed such that entering the laboratory values will automatically generate blood-free– calculated values without having to resort to using a calculator every time (Tables 1 and 2). 3. Discussion The mathematical models used above were generated in a two-part study.8 Briefly, peripheral venous blood and SF was obtained from 10 adult horses. The SF samples were split (into six) and subsequently contaminated with the use of autologous blood added in 10% increments from 0% to 50%. In some horses, autologous plasma was also added to artificially raise the baseline total protein concentration. An automated cell count was obtained followed by direct smears in both the SF and venous blood samples. The packed cell volume (PCV) and the TP concentration were measured with a hand-held refractometer after sample centrifugation. Dummy variables (RatioHCT, WBCDIFF, TPDIFF, and NPDIFF) were created as the difference between the variable count of a sample with a known percentage of contamination and one with 0% contamination. With the use of a combination of best-subset regression and linear regression techniques, mathematical models were generated that accounted for the change in TP concentration and WBC counts as a function of contamination. The models were then tested in five additional horses. Samples were contaminated by one author and the results presented to another author in blinded fashion. When noncontaminated TP and WBC counts were calculated and compared statistically with actual measured values, there was no significant difference (P ⫽ 0.3 and P ⫽ 0.53, respectively), despite calculated TP concentrations being 0.14 g/L higher and the calculated WBC count being 0.07*109/L lower than measured. In the original research, several interesting observations were made. First, it took almost 50% blood contamination (of normal synovial fluid) to raise the synovial fluid TP concentration toward the reported septic “cut-off value” of 2.5 g/dL. Adding blood also raised the neutrophil percentage; however, after the initial addition, further contamination did not change the percentage significantly and thus a “corrected” neutrophil percentage could not be calculated. Second, even in the most heavily contaminated samples, maximum values for synovial TP and WBC were 3.23 g/dL and 5.03 * 109/L, respectively. This would indicate that even in blood-contaminated samples, a total WBC and TP above 20 ⫻ 109/L and 4 g/dL, respectively, would be highly suggestive of a septic process. Finally, our opinion that it is unlikely that SF harvested in a clinical situation would be submitted for subsequent laboratory analysis if ⬎10% contaminated because at this point the sample is visually indistinguishable from blood. The methods presented above are used clinically to calculate noncontaminated SF TP concentration and WBC counts from blood-contaminated samples. However, diagnosing a potential septic arthritis is Table 2. Formula for Calculation of Uncontaminated White Blood Cell Count From a Blood-Contaminated Synovial Fluid Sample Laboratory Values 9 Synovial WBC, *10 /L Synovial TP, g/L Blood WBC, *109/L Blood TP, g/L Synovial hematocrit, % Blood hematocrit, % Calculated values Uncontaminated TP, g/L Uncontaminated WBC, *109/L Horse Name/Number 3.49 2.90 9.92 7.00 11.10 30.50 1.25 0.78 AAEP PROCEEDINGS Ⲑ Vol. 59 Ⲑ 2013 63 AMBULATORY PRACTICE still a challenge when results are above normal but are not above the published cut-off values for septic arthritis. We believe that in these circumstances, use of other diagnostic tools, including cytology as well as the clinical picture and clinician experience, cannot be overrated. References and Footnote 1. Bertone AL. Infectious arthritis. In: McIlwraith CW, Trotter GW, editors. Joint Disease in the Horse. Philadelphia: WB Saunders; 1996:397– 408. 2. Bertone AL, McIlwraith CW, Jones RL, et al. Comparison of various treatments for experimentally induced equine infectious arthritis. Am J Vet Res 1987;48:519 –529. 3. Steel CM, Equine Synovial Fluid Analysis. Vet Clin North Am Equine Pract 2008;24:437– 454. 64 2013 Ⲑ Vol. 59 Ⲑ AAEP PROCEEDINGS 4. Frisbie DD. Synovial joint biology and pathobiology. In: Auer JA, Stick JA, editors. Equine Surgery. 3rd edition. Philadelphia: Saunders Elsevier; 2006:1036 –1055. 5. Schneider RK. Synovial and osseous infections. In: Auer JA, Stick JA, editors. Equine Surgery. 3rd edition. Philadelphia: Saunders Elsevier; 2006:1121–1130. 6. Trotter GW, Mcllwraith CW. Clinical features and diagnosis of equine joint disease. In: McIlwraith CW, Trotter GW, editors. Joint Disease in the Horse. Philadelphia: WB Saunders; 1996:120 –144. 7. Schneider RK, Bramlage LR, Moor RM, et al. A retrospective study of 192 horses affected with septic arthritis/tenosynovitis. Equine Vet J 1992;24:436 – 442. 8. Roquet I, Hendrick S, Carmalt JL. The effect of blood contamination on equine synovial fluid analysis. Vet Comp Orthop Traumatol 2012;25:460 – 465. a Microsoft Excel, Microsoft Corporation. One Microsoft Way, Redmond, WA 98052– 6399.