Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy

Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes

Assessment of kidney function : clinical indications for measured GFR

We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charite´– Universitätsmedizin Berlin. Publisher Copyright: © The Author(s) 2021.In the vast majority of cases, glomerular filtration rate (GFR) is estimated using serum creatinine, which is highly influenced by age, sex, muscle mass, body composition, severe chronic illness and many other factors. This often leads to misclassification of patients or potentially puts patients at risk for inappropriate clinical decisions. Possible solutions are the use of cystatin C as an alternative endogenous marker or performing direct measurement of GFR using an exogenous marker such as iohexol. The purpose of this review is to highlight clinical scenarios and conditions such as extreme body composition, Black race, disagreement between creatinine- and cystatin C-based estimated GFR (eGFR), drug dosing, liver cirrhosis, advanced chronic kidney disease and the transition to kidney replacement therapy, non-kidney solid organ transplant recipients and living kidney donors where creatinine-based GFR estimation may be invalid. In contrast to the majority of literature on measured GFR (mGFR), this review does not include aspects of mGFR for research or public health settings but aims to reach practicing clinicians and raise their understanding of the substantial limitations of creatinine. While including cystatin C as a renal biomarker in GFR estimating equations has been shown to increase the accuracy of the GFR estimate, there are also limitations to eGFR based on cystatin C alone or the combination of creatinine and cystatin C in the clinical scenarios described above that can be overcome by measuring GFR with an exogenous marker. We acknowledge that mGFR is not readily available in many centres but hope that this review will highlight and promote the expansion of kidney function diagnostics using standardized mGFR procedures as an important milestone towards more accurate and personalized medicine.Peer reviewe

Performance of creatinine-based equations to estimate glomerular filtration rate in White and Black populations in Europe, Brazil and Africa.

peer reviewed("[en] BACKGROUND: A new Chronic Kidney Disease Epidemiology Collaboration equation without the race variable has been recently proposed (CKD-EPIAS). This equation has neither been validated outside USA nor compared with the new European Kidney Function Consortium (EKFC) and Lund-Malmö Revised (LMREV) equations, developed in European cohorts. METHODS: Standardized creatinine and measured glomerular filtration rate (GFR) from the European EKFC cohorts (n = 13 856 including 6031 individuals in the external validation cohort), from France (n = 4429, including 964 Black Europeans), from Brazil (n = 100) and from Africa (n = 508) were used to test the performances of the equations. A matched analysis between White Europeans and Black Africans or Black Europeans was performed. RESULTS: In White Europeans (n = 9496), both the EKFC and LMREV equations outperformed CKD-EPIAS (bias of -0.6 and -3.2, respectively versus 5.0 mL/min/1.73 m², and accuracy within 30% of 86.9 and 87.4, respectively, versus 80.9%). In Black Europeans and Black Africans, the best performance was observed with the EKFC equation using a specific Q-value (= concentration of serum creatinine in healthy males and females). These results were confirmed in matched analyses, which showed that serum creatinine concentrations were different in White Europeans, Black Europeans and Black Africans for the same measured GFR, age, sex and body mass index. Creatinine differences were more relevant in males. CONCLUSION: In a European and African cohort, the performances of CKD-EPIAS remain suboptimal. The EKFC equation, using usual or dedicated population-specific Q-values, presents the best performance in the whole age range in the European and African populations included in this study.","[en] ",""

Retracted: The clinical anatomy of the insertion of the rotator cuff tendons

2nd March 2017 - Article RetractedThe Article THE CLINICAL ANATOMY OF THE INSERTION OF THE ROTATOR CUFF TENDON has been retracted from the Anatomy Journal of Africa on author request, and on the understanding that it was concurrently submitted and published in another journal.Managing EditorThe rotator cuff (RC) insertions according to most anatomical texts are described as being separate. However, clear fusion of the RC tendon fibres exist with prior studies showing an interdigitation forming a common, continuous insertion onto and around the lesser- and greater tubercles (LT & GT) of the humerus. Current surgical repair methods (arthroscopic techniques), rarely mention or consider these connections during repair. Rotator cuff surgery remains a controversial subject, due to various available techniques, surgeon experience and preference and, the contradicting success rates. Therefore, the purpose of this project was to visualise and define the RC footprint and extension insertions with the aim of enhancing and improving knowledge of the basic anatomy in the hope that this will be considered during orthopaedic repair. Twenty shoulders (16 cadaveric & 4 fresh) were used in the study. The fresh shoulders were received from the National Tissue Bank and ethical clearance was obtained (239/2015). Reverse dissection was performed to visualise the RC unit exposing the interdigitated rotator hood (extension insertions), as well as the complete RC unit (tendons + internal capsule) separated from the scapula and humerus. Once the insertions were exposed and documented, the RC muscle footprint (articular surface area) was measured and recorded, using AutoCAD 2016. No statistical significant difference between left and right (p = 0.424) was noted, but a significant difference between males and females (p = 0.000) was. These findings indicate evidence that the RC tendons and the internal capsule are one complete and inseparable unit. The fact that the RC unit is more complex in its structure and attachment places importance on the biomechanical stresses encountered after repair. Functions of one RC muscle are not necessarily isolated but can be influenced by surrounding muscles as well. These findings also provide clarity for surgeons with the goal of improving and enhancing surgical methods for better post-operative patient outcome.Keywords: Orthopaedic; arthroscopic; re-tear; complex; reverse dissection; capsul

#4179 DIFFERENCES IN PROTOCOLS FOR MEASURING GLOMERULAR FILTRATION RATE USING IOHEXOL

peer reviewedAbstract Background and Aims When assessing kidney function, measurement of glomerular filtration rate (mGFR) using an exogenous marker such as iohexol is the gold standard. In this study, we aim to identify similarities and differences between iohexol-based mGFR protocols. Method Detailed data on iohexol measurement protocols were obtained using a standardized survey sent to centres in Europe and the US. It was completed by 15 participants. Data are reported as number and percentage (n, %). Results In the participating centres, measurements are performed after referral by a nephrologist (n=6, 40%), other specialties (n=5, 33%) or for research purposes (n=4, 27%). Most common indications for mGFR are evaluation of kidney donors (n=5, 33%), drug dosing (n=4, 27%), abnormal body composition (n=3, 20%) and transplant evaluation (n=4, 27%). Most participants perform measurements in the morning (n=10, 67%), with patients withholding caffeine (n=5, 33%), fasting (n=4, 27%) or avoiding heavy meals (n=3, 20%). Most centres use an IV iohexol dose of 5 mL 300 mg I/mL (n=10, 67 %) or a dose based on weight (n=2, 13%). The timing of sample collection is shown in Figure 1, most often a single sample per time point (n=12, 80%). Iohexol is measured by LC-MS (n=8, 53%) or LC-UV (n=7, 47%). Within-assay variability ranges between <2% (n=3, 20%) and 6-8% (n=1, 7%) and the between-assay variability ranges between <2% (n=2, 13%) and 6-8% (n=2, 13%). When asked about assumptions and corrections, most centres make a one-compartment assumption in their PK model (n=8, 53%), others making two-compartment (n=2, 13%) or measurement-dependent (n=1, 7%) assumptions. mGFR is standardized for body surface area according to the DuBois-formula (n=6 (40%), Haycock and Schwarz formula (n=2, 13%) or not-specified (n=7, 47%). Some participants correct their measurements for eGFR (n=9, 60 %). Most centres participate in external quality control (Equalis, n=12, 80%). Conclusion There is a large variation in protocols for iohexol-based mGFR, which highlights the need for a standardized mGFR protocol before widespread use in clinical routine

Genetic structure of natterjack toad (Epidalea calamita) populations in Flanders, Belgium, and its implications for conservation

Unique evolutionary potential could be lost when a population goes extinct or when individuals are translocated to other existing populations. Therefore, in order to identify priorities and to predict the efficiency and consequences of conservation actions, information is needed on the genetic structure of natural populations. In the urbanized and diverse landscapes of Flanders, Belgium, natterjack toad (Epidalea calamita) populations have been declining over the last decades. Therefore, this species is subjected to a wide range of different types of conservation measures (e.g. habitat management, corridor development, translocations). However, more information is needed on its genetic population structure. In this study, we sampled egg clutches from six populations and studied their genetic structure with six microsatellite markers. In total, 184 samples from 99 different egg strings were genotyped. Observed heterozygosity was generally high, even for the small and isolated populations (overall mean HO = 0.43). The weak clustering by the Bayesian analyses (STRUCTURE, Adegenet and BAPS) does not allow us to make strong conclusions on the population structure. However, the significant φST values between the populations underline the importance of genetic information when conservation priorities are discussed. Unique evolutionary potential could be lost when one or more natterjack toad populations would go extinct, and translocation of individuals to other existing populations should be considered with caution

Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy

Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes

Leukocyte Activation Profile Assessed by Raman Spectroscopy Helps Diagnosing Infection and Sepsis.

OBJECTIVES: Leukocytes are first responders to infection. Their activation state can reveal information about specific host immune response and identify dysregulation in sepsis. This study aims to use the Raman spectroscopic fingerprints of blood-derived leukocytes to differentiate inflammation, infection, and sepsis in hospitalized patients. Diagnostic sensitivity and specificity shall demonstrate the added value of the direct characterization of leukocyte’s phenotype. DESIGN: Prospective nonrandomized, single-center, observational phase-II study (DRKS00006265). SETTING: Jena University Hospital, Germany. PATIENTS: Sixty-one hospitalized patients (19 with sterile inflammation, 23 with infection without organ dysfunction, 18 with sepsis according to Sepsis-3 definition). INTERVENTIONS: None (blood withdrawal). MEASUREMENTS AND MAIN RESULTS: Individual peripheral blood leukocytes were characterized by Raman spectroscopy. Reference diagnostics included established clinical scores, blood count, and biomarkers (C-reactive protein, procalcitonin and interleukin-6). Binary classification models using Raman data were able to distinguish patients with infection from patients without infection, as well as sepsis patients from patients without sepsis, with accuracies achieved with established biomarkers. Compared with biomarker information alone, an increase of 10% (to 93%) accuracy for the detection of infection and an increase of 18% (to 92%) for detection of sepsis were reached by adding the Raman information. Leukocytes from sepsis patients showed different Raman spectral features in comparison to the patients with infection that point to the special immune phenotype of sepsis patients. CONCLUSIONS: Raman spectroscopy can extract information on leukocyte’s activation state in a nondestructive, label-free manner to differentiate sterile inflammation, infection, and sepsis