Biochemical Analysis of Leukocytes after In Vitro and In Vivo Activation with Bacterial and Fungal Pathogens Using Raman Spectroscopy

Abstract

Biochemical information from activated leukocytes provide valuable diagnostic information. In this study, Raman spectroscopy was applied as a label-free analytical technique to characterize the activation pattern of leukocyte subpopulations in an in vitro infection model. Neutrophils, monocytes, and lymphocytes were isolated from healthy volunteers and stimulated with heat-inactivated clinical isolates of Candida albicans, Staphylococcus aureus, and Klebsiella pneumoniae. Binary classification models could identify the presence of infection for monocytes and lymphocytes, classify the type of infection as bacterial or fungal for neutrophils, monocytes, and lymphocytes and distinguish the cause of infection as Gram-negative or Gram-positive bacteria in the monocyte subpopulation. Changes in single-cell Raman spectra, upon leukocyte stimulation, can be explained with biochemical changes due to the leukocyte’s specific reaction to each type of pathogen. Raman spectra of leukocytes from the in vitro infection model were compared with spectra from leukocytes of patients with infection (DRKS-ID: DRKS00006265) with the same pathogen groups, and a good agreement was revealed. Our study elucidates the potential of Raman spectroscopy-based single-cell analysis for the differentiation of circulating leukocyte subtypes and identification of the infection by probing the molecular phenotype of those cells.

Understanding viruses and viral infections by biophotonic methods

“…Similarly, Raman spectroscopy was applied to study in vitro adenovirus infection of human embryonic kidney 293 cells [185], herpes virus infection of Vero cells [186], Epstein-Barr virus infection in glial cells [187] and COVID-19 detection based on biochemical profile of patients' serum sample [188]. Raman spectroscopy-based investigations of activated lymphocytes' chemical profile during viral infection in patients significantly differed compared with the healthy donors [2,189]. The analysis of saliva samples as shown in Figure 18C indicated clear differences in the Raman spectral profile of COVID positive and negative subjects [165].…”

“…Raman spectroscopy is another promising option for investigating the sum parameters of complex samples [1–3, 155, 166–174]. For detecting HBV and HCV infections serum samples are well suited [5, 175, 176].…”

“…State-of-the-art microscopy techniques have the capability to observe interactions between viruses and their host's immune cells, capture evasive mechanisms employed by the virus and trigger of different hosts' immune responses. Further, biophotonic methods such as vibrational spectroscopy, atomic force microscopy, and so on, have enabled to apprehend the overall host response thus allowing the diagnosis of infectious diseases and detecting the causative agents [1][2][3][4][5][6][7][8].…”

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy

“…Cells could respond to different steps of viral infection, including virus entry, trafficking through the cell organelles for replication and egress of viral particles, or their entry into lysosomes for degradation of viral proteins 10 , 11 . As Raman microscopy allows the analysis on the single-cell level 12 – 14 , the biochemical modifications due to the cell reaction to the virus could be detected in the early stage. The conventional diagnostic methods are applicable after symptoms appear which might be too late to avoid serious consequences.…”

Specific Intracellular Signature of SARS-CoV-2 Infection Using Confocal Raman Microscopy

“…Cells could respond to different steps of viral infection, including virus entry, tra cking through the cell organelles for replication and egress of viral particles or their entry into lysosomes for degradation of viral proteins 11,12 . As Raman microscopy allows the analysis of on the single-cell level [13][14][15] , the biochemical modi cations due to the cell reaction to the virus could be detected in the early stage. The conventional diagnostic methods are applicable after symptoms appearance which might be too late to avoid serious consequences.…”