Plasma endotoxin concentration in horses: a methods study.
Abstract: 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.
Publication Date: 2004-03-30 PubMed ID: 15048624DOI: 10.1111/j.1939-165x.2004.tb00346.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article studied the best methods to measure endotoxin concentration in horse blood. They found that platelet-rich plasma generated from whole blood provided a more accurate endotoxin measurement than platelet-poor plasma and that it was crucial to process the blood samples within 15 minutes.
Methods of the study
- The researchers collected whole blood from five healthy horses using an anticoagulant of 3.8% (0.12 M) sodium citrate.
- The blood samples were divided into three blood fractions: whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP).
- Each fraction was spiked with 2 EU/mL endotoxin for testing purposes.
- Endotoxin concentrations in these spiked samples were then measured using a Limulus amoebocyte assay at five different time intervals: at the time of extraction (0 minutes), and 15, 30, 45 and 60 minutes after extraction.
- All the tests were performed in triplicate and the results were statistically analyzed using the Student’s t-test, with a significance threshold set at P<.05 (anything less than this value was considered significant).
Findings of the study
- The study found that the mean endotoxin concentrations of 2 EU/mL spiked WB were significantly different from those of PPP at all time points.
- The recovery of endotoxin in PRP generated from WB decreased significantly after 15 minutes.
- The researchers concluded that PRP generated from WB is significantly more reliable than PPP for determining endotoxin concentration ex vivo (outside the body).
- Based on their findings, the team identified a time frame of 15 minutes within which blood samples should be processed for endotoxin analysis to obtain accurate results.
Conclusions of the study
- This study provides important guidelines for accurately measuring endotoxin concentration in horse blood. These guidelines are critical for cellular and molecular studies of endotoxemia in horses and potentially other species too.
- These findings highlight the importance of the quick processing of blood samples, especially when testing for endotoxins.
Cite This Article
APA
Peek SF, Borah S, Semrad S, McGuirk S, Slack JA, Patton E, Coombs D, Lien L, Darien BJ.
(2004).
Plasma endotoxin concentration in horses: a methods study.
Vet Clin Pathol, 33(1), 29-31.
https://doi.org/10.1111/j.1939-165x.2004.tb00346.x Publication
Researcher Affiliations
- Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA.
MeSH Terms
- Animals
- Blood Chemical Analysis / methods
- Blood Chemical Analysis / veterinary
- Blood Platelets / metabolism
- Blood Specimen Collection / methods
- Blood Specimen Collection / veterinary
- Endotoxemia / blood
- Endotoxemia / diagnosis
- Endotoxemia / veterinary
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horses
- Indicators and Reagents
- Limulus Test / veterinary
- Reproducibility of Results
- Sensitivity and Specificity
- Time Factors
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