The plasma sample, which contains almost no leukocytes, is generally used to measure endotoxin levels in blood. However, it is known that the diagnostic ability is not high. We assumed that most endotoxins exist in a state trapped by leukocytes, and then we have devised a method (LRP37 method) to obtain leukocyte-rich plasma (LRP) for measuring blood endotoxins. In this study, we selected the following two methods for measuring endotoxins in the LRP; the conventional turbidimetric kinetic assay (TKA) and the semi-automated luminescent substrate method (SALS) using the leukocyte fraction obtained the LRP. The endotoxin levels obtained from these methods were compared with the diagnostic ability of existing sepsis markers. The target cases were 40 who were admitted to our institution. Comparing the non-infected group with the Gram-negative infected group, the area under the curve of TKA and SALS were 0.94 and 0.86, respectively, and these diagnostic capabilities were the best among all the markers tested.
Article
Comparison of diagnostic ability of new blood endotoxin measurement method using leukocyte-rich plasma sample and existing sepsis markers
https://doi.org/10.21203/rs.3.rs-2961692/v1
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Leukocytes
Lipopolysaccharide
Sepsis
Luminescent substrate method
Turbidimetric kinetic assay
Procalcitonin
Presepsin
Interleukin-6
Endotoxins are cell wall component of Gram-negative bacteria and have various biological activities. The pathophysiological role of endotoxin in sepsis is of great importance. Endotoxins in blood can be measured by the Limulus test, which relies on the gelation reaction in horseshoe crab amebocyte lysates [1]. As a measurement method using this principle, the quantitative synthetic chromogenic substrate method [2] and the turbidimetric kinetic assay (TKA) [3] were developed. In Japan, an endotoxin-specific assay designed not to react with β-D glucan is used [3, 4]. TKA is approved for reimbursement by the Japanese National Health Insurance as a method for measuring endotoxin in human blood. This assay detects rising turbidity and has excellent detection limits. This method, which is widely used in Japan, uses a plasma sample obtained from heparinized. The measurement sensitivity of this sample is around 50% as far as we have examined [4, 5]. We believe that the reason for this low measurement sensitivity is that, many of endotoxins exist in a state bound to or taken up by leukocytes, and these endotoxins cannot be measured by this measurement method [4]. In fact, our studies to date have shown that the sensitivity of endotoxin measurement is improved by removing only red blood cells using dextran without centrifugation of whole blood [5]. We have previously reported this sampling method as the LRP37 method [5]. Therefore, the LRP37 method was adopted for this study.
The disadvantage of TKA is that it can take up to two hours at most. Therefore, we have used a new measuring system called semi-automated luminescent substrate method (SALS) [6] as well. For TKA, a 10-fold dilution of LRP is sufficient, but for SALS, it must be diluted to at least 75-fold because of the strong inhibitory factor activity in the plasma. Therefore, we centrifuged the LRP to obtain only the leukocyte fraction and prepared samples for SALS༎Thus, in this study, we used a method of measuring endotoxin in human blood by TKA for samples obtained by the LRP37 method (TKA with LRP) and a method of measuring only the leukocyte fraction by SALS (SALS with leukocytes). In addition, c-reacting protein (CRP), white blood cell (WBC), procalcitonin (PCT), presepsin (PSEP), and interleukin-6 (IL-6) were selected as existing sepsis markers, and their diagnostic abilities were compared.
1. Blood samples
Blood samples were obtained from 57 patients. Patients who were transported to the Iwate Advanced Critical Care Center from July 14, 2020 to December 31, 2022 were enrolled in this study. There were 17 non-infected cases such as trauma, 13 Gram-positive infections, and 27 Gram-negative infections. Written informed consent was obtained from all patients or their families for specimen collection. The International Consensus Definition of Sepsis and Septic Shock, 3rd Edition (Sepsis-3) was used to determine sepsis [7]. This study obtained ethical approval from the Ethics Committee of Iwate Medical University (Ethics Committee No. MH2020-004) and all procedures in this study were performed in accordance with the Declaration of Helsinki (World Medical Association 2013).
2.Measuement reagent
Pretreatment solution for plasma
A pretreatment solution for plasma endotoxin assay (0.02% Triton X-100 solution, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used [3].
Limulus amebocyte lysate (LAL)
Lyophilized LAL; Endotoxin Single Test WAKO (FUJIFILM Wako) was used. This reagent is specific for endotoxin by adding glucan [3].
Dextran T500
Six %(W/V) Dextran T-500 (Pharmacosmo, Holbaek, Denmark), an average molecular weight of 500,000 Da, was prepared in physiological saline for injection (Otsuka Pharmaceutical Co., Ltd., Naruto, Japan) and autoclaved it at 121°C for 90 min to ensuring to be endotoxin-free. In other words, no gelation occurred even after the maximum measurement time (200 min) of incubation in TKA (endotoxin content is calculated to be less than 0.05 pg/ml).
Preparation of LRP by the LRP37 method
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 [5]. The upper layer was collected as the LRP, whereas the lower layer was red blood cells layer aggregated by dextran. When measuring endotoxin by TKA, the specimen obtained by this method was used. When the specimen obtained by this method was examined under a microscope༎the recovery rate of leukocytes was approximately 96% of the peripheral white blood cell count. (Fig. 1A) Also, no leukocytes were detected in the plasma obtained by centrifugation of whole blood at 3000 rpm for 40 seconds. (Fig. 1B)
Preparation of leukocytes from LRP
LRP was centrifuged at 3000 rpm for 10 minutes in a centrifuge to obtain a precipitate as a leukocyte fraction. The volume of the liquid portion of LRP is diluted from the plasma volume by the volume of hematocrit and the dextran solution, the dilution factor being [(A + amount of dextran)/A)] (where A = blood volume - blood volume x hematocrit/100). For example, if the blood volume is 800 µL and the hematocrit value is 30, A is 560 (800–800 × 0.3) and the dilution factor is calculated as (560 + 400)/560 = 1.71 because the volume of dextran is 400 µL. That is, when 510 µL of LRP was centrifuged, 300 µL of water was added to the precipitate (500/1.71 = 298 µL) to obtain a specimen. In this case, the mixture was vigorously mixed with a vortex mixer (MS-3, IKA Japan, Osaka, Japan) to confirm destruction of leukocytes. This sample was used for endotoxin measurement by SALS.
3. Actual measurement method
TKA
LAL was dissolved with 200 µL of the pretreated LRP and placed the test tube to Toxinometer ET-5500 (FUJIFILM Wako), and measured endotoxin as described previously (3, 4). We then calculated the amount of endotoxin in the sample from the standard curve that had been plotted by using E. coli O111:B4 LPS. LRP were diluted 10-times with the pretreatment solution.
SALS
SALS developed for the endotoxin detection of dialysate in blood dialysis, by DKK-TOA Corporation (Tokyo, Japan) was used. The detail of this method was already described (6, 8, 9). Briefly, system consists of LAL tube, bioluminescence (BL) tube and a specially made luminometer Luminites®-ET connected with Windows PC. The LAL tube contains the LAL reagent (FUJIFILM WAKO). The BL tube, which is smaller in diameter than the LAL tube, contains ATP, luciferase and synthetic luminescent substrate Benzoyl-Leu-Gly-Arg-aminolluciferin, which are automatically added to the reaction mixture in the LAL tube, when the reaction is completed. High intensity-luminescence mutant firefly luciferase was isolated by DKK-TOA Corporation for this assay.
4. Measurement of infection markers
Procalcitonin (BioVendor R&D, Brno, Czech Republic) and interleukin 6 (R&D Systems, Minnesota, USA) were measured by Enzyme-linked immunosorbent assay, and presepsin (LSI Medience, Tokyo, Japan) was measured by Chemiluminescent enzyme immunoassay. Plasma samples obtained by centrifugation was used for the measurement.
5.Statistical analysis
Comparisons between two groups were made using the Mann–Whitney U test. The Spearman rank order correlation coefficient was employed for the analysis of correlations. We used the cutoff value obtained by receiver operating characteristic (ROC) analysis. A p-value < 0.05 was considered statistically significant. Two-group comparisons, ROC analysis, correlations, and multiple liner regression analyses were performed using JMP software (SAS Institute, Cary, NC, USA).
The measured values of each marker tended to be higher in the infected group than in the non-infected group, and the values of CRP, PCT, and PSEP were significantly higher in the infected group. No significant difference was observed in WBC, IL-6, TKA with LRP, and SALS with leukocytes. When the diagnostic ability was compared by the area under the curve (AUC), the AUC of PCT was the best at 0.83. Comparing the Gram-positive and Gram-negative groups, excluding the non-infected group, there was a tendency for Gram-negative bacteria to be high in SALS with leukocytes and TKA with LRP, and a significant difference was observed especially in TKA with LRP (Table 1,2). When comparing the non-infected group and the Gram-negative bacteria group, excluding the Gram-positive bacteria group, all markers were significantly higher in the Gram-negative group, and the AUC of TKA method with LRP was the best at 0.94. The AUC for SALS was 0.86, which was similar to that for PCT but better than the other parameters(Table 3). When the evaluation of the severity of each marker was compared by logistic regression analysis, a significant correlation was observed between the SOFA score and CRP, and a significant correlation was recognized between the APACHE II score and SALS with leukocytes(Table 4).
| Non-infected cases (n = 17) | Gram-positive infected cases (n = 13) | Gram-negative infected cases (n = 27) | |
|---|---|---|---|
| WBC (/µL) | 10.68 ± 2.09 | 10.37 ± 2.58 | 17.19 ± 1.69 |
| CRP (mg/dL) | 3.96 ± 2.68 | 8.95 ± 3.42 | 16.26 ± 2.23 |
| PCT (ng/mL) | 1.06 ± 2.81 | 7.93 ± 3.82 | 10.6 ± 2.05 |
| IL-6 (pg/mL) | 110.88 ± 1439.17 | 52375 ± 1892.32 | 4882.0 ± 1246.12 |
| PSEP (pg/mL) | 468.09 ± 1050.21 | 4596.04 ± 1414.9 | 2457.76 ± 599.36 |
| TKA with LRP (mEU/mL) | 13.57 ± 169.53 | 16.74 ± 224.67 | 456.67 ± 148.28 |
| SALS with leukocytes (mEU/mL) | 59.69 ± 143.41 | 14.54 ± 183.52 | 506.08 ± 122.03 |
| WBC: white blood cell CRP: C-reacting protein PCT: procalcitonin IL-6: intereukin-6 PSEP: presepsin | |||
| TKA: turbidimetric kinetic assay LRP: leukocyte rich plasma SALS: semi-automated luminescent method | |||
| Non-infected cases | Infected cases | ཐvalue | Area under curve | Cut-off value | Sensitivity | Specificity | |
|---|---|---|---|---|---|---|---|
| WBC (/µL) | 10.68 ± 2.09 | 14.97 ± 1.36 | 0.09 | 0.60 | 18.31 | 0.94 | 0.33 |
| CRP (mg/dL) | 3.96 ± 2.68 | 13.88 ± 1.75 | 0.03※ | 0.81 | 4.04 | 0.71 | 0.80 |
| PCT (ng/mL) | 1.06 ± 2.81 | 9.73 ± 1.82 | 0.01※ | 0.83 | 0.67 | 0.88 | 0.73 |
| IL-6 (pg/mL) | 110.88 ± 1439.17 | 3465.9 ± 938.24 | 0.06 | 0.77 | 8.0 | 0.47 | 0.95 |
| PSEP (pg/mL) | 468.09 ± 1050.21 | 3152.7 ± 684.73 | 0.03※ | 0.81 | 1136 | 1.00 | 0.50 |
| TKA with LRP (mEU/mL) | 13.57 ± 169.53 | 292.51 ± 110.57 | 0.15 | 0.80 | 33.6 | 0.94 | 0.60 |
| SALS with leukocytes (mEU/mL) | 59.69 ± 143.41 | 308.33 ± 93.49 | 0.17 | 0.67 | 22.7 | 0.70 | 0.65 |
| WBC: white blood cell CRP: C-reacting protein PCT: procalcitonin IL-6: intereukin-6 PSEP: presepsin | |||||||
| TKA: turbidimetric kinetic assay LRP: leukocyte rich plasma SALS: semi-automated luminescent method | |||||||
| ཐvalue | Area under curve | Cut-off value | Sensitivity | Specificity | |
|---|---|---|---|---|---|
| WBC (/µL) | 0.02※ | 0.67 | 10.74 | 0.64 | 0.71 |
| CRP (mg/dL) | < 0.01※ | 0.83 | 4.95 | 0.76 | 0.78 |
| PCT (ng/mL) | < 0.01※ | 0.86 | 0.69 | 0.88 | 0.82 |
| IL-6 (pg/mL) | 0.02※ | 0.80 | 136.18 | 0.88 | 0.60 |
| PSEP (pg/mL) | 0.04※ | 0.80 | 1136 | 0.94 | 0.49 |
| TKA with LRP (mEU/mL) | 0.07 | 0.94 | 33.6 | 0.89 | 0.89 |
| SALS with leukocytes (mEU/mL) | 0.02※ | 0.86 | 43.8 | 0.82 | 0.85 |
| WBC: white blood cell CRP: C-reacting protein PCT: procalcitonin IL-6: intereukin-6 PSEP: presepsin | |||||
| TKA: turbidimetric kinetic assay LRP: leukocyte rich plasma SALS: semi-automated luminescent method | |||||
Table4. Logistic regression analysis of severity scores and each infection markers
WBC: white blood cell CRP: C-reacting protein PCT: procalcitonin IL-6: intereukin-6 PSEP: presepsin
TKA: turbidimetric kinetic assay LRP: leukocyte rich plasma SALS: semi-automated luminescent method
A comparison of the diagnostic ability of existing sepsis markers such as CRP, PCT, IL-6, and PSEP has been reported so far, but no clear difference has been observed yet. Bacil et al [11]. assessed the diagnostic performance of sepsis in 33 ICU-admitted patients and reported that PCT was superior to IL-6. Meynaar et al [12]. also reported that PCT was superior to IL-6 and CRP. However, Du et al [13]. reported no difference in diagnostic performance between IL-6 and PCT, and Takahashi et al [14]. reported that IL-6 was the best for diagnosing sepsis. In recent years, there have been reports that PSEP [15, 16, 17] is the best, and a large-scale study on this topic is awaited. In this study, comparing the non-infected group and the infected group, the AUC of PCT was the highest, but the difference with CRP and PSEP was slight and not much different from previous reports. However, when the infected group was limited to Gram-negative cases, the diagnostic ability of TKA with LRP to distinguish between the uninfected group and the Gram-negative bacterial infection was excellent. This is considered to be the first finding, as there is no paper that examines the sepsis diagnostic ability of TKA with LRP. We believe that most endotoxins in blood are bound to leukocytes. Therefore, it is possible that the conventional endotoxin measurement method using plasma that contains almost no white blood cells cannot accurately evaluate endotoxin. In the sample obtained by actually centrifuging heparinized whole blood for 40 seconds, no leukocytes were found in the plasma, and it was confirmed that most leukocytes were recovered in the sample obtained by the LRP37 method. Based on these facts, we believe that the LRP37 method should be used for the samples used to measure blood endotoxins. In this research, we also examined specimens containing only the leukocyte for the clinical application of SALS. As a result, AUC of SALS with leukocytes was 0.86, which is slightly inferior to TKA with LRP. But we think this value is not so low. There is some variation in the measured values, and the measurement method needs further improvement. However, SALS has two advantages: most of the operation is automatic and the measurement time is only 20 minutes. These are very useful for diagnosing sepsis, which requires speed and convenience. From that point of view, we would like to continue the study of SALS.
It has long been thought that blood endotoxin levels do not correlate with severity. In this study, a correlation was found between the SOFA score and both endotoxin measurement methods, and a correlation was found between the APACHE II score and SALS. When all markers were analyzed by multivariate analysis, CRP had the strongest correlation with SOFA score, and SALS had a significant correlation with APACHE II. Opinions are also divided on severity assessment by infectious disease markers. In this study, the t-value was not high, so we think it is necessary to investigate this point by increasing the number of cases.
In this study, 1) PCT was superior in diagnosing non-infected and infected groups. 2) TKA with LRP was superior in diagnosing the non-infected group and the Gram-negative infection group. 3) There was a significant correlation between the SOFA score and CRP, and a significant correlation between APACHE II and SALS with leukocytes. In both cases, we plan to increase the number of cases and conduct further studies.
We believe that the TKA with LRP is the best method for measuring endotoxin, but considering the ease of measurement and the measurement time, we believe that SALS has sufficient value for clinical application.
Acknowledgements
None
Author contributions
Daisuke Yoshinao, Gaku Takahashi: Conceptualization, data collection, data analysis and interpretation, drafting of the article. Katsuya Inada, Tomohiko Mase: Data collection, data analysis and interpretation, critical revision of the article. Shigenori Kan, Maria Nonoguchi: Data collection, data analysis and interpretation, critical revision of the article. All authors: Final approval of the article.
Competing interests
The authors declare no competing interests.
Data availavility
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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No competing interests reported.