Sepsis is a serious clinical syndrome triggered by a pathogen invasion of an organism and the resulting inflammatory response of the body's immune system to it. For a long time now, the advanced method of infection diagnosis has been based on blood culture, whose processing time (7 days) significantly delayed clinical decision-making and prevented timely administration of antibiotics. We developed an inflamed endothelium-mimicking surface by functionalizing the aldehyde-coated glass substrate in the microchannel with a set of the cell adhesion molecules (CAMs) (P-selectin, E-selectin, ICAM-1).
We found that perfusion with the infected blood samples captured a higher percentage of leukocytes on the biomimetic surface than when perfused with the healthy blood samples. The usefulness of the proposed microfluidic assay and clinical blood culture techniques in detecting infection from rat blood samples 12 hours after E.
Technical Terms and Abbreviations
Introduction
The successful development of the body's immune response to an infection by a pathogen is possible due to timely recruitment of immune cells to the sites of infection. This multi-step process is regulated by the formation of highly specific interactions between the cell adhesion molecules expressed by the endothelium of the infected vessel and the corresponding receptors of the leukocytes[1]. The extravasation cascade is preceded by upregulation of the endothelial expression of cell adhesion molecules, such as P-selectin, E-selectin and ICAM-1, which together orchestrate the series of transient cell-to-cell interactions that ultimately lead to migration of the leukocytes from blood to the infected tissue[2].
Constitutive expression of ligands for P-selectin and E-selectin on the leukocytes, named PSGL-1 and ESL-1, respectively, has been reported to be responsible for initiating the instantaneous recruitment of the leukocytes from blood to the endothelium of the underlying barrel. an infected tissue[3]. After completing the selectin-dependent role, leukocytes firmly adhere to the endothelium through the formation of strong bonds between the integrin LFA-1 of the leukocytes and endothelial ICAM-1[4]. Although the components of the immune system participating in leukocyte recruitment and extravasation have been well studied, the development of an ex vivo microfluidic system for monitoring the leukocyte-endothelial interactions would further advance the works targeting the development of new sepsis diagnosis methods.
The importance of early diagnosis of infection and timely prescription of appropriate treatment is further emphasized by the fact that sepsis mortality increases by 8% for every hour of delay in antibiotic administration[6]. As such, the delay in appropriate therapeutics to treat sepsis since the golden hour is estimated to be responsible for the vast majority of in-hospital mortality, affecting the lives of 31.5 million people worldwide [9]. Timely clinical decision-making in these settings is severely limited by the relatively low sensitivity of commonly performed PCR and CT tests and their unavailability for on-site testing needs.
We report for the first time that PSGL-1, a primary ligand of the P-selectin, is upregulated by the onset of infection in rat infection models, allowing assessment of the activated state of the leukocytes based on the different frequency of their adhesion. to the functionalized surface of the CAMs.
Materials and Methods
A schematic of the Schiff-based bond-based chemical bonding of the PDMS device to glass substrate coated with aldehyde group and the subsequent attachment of the CAMs onto it. Based on a previous work where we showed that proteins are effectively immobilized on the aldehyde-functionalized glass slides for immunoassay purposes, we used aldehyde-coated glass as a substrate in our microfluidic device.[10] Since plasma treatment was reported to cause irreversible damage to aldehyde-functionalized surfaces, we developed an alternative approach for irreversible chemical bonding of the PDMS device to glass slides. After the surface of the PDMS plate containing the microfluidic pattern is coated with APTES to generate free amino groups, it is pressed against the aldehyde-coated glass slide and the assembly is placed at 65° for 5 h to facilitate a Schiff base coupling between the amino groups and.
The experimental setup (Figure 1b) consists of a microfluidic device for blood analysis, pumps and gas-tight syringes that control the perfusion of samples into the device via tubing, and a microscope to instantly acquire microchannel images for cell quantification. The microfluidic device consists of four identical microfluidic channels with one inlet and one outlet (Figure 1c). Schematic of the fluidic component that infuses the sample into the microfluidic device, allowing leukocytes to roll across the microchannel substrate.
Microscopic images of leukocytes rolling down the channel were superimposed at a time interval of 20 seconds. To distinguish the infection from the healthy condition based on the frequency of leukocyte capture on the CAM-functionalized substrate, 100 μl of lysed blood samples from the respective experimental groups were flowed into the proposed microfluidic device. The frequency of leukocyte entrapment was estimated as the percentage of the total number of leukocytes that adhered to the channel substrate upon perfusion, while the total number of leukocytes perfused through the channel was counted by Hoechst immunostaining of blood samples and subsequent imaging in a hemocytometer.
In order to investigate how the percentage of neutrophils among captured leukocytes changes in response to infection, we performed immunocytochemical staining of captured leukocytes with neutrophil-.
Results and Discussion
To study how the infection affects the expression levels of the adhesion receptors, we used a. Moreover, this finding is consistent with the previous study of the effect of systemic infection on the expression of cell adhesion receptor in mice, which reported a significant increase in PSGL-1 expression [ 13 ]. Interestingly, in addition to the significantly increased MFI of the stained PSGL-1, the proportion of PSGL-1-expressing leukocytes in blood samples from infected rats was significantly increased compared to that of the healthy rats (Figure 4d).
LFA-1, on the other hand, showed no changes in either the expression level or the percentage of. Since PSGL-1 and ESL-1 are directly involved and actively facilitate leukocyte rolling and adhesion in the extravasation cascade, we hypothesized that the changes in their expression levels and the elevated percentage of the expressing cells would significantly affect the frequency of leukocyte adhesion to CAM's functionalized substrate. a and b). Fluorescence microscopy images of the leukocytes from healthy and infected blood samples after their immunochemical staining with fluorescently labeled anti-PSGL-1 (n = 3; a) and anti-ESL-1 antibodies (n = 3; b). c to h).
Although there are numerous reports that PSGL-1 and ESL-1 are expressed by the majority of leukocytes regardless of their type[14], we predicted that neutrophils, accounting for the primary role they play in the initial inflammatory response , would represent higher percentage of leukocytes expressing PSGL-1 and ESL-1 compared to the healthy rats[15]. Immunocytochemical staining of the leukocytes with anti-myeloperoxidase antibody revealed that the percentage of neutrophils increases about threefold as a result of infection (Figure 5). The small fraction of rolling leukocytes eventually adhered to the surface via formation of interactions between LFA-1 and ICAM-1, respectively, of the leukocyte and the CAMs-coated microchannel.
Functionalization of the microchannel with E-selectin and ICAM-1 showed significant differentiation of the sepsis from healthy state, but it captured significantly lower percentage of the leukocytes compared to the presented state 1 (Figure 6b).
Healthy Infection
Conclusion
One of the initial reactions of the organism during the systemic sepsis is the activation of the endothelium at the sites of infection via expression of cell adhesion molecules, such as P-selectin, E-selectin and ICAM-1. Although the process of up-regulation of these molecules is well investigated in the previous works, the mechanism that modulates the recruitment of the leukocytes from the blood stream to the infected tissue, as well as the regulation of the expression of receptors complementary to endothelial cell adhesion molecules have been left unexplored. We found that the recruitment of the leukocytes for extravasation is regulated not only by the upregulation of the adhesion molecules on the endothelium, but also by the modulation of expression of their respective receptors on the leukocytes.
As such, we found that the population of leukocytes actively expressing the primary ligands of P-selectin and E-selectin, PSGL-1 and ESL-1, is significantly increased as a result of infection, respectively. We used these findings to design a new microfluidic device for diagnosing infections using a drop of blood. Although several works have been published in recent years proposing microfluidic methods for the detection of infection based on changes in the expression of a cluster of differentiation (CD) [21–22], our method stands out for its ability to detect sepsis as detectable as early as possible. as one hour after infection, which is significantly faster compared to traditional methods, such as blood culture and PCR.
In addition to on-chip blood analysis, the proposed method requires performing off-chip procedures such as in-tube RBC lysis and leukocyte quantification in a hemocytometer. One way to avoid the need for these procedures would be an integration of a microfluidic module for the preprocessing of the blood samples, which would significantly simplify the method and reduce the operative time. We have shown that the expensive fluorescent imaging component of the method can be omitted, as we have validated that leukocyte quantification is possible with bright-field imaging.
This significantly simplifies the process of optimizing the proposed device for the development of a point-of-care testing system.
Ethyl acetate extracts of alfalfa (Medicago sativa L.) sprouts inhibit lipopolysaccharide-induced inflammation in vitro and in vivo. Multiparameter Affinity microarray for early sepsis diagnosis based on CD64 and CD69 expression and cell capture.
Acknowledgement
