Plasma endotoxin concentration in horses: a methods study.
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.


CONCLUSIONS
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.
Peek
Simon F
SF
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA.
Borah
Sarah
S
Semrad
Susan
S
McGuirk
Sheila
S
Slack
Jo Ann
JA
Patton
Elisabeth
E
Coombs
Dane
D
Lien
Laura
L
Darien
Benjamin J
BJ
D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods / Q000662:veterinary*; D001792:Blood Platelets / Q000378:metabolism*; D001800:Blood Specimen Collection / Q000379:methods / Q000662:veterinary*; D019446:Endotoxemia / Q000097:blood / Q000175:diagnosis / Q000662:veterinary*; D006734:Horse Diseases / Q000097:blood* / Q000175:diagnosis; D006736:Horses; D007202:Indicators and Reagents; D008033:Limulus Test / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D007202:Indicators and Reagents