In May it was reported that, owing to an IT error, from 2009 to 2018, approximately 450,000 women aged between 68 and 71 were not recalled for their final mammogram appointments in England. Jeremy Hunt, the health secretary has been quoted as saying that between 135 and 270 women “may have had their lives shortened as a result”.  The panic-quelling response came very quickly. The Guardian newspaper reported that Sir
Richard Peto, a professor of medical statistics at Oxford University, had written that there is still substantial uncertainty about the exact ages that mammographic screening should start and end. Additionally, a group of academics and GPs wrote a letter to The Times newspaper saying that the women should not be concerned unless they notice a lump or other symptoms and that the breast cancer screening programme mostly causes more unintended harm than good; many women and doctors avoid breast screening as it has no impact on all-cause death; and that the most dangerous and advanced cancers are not prevented by screening programmes. Breast cancer charities retorted that mammographic screening remains the best tool available for detecting breast cancer at an early and therefore more easily treatable stage and we must not forget that the programme does save lives. The UK’s NHS breast screening programme began in 1988 and national coverage was reached in the mid-1990s. However, over twenty years on, there seems to be an increasing body of data to suggest that the ‘accidental’ harm resulting from mammography because of over-detection and over-treatment of clinically unimportant lumps has been underestimated. The often-quoted figure is that for every woman whose life is extended, three receive unnecessary surgery, chemotherapy or radiotherapy. Hence our ‘best tool available’ seems to be a rather blunt tool. Biomarkers, surely, could provide the refinement needed to stratify patients according to therapy response. This will enable the delivery of individually tailored treatment plans and so will, crucially, prevent the unnecessary administration of chemotherapy and radiotherapy. Work on this is, of course, underway. The EU-funded RESPONSIFY study in Germany has already led to two parameters being included into German breast cancer guidelines for the treatment of HER2-positive breast cancer. Additionally, a gene-expression panel that predicts whether chemotherapy will be beneficial for preventing recurrence is already being used, with some success for the low and high scores. Further work is needed, but perhaps the day is in sight where women will no longer undergo unnecessary chemotherapy.

Extracellular vesicles as markers and mediators in sepsis
^{Raeven P, Zipperle J, Drechsler S. Theranostics 2018; 8(12):3348–3365}

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It remains a highly lethal condition in which current tools for early diagnosis and therapeutic decision-making are far from ideal. Extracellular vesicles (EVs), 30 nm to several micrometers in size, are released from cells upon activation and apoptosis and express membrane epitopes specific for their parental cells. Since their discovery two decades ago, their role as biomarkers and mediators in various diseases has been intensively studied. However, their potential importance in the sepsis syndrome has gained attention only recently. Sepsis and EVs are both complex fields in which standardization has long been overdue. In this review, several topics are discussed. First, we review current studies on EVs in septic patients with emphasis on their variable quality and clinical utility. Second, we discuss the diagnostic and therapeutic potential of EVs as well as their role as facilitators of cell communication via micro RNA and the relevance of microorganism-derived EVs. Third, we give an overview over the potential beneficial but also detrimental roles of EVs in sepsis. Finally, we focus on the role of EVs in selected intensive care scenarios such as coagulopathy, mechanical ventilation and blood transfusion. Overall, the prospect for EV use in septic patients is bright, ranging from rapid and precise (point-of-care) diagnostics, prevention of harmful iatrogenic interventions, to using EVs as guides of individualized therapy. Before the above is achieved, however, the EV research field requires reliable standardization of the current methods and development of new analytical procedures that can close the existing technological gaps.

Diagnostic accuracy of lipopolysaccharide-binding protein for sepsis in patients with suspected infection in the Emergency Department
^{García de Guadiana Romualdo L, Albaladejo Otón MD, Rebollo Acebes S, Esteban Torrella P, Hernando Holgado A, et al. Ann Clin Biochem 2018; 55(1): 143–148}

BACKGROUND: Biomarkers can facilitate the diagnosis of sepsis, enabling early management and improving outcomes. Lipopolysaccharide-binding protein (LBP) has been reported as a biomarker for the detection of infection, but its diagnostic value is controversial. In this study, we assessed the diagnostic accuracy of LBP for sepsis in the Emergency Department (ED) patients, comparing it with more established biomarkers of sepsis, including procalcitonin (PCT) and C-reactive protein (CRP).

METHODS: LBP and other sepsis biomarkers, including PCT and CRP, were measured on admission in 102 adult patients presenting with suspected infection. Classification of patients was performed using the recently updated definition for sepsis (Sepsis-3). The diagnostic accuracy of LBP, CRP and PCT for sepsis was evaluated by using receiver operating characteristic curve (ROC) analysis.

RESULTS: A total of 49 patients were classified as having sepsis. In these patients, median (interquartile range) LBP (41.8 [41.1] µg/dL vs 26.2 [25] µg/dL), CRP (240 [205] mg/L vs 160 [148] mg/dL) and PCT (5.19 [13.68] µg/L vs 0.39 [1.09] µg/L) were significantly higher than in patients classified as not having sepsis (P<0.001 for all three biomarkers). ROC curve analysis and area under curve (AUC) revealed a value of 0.701 for LBP, similar to CRP (0.707) and lower than that for PCT (0.844) (P=0.012).

CONCLUSION: In adult ED patients with suspected infection, the diagnostic accuracy for sepsis of LBP is similar to that of CRP but lower than that of PCT.

An innovative approach for the integration of proteomics and metabolomics data in severe septic shock patients stratified for mortality
^{Cambiaghi A, Díaz R, Martinez JB, Odena A, Brunelli L, et al. Sci Rep 2018; 8(1):6681}

In this work, we examined plasma metabolome, proteome and clinical features in patients with severe septic shock enrolled in the multicentre ALBIOS study. The objective was to identify changes in the levels of metabolites involved in septic shock progression and to integrate this information with the variation occurring in proteins and clinical data. Mass spectrometry-based targeted metabolomics and untargeted proteomics allowed us to quantify absolute metabolite concentration and relative protein abundance. We computed the ratio D7/D1 to take into account their variation from day 1 (D1) to day 7 (D7) after shock diagnosis. Patients were divided into two groups according to 28-day mortality. Three different elastic net logistic regression models were built: one on metabolites only, one on metabolites and proteins and one to integrate metabolomics and proteomics data with clinical parameters. Linear discriminant analysis and partial least squares discriminant analysis were also implemented. All the obtained models correctly classified the observations in the testing set. By looking at the variable importance (VIP) and the selected features, the integration of metabolomics with proteomics data showed the importance of circulating lipids and coagulation cascade in septic shock progression, thus capturing a further layer of biological information complementary to metabolomics information.

Validity of biomarkers in screening for neonatal sepsis – a single-center hospital-based study
^{Rashwan NI, Hassan MH, Mohey El-Deen ZM, Ahmed AE. Pediatr Neonatol 2018; doi: 10.1016/j.pedneo.2018.05.001 [Epub ahead of print]}

BACKGROUND: The diagnosis of neonatal sepsis still considered to be a challenge for both clinicians and the laboratory owing to the non-specific clinical presentations. The present study aimed to compare and assess the diagnostic and prognostic values of C-reactive protein (CRP), high sensitivity CRP (hsCRP), presepsin, interleukin-6 (IL-6) and procalcitonin (PCT) in neonatal sepsis separately and in combination.

METHODS: This hospital-based cross-sectional study has been conducted on 168 neonates recruited from the Neonatal Intensive Care Unit of Qena University Hospitals, Upper Egypt. Measurements of CRP using the latex agglutination test, hsCRP, presepsin, IL6 and PCT assays using commercially available ELISA assay kits were done to all included neonates.

RESULTS: There were significantly higher serum levels of CRP among late onset versus early onset sepsis group with significantly higher serum levels of hsCRP and presepsin among early onset compared with the late onset sepsis group (P<0.05 for all). There were significantly higher hsCRP, presepsin and PCT serum levels in proven versus probable sepsis group (P<0.05 for all). Significantly higher serum levels of presepsin and PCT were noted among survivors versus non-survivors sepsis group (P<0.05 for all). The cut-off value of the serum level of CRP >6 mg/dL showed lower sensitivity and specificity than that of hsCRP at cut-off >140 ng/mL in diagnosing neonatal sepsis. The cut-off value of presepsin >200 ng/mL showed equal sensitivity and specificity to IL-6 at cut-off >22 pg/mL. The cut-off value of PCT at >389 pg/mL showed sensitivity and specificity approximate to that of hsCRP.

CONCLUSIONS: CRP could be a helpful prognostic marker in late onset neonatal sepsis. hsCRP and PCT have higher diagnostic accuracy in neonatal sepsis in comparison to other studied markers. Both IL-6 and presepsin have equal diagnostic utility in neonatal sepsis, but presepsin could be a helpful diagnostic marker in early onset neonatal sepsis.

Plasma miRNA-223 correlates with risk, inflammatory markers as well as prognosis in sepsis patients
^{Wu X, Yang J, Yu L, Long D. Medicine 2018; 97(27):e11352}

The purpose was to evaluate the role of plasma microRNA-223 (miRNA-223) in risk and prognosis in sepsis patients, and its correlation with inflammatory markers. In this study, 187 sepsis patients from July 2015 to December 2016 were consecutively enrolled. Blood samples from septic patients and healthy controls (HCs) were collected, and plasma was separated for miRNA-223 expression detected by quantitative real-time PCR (qPCR). Enzyme-linked immune sorbent assay (ELISA) was performed to detect inflammatory markers. The results were as follows: miRNA-223 was highly expressed in sepsis patients compared to HCs (P<0.001). Receiver operating characteristic (ROC) curve revealed miRNA-223 disclosed a good diagnostic value of sepsis with area under curve (AUC) of 0.754, 95 % CI: 0.706–0.803. Sensitivity and specificity were 56.6 % and 86.6 % at the best cut-off point, respectively. Multivariate logistic analysis indicated that miRNA-223 could predict sepsis risk independently. Spearman’s correlation disclosed that miRNA-223 relative expression positively correlated with APCHE II score (r=0.459, P<0.001), CRP (r=0.326, P<0.001), TNFα (r=0.325, P<0.001), IL-1β (r=0.165, P=0.024), IL-6 (r=0.229, P=0.002) and IL-8 (r=0.154, P=0.035), while it negatively correlated with IL-10 (r=−0.289, P<0.001). miRNA-223 expression in non-survivors was higher than that in survivors (P<0.001). ROC curve revealed miRNA-223 could distinguish sepsis non-survivors from survivors with AUC of 0.600, 95 % CI: 0.505–0.695. Sensitivity and specificity were 83.5 % and 38.9 % respectively at the best cut-off point. In conclusion, plasma miRNA-223 correlates with disease severity and inflammatory markers levels, and it might serve as a novel diagnostic and prognostic biomarker in sepsis patients.

Biomarkers of endothelial dysfunction predict sepsis mortality in young infants: a matched case-control study
^{Wright JK, Hayford K, Tran V, Al Kibria GM, Baqui A, et al. BMC Pediatr 2018; 18(1):118}

BACKGROUND: Reducing death due to neonatal sepsis is a global health priority, although there are limited tools to facilitate early recognition and treatment. We hypothesized that measuring circulating biomarkers of endothelial function and integrity (i.e. Angiopoietin-Tie2 axis) would identify young infants with sepsis and predict their clinical outcome.

METHODS: We conducted a matched case-control (1:3) study of 98 young infants aged 0–59 days of life presenting to a referral hospital in Bangladesh with suspected sepsis. Plasma levels of Ang-1, Ang-2, sICAM-1, and sVCAM-1 concentrations were measured at admission. The primary outcome was mortality (n=18); the secondary outcome was bacteremia (n=10).

RESULTS: Ang-2 concentrations at presentation were higher among infants who subsequently died of sepsis compared to survivors (aOR 2.50, P=0.024). Compared to surviving control infants, the Ang-2:Ang-1 ratio was higher among infants who died (aOR 2.29, P=0.016) and in infants with bacteremia (aOR 5.72, P=0.041), and there was an increased odds of death across Ang-2:Ang-1 ratio tertiles (aOR 4.82, P=0.013).

CONCLUSIONS: This study provides new evidence linking the Angiopoietin-Tie2 pathway with mortality and bacteremia in young infants with suspected sepsis. If validated in additional studies, markers of the angiopoietin-Tie2 axis may have clinical utility in risk stratification of infants with suspected sepsis.

Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis
^{van de Groep K, Bos MP, Savelkoul PHM, Rubenjan A, Gazenbeek C, et al. Eur J Clin Microbiol Infect Dis 2018; 37(7):1333–1344}
Molecular tests may enable early adjustment of antimicrobial therapy and be complementary to blood culture (BC) which has imperfect sensitivity in critically ill patients. We evaluated a novel multiplex real-time PCR assay to diagnose bloodstream pathogens directly in whole blood samples (BSI-PCR). BSI-PCR included 11 species- and four genus-specific PCRs, a molecular Gram-stain PCR, and two antibiotic resistance markers. We collected 5 mL blood from critically ill patients simultaneously with clinically indicated BC. Microbial DNA was isolated using the Polaris method followed by automated DNA extraction. Sensitivity and specificity were calculated using BC as reference. BSI-PCR was evaluated in 347 BC-positive samples (representing up to 50 instances of each pathogen covered by the test) and 200 BC-negative samples. Bacterial species-specific PCR sensitivities ranged from 65 to 100 %. Sensitivity was 26 % for the Gram-positive PCR, 32 % for the Gram-negative PCR, and ranged 0 to 7 % for yeast PCRs. Yeast detection was improved to 40 % in a smaller set-up. There was no overall association between BSI-PCR sensitivity and time-to-positivity of BC (which was highly variable), yet Ct-values were lower for true-positive versus false-positive PCR results. False-positive results were observed in 84 (4 %) of the 2200 species-specific PCRs in 200 culture-negative samples, and ranged from 0 to 6 % for generic PCRs. Sensitivity of BSI-PCR was promising for individual bacterial pathogens, but still insufficient for yeasts and generic PCRs. Further development of BSI-PCR will focus on improving sensitivity by increasing input volumes and on subsequent implementation as a
bedside test.

From traditional biochemical signals to molecular markers for detection of sepsis after burn injuries
^{Muñoz B, Suárez-Sánchez R, Hernández-Hernández O, Franco-Cendejas R, Cortés H, Magaña JJ. Burns 2018; doi: 10.1016/j.burns.2018.04.016 [Epub ahead of print]}

Sepsis is a life-threatening organ-dysfunction condition caused by a dysregulated response to an infectious condition that can cause complications in patients with major trauma. Burns are one of the most destructive forms of trauma; despite the improvements in medical care, infections remain an important cause of burn injury-related mortality and morbidity, and complicated sepsis predisposes patients to diverse complications such as organ failure, lengthening of hospital stays, and increased costs. Accurate diagnosis and early treatment of sepsis may have a beneficial impact on clinical outcome of burn-injured patients. In this review, we offer a comprehensive description of the current and traditional markers used as indicative of sepsis in burned patients. However, although these are markers of the inflammatory post-burn response, they usually fail to predict sepsis in severely burned patients because they do not reflect the severity of the infection. Identification and measurement of biomarkers in early stages of infection is important in order to provide a timely response and the effective treatment of burned patients. Therefore, we compiled important experimental evidence, demonstrating novel biomarkers, including molecular markers such as genomic DNA variations, alterations of transcriptome profiling (mRNA, miRNAs, lncRNAs and circRNAs), epigenetic markers, and advances in proteomics and metabolomics. Finally, this review summarizes next-generation technologies for the identification of markers for detection of sepsis after burn injuries.

According to the World Health Organization, depression affects more than 300 million people and is the leading cause of ill health and disability worldwide. Currently, diagnosis of depression involves the use of questionnaires about the patient’s general health, the way they are feeling and how this is affecting them. Blood tests are carried out during diagnosis, but are for the purpose of excluding other conditions, such as thyroid disease or vitamin D deficiency, that can give rise to symptoms similar to depression. There is no physical test for depression per se. Treatment for depression ranges from ‘wait and see’ and exercise for very mild forms through to self-help groups, talking therapies, such as counselling and cognitive behavioural therapy, for mild to moderate depression, as well as antidepressant medication for the more severe end of the spectrum. There are several classes of antidepressant drugs and treatment is largely through a process of trial and error in order to determine what does or does not work for certain patients, as it is recognized that there is a large variation in the way individuals respond to the different medications. Additionally, although numbers vary, conservative estimates suggest at least 30% of patients do not respond to antidepressant medication, and suffer from what is termed treatment-resistant depression.
However, recently, a line of research about one cause of depression has been gaining traction: the role of inflammation. Recent work suggests that an overactive immune system causing higher levels of inflammation results in an increased risk of depression and that these patients are less likely to respond to antidepressants; perhaps, therefore, the cause of treatment-resistant depression. It has also been noticed that patients taking anti-inflammatory medication for rheumatoid arthritis experience improvements in mood that are more profound than just feeling happier because of reduced pain; changes that have been confirmed by brain scans. Professor Ed Bullmore, Head of the Department of Psychiatry at the University of Cambridge, is certain that inflammation can cause depression and his new book, The Inflamed Mind: A radical new approach to depression, is about to bring these ideas to the attention of a much more general audience. The exciting relevance of this research for clinical lab diagnostics is the thought that a blood test for biomarkers of inflammation will help in an objective diagnosis of a certain type of depression and that treatment will be much better tailored to the individual – perhaps the individuals who fail to respond to current antidepressants. Even if this benefits only a small proportion of people with depression, because of the prevalence of the condition a large number of people will benefit.

The increasing global burden of sepsis in healthcare calls for better diagnostic tests that allow earlier detection of sepsis and infections that could lead to sepsis. The major problem for patients at risk for sepsis is an immunological imbalance. Cells of the innate immune system, such as monocytes and neutrophils, are the first-line of defence against infections. In the presence of sepsis, these cells produce a flood of inflammatory cytokines, causing widespread inflammation that can lead to death. Monocytes perform multiple immunological functions, and play a role in the development of sepsis-induced inflammation and immunosuppression. Monocyte subpopulations with different functions and morphologies vary in number over the course of the inflammatory response. The monocyte distribution width (MDW) is a novel cellular marker of monocyte anisocytosis that can add significant value to the white blood cell (WBC) count and help detect sepsis in patients entering the emergency department (ED).

by Elena A. Sukhacheva

Sepsis epidemiology and definitions
Sepsis is a major healthcare burden and, despite progress in diagnostic and treatment options, mortality from sepsis remains unacceptably high. The number of septic patients in the U.S., UK and EU is increasing [1–4]. Clearly, there is an unmet need for better diagnostic tests that can provide both the early detection of sepsis and the detection of severe infections that may progress to sepsis, if not diagnosed early enough. Global increases in sepsis frequency may be related to the aging population, as the incidence of sepsis is disproportionately increased in elderly adults, and age is an independent predictor of mortality [5]. Furthermore, immunosuppressive drugs, which are increasingly being used for diverse conditions, may result in more severe infections and increased sepsis frequency [6].
The definition of sepsis has recently been changed from the previous Sepsis-2 definition of a systemic inflammatory response (SIRS) in the presence of an infection [7], to the current Sepsis-3 definition of a life-threatening organ dysfunction caused by a dysregulated host response to infection [8].  The new Sepsis-3 definition reflects newfound understanding that the immune response in sepsis is more complex than previously thought, comprising both pro- and anti-inflammatory mechanisms.

Immune response in sepsis
It is now clear that the major problem for patients with sepsis, or at high risk of developing sepsis, is immunological imbalance, and dysregulation of the mechanisms of innate and adaptive immunity. Sepsis occurs when the immune system begins, in one way or another, to lose the battle against severe infection. After sepsis onset, the production of pro-inflammatory cytokines (IL-1β, IL-6, and tumour necrosis factor [TNFα]) by the cells of the innate immune system (neutrophils and monocytes) may result in a “cytokine storm” that produces overwhelming inflammation, which can lead to blood pressure collapse, coagulation abnormalities and, ultimately, organ failure and death. In the later stages of disease, patients who survive the cytokine storm may die from sepsis-related immunosuppression and an inability of the immune system to combat infection efficiently [9]. Inflammatory and immunosuppressive processes may overlap in sepsis [10,11], further complicating the biology of this fatal condition whose mechanisms are still poorly understood by scientists. Figure 1 shows the current understanding of immune imbalance in sepsis [12]. While all immune cells are involved in the immune response in sepsis [13–16] (Figure 2), this document is mainly focused on changes in monocytes, with other cell populations discussed only briefly.

Under normal conditions, neutrophils usually stay in the circulation for only a few hours and undergo apoptosis within 24 hours of release from the bone marrow. In sepsis, the delay in neutrophil apoptosis [17,18], combined with the increased neutrophil production in the bone marrow, results in neutrophilia. The function of these neutrophils, however, is impaired [19], with decreased chemotactic activity [20,21], decreased antibacterial function and increased production of anti-inflammatory cytokine interleukin 10 (IL-10) [22].
Sepsis also has a profound effect on all the main lymphocyte subpopulations [14]: CD4+ T-cells, CD8+ T-cells and B-cells undergo increased apoptosis; T-regulatory cells are more resistant to sepsis-induced apoptosis, leading to an increased proportion of T-regulatory cells and an immunosupressive phenotype. T-helper cell polarization from a pro-inflammatory Th1 phenotype towards an anti-inflammatory Th2 phenotype also contributes to increased immunosuppression in sepsis.

Monocytes also undergo multiple changes in sepsis, but before discussing these phenomena, it is important to discuss some basic information about the biology and classification of monocytes.

Monocytes’ biology and classification
Monocytes are cells of the innate immune system, the body’s first-line of defence against infection. Other cells of this system include neutrophils, basophils, eosinophils, mast cells, as well as certain types of lymphocytes such as γδ-T-cells and natural killer cells. The innate immune response develops during the first hours and days after pathogen invasion, and the majority of pathogens entering the human body usually are inactivated by this response and do not require adaptive mechanisms with lymphocyte involvement.

Myeloid precursors in the bone marrow differentiate into promonocytes and then into mature monocytes that enter the peripheral blood. These monocytes stay in the circulation for one to three days, after which they migrate into tissues and organs, where they turn into macrophages and dendritic cells. Morphologically, monocytes are large cells measuring 10 to 18 µm in diameter, with convoluted nuclei and azurophilic granules in their cytoplasm.

Monocytes and dendritic cells perform multiple immunological functions that include phagocytosis, antigen presentation and cytokine production. The function of these cells is regulated by a number of cell surface receptors:

• CD14, the receptor for complexes of bacterial lipopolysaccharides and human serum proteins
• Receptors such as CD163 that scavenge membrane fragments and other components of damaged cells
• Multiple receptors for the Fc regions of IgG: CD64 (FcγR1, high-affinity receptor), CD32 (FcγR2, medium-affinity receptor) and CD16 (FcγR3, present only on subpopulations of so-called pro-inflammatory monocytes)
• Other receptors necessary for interaction with lymphocytes and receptors for cytokines

Three subpopulations of monocytes have been characterized in peripheral blood [23–25]. Classical monocytes make up the main monocyte population. Expressing high level CD14 and no CD16 (CD14++CD16-), they represent 80–90% of monocytes in peripheral blood. “Intermediate” monocytes expressing CD16 (CD14++CD16+) are normally found at low numbers, but increase with cytokine stimulation and inflammation. Nonclassical monocytes display decreased expression of CD14 and increased expression of CD16 (CD14+CD16++), and comprise 9%+/-5% of all monocytes, with an average count in healthy donors of approximately 45+/-22 cells/µL [26].

In the literature, nonclassical monocytes are sometimes referred to as inflammatory or pro-inflammatory monocytes; however, published recommendations for the nomenclature of monocytes and dendritic cells in the blood clearly advocate avoiding functional terminology, “because this leads to confusion as the label ‘inflammatory’ has been used for different subpopulations in humans and mice [24].” Also, “these terms may prematurely ascribe functional attributes to cells based on ex vivo studies while they largely remain to be functionally characterized in vivo [24].” Subsets of nonclassical monocytes are expanded dramatically in several pathological conditions including sepsis [26–28], HIV-1 infection [29–33], diabetes [34–35], tuberculosis [36] and other disease states [37].

The recent detailed analysis performed by Mukherjee et al. [28] revealed the functions of monocyte subsets as follows: classical monocytes are phagocytic with no inflammatory attributes, nonclassical subtypes display inflammatory characteristics on activation and display properties for antigen presentation, and intermediate subtypes appear to have both phagocytic and inflammatory functions [28]. In 2017, research based on single-cell RNA sequencing discovered even more subtypes, describing six subpopulations of dendritic cells and four monocyte subpopulations [39]. This classification was based solely on transcriptional activity, and further studies will be needed to understand function and describe the phenotype of all cell subpopulations. Nonetheless, it is clear that morphologically similar cells that we call monocytes may actually have very different functions in human immunity.

Monocytes in sepsis
Monocytes, as cells of first-line defence against infection, are involved in the immune response from very early stages. Abundant literature exists on monocytes and the changes they undergo in sepsis.

A recent study on the dynamics of monocyte subpopulations in peripheral blood at the onset of infection has demonstrated a decrease in the number of peripheral blood monocytes during the early stages of lipopolysaccharide (LPS)-induced acute inflammation in humans. This loss may be due to the migration of monocytes from the blood into tissues, where they differentiate into macrophages and dendritic cells, or it may reflect an increase in monocytes residing in the marginal pool or rolling on the vessel walls [40]. For all three subpopulations of monocytes, the number of cells was decreased at one to two hours after LPS injection. This decrease was followed by a return to the baseline count, but with differences in timing for the three monocyte subsets. This difference in timing means that the early stages of infection, before the appearance of any clinical symptoms, are characterized by differences in the proportions of monocyte subpopulations relative to baseline pre-infection proportions.

Functional changes in monocytes and, in parallel, changes in their cellular morphology, have been demonstrated in the past for a human THP-1 monocytic cell line infected with viable C. pneumonia bacteria [41]. The differentiation of infected cells into macrophages was accompanied by a change to an ameboidor diffused morphology as assessed by microscopy after Giemsa staining.

Multiple studies have demonstrated the importance of HLA-DR expression on monocytes as a prognostic marker in septic patients. A decreased level of HLA-DR expression on monocytes has been found to be a negative prognostic indicator [42–44] and may be used to evaluate the functional activity of the immune system [45,46]. Decreased HLA-DR, as a marker of monocyte anergy, correlates with decreased antigen presentation capacity and decreased pro-inflammatory cytokine release. This has been analyzed mainly by flow cytometry, but, recently, new methods based on real-time PCR have emerged [47,48].
Another monocyte marker, CD16, plays an important role in orchestrating the response of monocytes to Gram-negative sepsis. It has been demonstrated that CD16 on human monocytes is a key regulator of the TRIF-dependent TLR4 signalling pathway, and this pathway is preferentially activated in the CD16+ monocyte subset [49]. Recent publications suggest the variability of monocyte properties in sepsis. Detailed analysis of gene expression in patient monocytes during sepsis and after recovery demonstrated plasticity of monocytes in the course of disease [50]. The significant up-regulation of pro-inflammatory cytokines (IL-1b, IL-6) and chemokines (CCL3 and CCL5) has been demonstrated in sepsis monocytes compared to monocytes after recovery. Transcriptional factor NF-kB, a central transcriptional regulator of the inflammatory response, was also activated in sepsis monocytes, supporting their involvement in severe inflammation. At the same time, anti-inflammatory cytokine IL-10 was found to be up-regulated in sepsis monocytes. These studies once again highlight the diversity of monocytes’ function in sepsis pathogenesis, and their key role in disease progression, with the possible polarization from a pro-inflammatory state to an immunosuppressive state.

More recently, Crouser et al. demonstrated that the morphological variability that occurs during monocyte activation in the early inflammatory response can be captured by measuring the monocyte distribution width (MDW), an indicator of monocyte anisocytosis. Investigators showed that MDW could be a novel cellular marker that may help detect sepsis early in patients admitted to the emergency department (ED) [51]. Multiple morphometric characteristics of monocytes were obtained using a DxH 800 cellular analysis system, which employs physical measurement of cell volume, conductivity and multiple angles of laser scatter to classify leukocytes into five sub-populations and detect the presence of abnormal cells. This study showed that anisocytosis of circulating monocytes provides significant added value to WBC count for the detection of sepsis in the ED population.

Conclusion
In summary, monocytes are a very heterogeneous population of cells that differ in phenotype, size, nuclear morphology, gene profile and function [52]. In sepsis, this diversity is even more pronounced due to functional changes of monocyte subsets, and is accompanied by a variation in monocyte morphology.

Morphological variability is just the tip of the iceberg of the underlying biological heterogeneity, and may be an important early marker of sepsis or severe infections with a high risk of progressing to sepsis. A recent publication from Crouser [51], together with previous research on sepsis using cellular morphometric parameters gathered using a DxH 800 analyser [53–56], may build the foundation for practical usage of MDW in combination with currently-used sepsis markers (WBC, PCT, CRP, IL-6) for early sepsis screening and diagnosis, leading to early initiation of appropriate therapy.

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The author
Elena A. Sukhacheva, Ph.D.
Senior Manager,
Global Scientific Affairs, Hematology, Beckman Coulter Diagnostics,
Miami, FL, USA