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Plasma endotoxin concentration in horses: a methods study
Author(s) -
Peek Simon F.,
Borah Sarah,
Semrad Susan,
McGuirk Sheila,
Slack Jo Ann,
Patton Elisabeth,
Coombs Dane,
Lien Laura,
Darien Benjamin J.
Publication year - 2004
Publication title -
veterinary clinical pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.537
H-Index - 51
eISSN - 1939-165X
pISSN - 0275-6382
DOI - 10.1111/j.1939-165x.2004.tb00346.x
Subject(s) - limulus amebocyte lysate , ex vivo , platelet poor plasma , whole blood , limulus , chemistry , in vivo , chromatography , platelet , platelet rich plasma , sodium citrate , in vitro , medicine , immunology , biology , biochemistry , lipopolysaccharide , pathology , microbiology and biotechnology , paleontology
Background: Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation. Objective: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses. Methods: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet‐rich plasma (PRP), and platelet‐poor plasma (PPP) were spiked with endotoxin (2 EU/mL). Endotoxin‐spiked WB samples were centrifuged immediately to generate PRP for measurement. Endotoxin concentration was subsequently measured by Limulus amebocyte assay at 0, 15, 30, 45, and 60 minutes. Assays were performed in triplicate and results were analyzed using Student's t ‐test, with significance set at P < .05. Results: Mean endotoxin concentrations in 2 EU/mL‐spiked WB were significantly different from those in PPP at all time points tested. Recovery of endotoxin in PRP generated from WB was significantly diminished after just 15 minutes. Conclusion: PRP generated from WB is significantly more reliable than PPP in determining endotoxin concentration ex vivo. Measurement of endotoxin in PRP generated from WB was significantly diminished after 15 min, identifying a time frame within which to process blood samples for endotoxin analysis.