Relationship between automated total nucleated cell count and enumeration of cells on direct smears of canine synovial fluid

Abstract

Canine osteoarthritis is a common condition seen in veterinary clinical practice and causes considerable morbidity in dogs as they age. Synovial fluid analysis is an important tool for diagnosis and treatment of canine joint disease and obtaining a total nucleated cell count (TNCC) is particularly important. The low volume of fluid obtained during arthrocentesis is often insufficient for obtaining an automated TNCC, thereby limiting sample interpretation. The aim of the present study was to investigate whether estimation of TNCC in canine synovial fluid could be achieved by performing manual cell counts on direct smears of fluid. Fifty eight synovial fluid samples, taken by arthrocentesis from 48 dogs, were included in the study. Direct smears of synovial fluid were prepared, and hyaluronidase added before cell counts were obtained using a commercial laser-based instrument. A protocol was established to count nucleated cells in a specific region of the smear, using a serpentine counting pattern; mean number of nucleated cells/400× field was then calculated. There was a positive correlation between the automated TNCC and mean manual cell count, with more variability at higher TNCC. Regression analysis was performed to estimate TNCC from manual counts. By this method, 78% of the samples were correctly predicted to fall into one of three categories (within the reference interval, mildly to moderately elevated, or markedly elevated) relative to the automated TNCC. Intra-observer and inter-observer agreement was good to excellent. The results of the study suggest that interpretation of canine synovial fluid samples of low volume can be aided by manual cell counting of direct smears.

Clinical features and pathological joint changes in dogs with erosive immune-mediated polyarthritis: 13 cases (2004–2012)

“…A prospective study in which a standard protocol is used to manage dogs with IMPA should be performed so that the clinical outcomes for dogs with erosive IMPA and dogs with nonerosive IMPA can be compared. The treatment protocol we recommend for such a study is the administration of leflunomide as a disease-modifying agent until the estimated synovial fluid TNCC is < 3,000 cells/μL (as determined on the basis of cytologic evaluation of serially collected synovial fluid samples) 1,16,18 and administration of an NSAID for analgesia as needed. The cytologic analyses for the dogs of the present study were limited because only direct smears of synovial fluid samples were available for review.…”

“…Direct smears were considered unsuitable for review when the synovial fluid was unevenly distributed, the base of the smear covered less than two thirds of the width of the slide, the smear lacked a complete feathered edge or contained >10% ruptured nucleated cells. 18 Direct smears of synovial fluid samples that were unsuitable or unavailable for review were excluded from statistical analyses.…”

“…Suitable direct smears of synovial fluid samples were reviewed by use of a validated method for TNCC estimation 18 by 1 investigator (MLS). Within each counting field, all nucleated cells were counted, including those with pyknotic and karyorrhectic nuclei, as well as cells within groups and naked nuclei.…”

“…After correction for the dimensions of the 400X field area, the TNCC was estimated by use of the following regression formula: y = 0.45x − 0.36), where y is the estimated TNCC and x is the mean number of nucleated cells/400X field. 18 The numbers of PMNLs, mononuclear cells, lymphocytes, and eosinophils were estimated on the basis of the differential for a count of 100 to 200 cells that was obtained from the medical record. When available, bacterial culture and antimicrobial sensitivity results for synovial fluid samples obtained at the time of IMPA diagnosis were reviewed and reported.…”

“…4,17 When an adequate volume of synovial fluid cannot be obtained for a complete analysis, evaluation of a direct smear can be used to estimate the TNCC. 18 Evaluation of radiographic images of joints can be useful for determining which joints are affected and whether bone lysis is present in those joints. 4 Additional diagnostic testing modalities such as bacterial culture may be indicated to rule out infectious causes of polyarthritis.…”