Clinical refractometer

Synovial fluid samples were obtained by routine aseptic technique. The sample was divided into 2 EDTA blood collection vials and 1 collection in a blood culture bottle (Oxoid Signal blood culture system, Oxoid microbiological products, Thermo Fisher, Hampshire, UK) or in a plain tube if the volume available was <10 ml. Cytology was performed within 12 h on 1 EDTA sample and the following parameters determined: NCC, %N, TP, and presence of intracellular bacteria. The NCC was determined using a Neubauer chamber after treating synovial fluid with hyaluronidase solution (Sigma Aldrich, UK). The other cytological parameters were determined by examination of direct smears and cytospin samples, stained with a modified Romanowsky stain, by a board-certified clinical pathologist. TP was quantified on EDTA samples by a clinical refractometer (Atago, Japan). Bacterial culture was performed on plain samples or blood culture samples using MacConkey and blood agar. Blood culture samples were processed according to the manufacturer's guidelines. The second EDTA sample was frozen for 1–2 months at −20°C until SAA quantification.

Over the very long observation period of this study, several hematology and chemistry analyzers were used. Blood samples for complete blood count (CBC) were collected in potassium-EDTA tubes and analyzed (Minos ST-Vet, Minos, Montpelier, France; Arcus, Abacus or Abacus Junior Vet, Diatron, Vienna, Austria; Advia 120 or 2120i, Siemens, Erfurt, Germany) within 30 min from collection. The packed cell volume (PCV) was measured manually by centrifuging whole blood in heparinized capillaries. Total plasma protein concentration was measured via refractometry (clinical refractometer, Atago, Tokyo, Japan). Blood samples for serum chemistry were collected in tubes with no anticoagulant; and with gel separators, allowed to clot, were centrifuged within 60 min from collection; and harvested sera were analyzed (Kone Progress Selective Chemistry Analyzer, Kone Corporation Instrument Group; Espoo, Finland; Maxmat SA PL, Maxmat, Montpellier, France; Cobas-Mira, Cobas Integra 400 Plus and Cobas 6000, Roche, Mannheim, Germany; at 37 °C) immediately, or stored at 4 °C pending analysis, performed within 12 h from collection. Blood samples for PT and aPTT were collected in 3.2% trisodium citrate tubes and centrifuged within 15 min from collection. Harvested plasma was then analyzed immediately (KC 1A micro, Amelung, Lemgo, Germany; ACL-200, ACL-9000 and ACL Top 300, IL, Milano, Italy).