A Dextran-Based Warming Method for Preparing Leukocyte-Rich Plasma and its Clinical Application for Endotoxin Assay

Abstract

We devised a method using dextran for obtaining leukocyte-rich plasma (LRP) to measure endotoxin in blood. In order to find the optimal temperature for obtaining LRP, the measurement results were examined using samples prepared at 37 and 0°C. Sample separation time of LRP was significantly shorter at 37°C than at 0°C. Endotoxin measurement values showed a strong correlation between the two groups but many of the LRPs made at 37°C had measurements above those of the LRPs prepared at 0°C. The diagnostic accuracy for gram-negative bacterial infection was superior for LRP prepared at 37°C, with sensitivity and specificity of 96.8 and 100%, respectively.

Clinical relevance of leukocyte-associated endotoxins measured by semi-automatic synthetic luminescent substrate method

“…From these points, we believe that SALS has sufficient potential for clinical application in the future. The method of using LRP instead of plasma as the target of endotoxin measurement has already been reported by Kan et al 14 , and a method of collecting LRP at 37 °C using dextran as an aggregating agent has recently been devised by us (LRP37 method) 10 . Samples obtained by this method were used for measurement of this study.The method to obtain LRP uses the aggregating agent dextran under 1 × g conditions to remove erythrocytes which interfere the limulus test, and leukocytes are still suspended in plasma.…”

“…Heparinized blood was collected and equilibrated at 37 °C for about 10 min. Next, 800 µL of the blood and 400 µL of the 37 °C-equilibrated 6% dextran T500 were mixed and incubated at 37 °C about 15 to 20 min until almost clear upper layer was obtained 10 . The upper layer was collected as the LRP, whereas the lower layer was red blood cells layer aggregated by dextran.…”

“…Plasma and LRP were diluted 10-times with the pretreatment solution. In the case of LRP, LRP was mixed vigorously with a vortex mixer (MS-3, IKA Japan, Osaka, Japan) to ensure that leukocytes were destroyed 10 . Then that was heated at 70 °C for 10 min to inactivate the interfering factors 5 .…”

“…www.nature.com/scientificreports/ For example, if the blood volume and hematocrit value are 800 μL and 30, respectively, A is calculated as 560 (800-800 × 0.3). As the dextran volume is 400 μL, a multiplying factor of 1.71 is obtained by (560 + 400)/560 10 .…”

“…First, we examined the effect of proteins in the blood on this device. We also reported that detection sensitivity is improved by using leukocyte-rich plasma (LRP) obtained by the dextran warming method 10 . This study uses these new methods to measure endotoxins.…”