University of Groningen Early onset sepsis in Suriname Zonneveld, Rens

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University of Groningen

Early onset sepsis in Suriname

Zonneveld, Rens

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

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Publication date: 2017

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Zonneveld, R. (2017). Early onset sepsis in Suriname: Epidemiology, Pathophysiology and Novel Diagnostic Concepts. Rijksuniversiteit Groningen.

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8

Analyzing Neutrophil Morphology,

Mechanics, and Motility in Sepsis:

Options and Challenges for Novel

Bedside Technologies

Rens Zonneveld, Grietje Molema, Frans B. Plötz Critical Care Medicine 2016, 44:218-28

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N O V EL A SP EC TS O F N EU TR O PH IL S I N S EP SIS

8 ABSTRACT

Objective

Alterations in neutrophil morphology (size, shape and composition), mechanics (deformability), and motility (chemotaxis and migration) have been observed during sepsis. We combine summarizing features of neutrophil morphology, mechanics, and motility that change during sepsis with an investigation into their clinical utility as markers for sepsis through measurement with novel technologies.

Data Sources

We performed an initial literature search in MEDLINE using search-terms ‘neutrophil’, ‘morphology’, ‘mechanics’, ‘dynamics’, ‘motility’, ‘mobility’, ‘spreading’, ‘polarization’, ‘migration’, ‘chemotaxis’. We then combined the results with ‘sepsis’ and ‘septic shock’. We scanned bibliographies of included articles to identify additional articles.

Study Selection and Data Extraction

Final selection was done after the authors reviewed recovered articles. We included articles based on their relevance for our review topic.

Data Synthesis

When compared to resting conditions, sepsis causes an increase in circulating numbers of larger, more rigid neutrophils that show diminished granularity, migration and chemotaxis. Combined measurement of these parameters could provide a more complete view on neutrophil phenotype manifestation. For that purpose, sophisticated automated hematology analysers, microscopy and bedside microfluidic devices provide clinically feasible, high throughput, and cost limiting means.

Conclusions

We propose that integration of features of neutrophil morphology, mechanics and motility with these new analytical methods can be useful as markers for diagnosis, prognosis and monitoring of sepsis and may even contribute to basic understanding of its pathophysiology.

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8 INTRODUCTION

Neutrophils play an essential role during infection and sepsis. While they actively remove invading pathogens during infection, they contribute to the development of septic shock and multi-organ failure through their excessive inflammatory activation, aberrant recruitment, and dysregulated interactions with the vascular endothelium [1-3]. Alterations in neutrophil morphology (size, shape and composition), mechanics (deformability), and motility (chemotaxis and migration) have been observed during sepsis in experimental and clinical studies. For example, during sepsis neutrophils become larger and less granular, which is associated with worse outcome [4-6]. Additionally, neutrophils from septic patients are more resistant to mechanical forces [7] and show slower and diminished migration and chemotaxis when compared to neutrophils from healthy controls [8,9]. To date, the exact roles of such alterations in neutrophils during sepsis remain unclear.

Novel technologies that facilitate detailed high-throughput measurement of these alterations are currently available. Combinatory use of such methods may help to integrate features of morphology, mechanics and motility into a model of specific neutrophil phenotypes. For example, during infection and sepsis, these phenotypes may correlate with clinical features, and predict outcomes. Based on the extent of change of these features, we may be able to distinguish a septic phenotype from a phenotype that corresponds with mere local infection. Ideally, the neutrophils then return to a baseline phenotype after successful treatment or recovery. Analysis of the above mentioned phenotypes could furthermore improve our understanding of neutrophil physiology and sepsis pathogenesis.

Therefore, we combined summarizing the current literature on features of neutrophil morphology, mechanics and motility that change during sepsis with an investigation into their clinical utility as markers for sepsis through measurement with novel technologies.

POTENTIAL FEATURES OF NEUTROPHIL PHENOTYPES

In this section we focus on experimental and clinical evidence for alterations in morphology, mechanics and motility of neutrophils during sepsis and correlations with severity of disease and mortality. Table 1 summarizes these properties, along with established and novel methodologies that can be used to measure them.

Morphology

Morphological features of neutrophils comprise size, shape and (intracellular) composition. The bone marrow contains and retains neutrophil precursor cells representing different stages of maturation (myeloblasts, promyelocyte, myelocyte, and metamyelocyte, respectively), and neutrophils with specific, morphologically distinguishable nuclear compositions, such as immature ‘band’ neutrophils, and mature polymorphonuclear ‘segmented’ neutrophils [3,10]. During sepsis, these cell types are all released into the circulation. An increased ratio of immature to mature neutrophils in the blood correlates with the presence of, and discriminates between, infection and sepsis [11,12]. Microscopic examination of traditional peripheral blood smears reveals that circulating neutrophil precursors and immature neutrophils are larger than senescent neutrophils.

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Larger neutrophils may also result from inflammatory activation of circulating neutrophils in the blood stream. For example, Hoffstein et al. found larger cell sizes after short in vitro incubation with bacterial derived Formyl-Methionyl-Leucyl-Phenylalanin (fMLP) [13], possibly reflecting neutrophil responses that also happen in vivo. This increase in cell size was due to fusion of granule membranes with the cell membrane after degranulation. The clinical relevance of increases in size, shape, volume and granularity, of neutrophils during sepsis is further revealed in studies using devices such as automated hematology analyzers or flow cytometers, which are discussed below.

Blood smears also reveal neutrophil changes in intracellular composition after inflammatory stimulation such as the occurrence of cytoplasmic vacuoles, Döhle bodies and toxic granules. Furthermore, both experimental and clinical studies suggest that based on morphological features of apoptosis (e.g., nuclear condensation, perinuclear chromatin aggregation and membrane blebbing), the degree of neutrophil apoptosis is inversely related to sepsis severity [14-16]. Additionally, a range of experimental studies and some clinical data show association of increased formation of Neutrophil Extracellular Traps (NETs) with sepsis [10,16-19]. NETs are thought to be a defense mechanism against invading pathogens and present extracellular presence of DNA, covered with histones, granular proteins and enzymes. Much of the evidence on NETs is based on ex vivo stimulation of neutrophils with cytokines (Tumor Necrosis Factor (TNF-β), IL-8 or IL1β), plasma from SIRS or septic patients, bacteria, or artificial stimulation with Phorbol 12-myristate 13-acetate (PMA) to form NETs [10,17]. Staining blood smears with immunofluorescently labeled antibodies (e.g., against neutrophil elastase or DNA) can identify NETs in patients [18,19]. At present, this analysis is not clinically feasible yet and data reported so far was not sufficient to discriminate sepsis from other causes of critical illness [18,19].

Table 1. Classic and novel methods for the assessment of neutrophil morphological, mechanical and dynamic

changes in sepsis

Cell Property

Measured Classic Methods Novel Methods Clinical Implementation

Morphology Size, Volume, Granularity Subcellular structures Immature count Blood smears EM Automated hematology analysers Standardized flow cytometry Microscopy Automated hematology analysers Standardized flow cytometry Apoptosis NETosis IF Microscopy EM

None IF measurement of NETs in

blood smears

Mechanics Deformability Sequestration

assays Micropipette assays

Magnetic twisting cytometry

Atomic Force Microscopy

None yet

Motility Migration

Chemotaxis

Transwell assays Time Lapse Microscopy

Microfluidic Devices, K.O.A.L.A.

Bedside Microfluidic Devices / K.O.A.L.A.

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8 Mechanics

In vivo, in order to pass through the microvasculature (diameter 5-6 μm), neutrophils (diameter

7-9 μm) have to be deformable (i.e., flexible or less rigid/stiff). Deformability of neutrophils was investigated in relation to their pulmonary microvascular sequestration in experimental mouse models of sepsis and acute respiratory distress syndrome (ARDS), and in vitro in micropore transit assays [20-25]. Collectively, these studies concluded that a decrease in deformability of activated neutrophils prevented them from traversing through the microvasculature or micropores, or prolonged their transit time in in vitro assays. Other determinants that alter with inflammatory activation and that contribute to increased transit time, such as adhesion (e.g., to endothelium in

vivo or to substrates coated with immobilized adhesion molecules in laboratory setting) were often

not taken into account. Only one in vivo study in rats described sequestration due to increased stiffness of neutrophils, which still occurred after pharmacological blocking of neutrophil integrins that are necessary for proper adhesion of neutrophils to the endothelium, indicating stiffness as the main determinant [25].

When deformability is measured ex vivo, some differences exist between different subtypes of neutrophils. First, immature neutrophils in the bone marrow are reported to be less deformable compared to circulating neutrophils [26]. Second, inflammatory stimulation of circulating neutrophils causes decreased deformability of these cells [27]. This implies that an overall decreased deformability of neutrophils in sepsis may be the resultant of increased rigidity of different subsets (i.e., immature or mature, quiescent or activated). In one clinical study neutrophils from septic patients showed substantially less deformability when compared to neutrophils from healthy volunteers [7]. Interestingly, neutrophils from patients with septic shock and ARDS were even less deformable than neutrophils from patients that had sepsis [7]. Neutrophils from septic neonates were shown to be substantially more rigid than those from healthy neonates [28]. Since also the percentages of circulating immature neutrophils in septic neonates were increased, the data implied that both immature and activated neutrophils were important contributors to overall stiffness of the whole population.

Motility

Upon inflammatory activation in vivo, neutrophils adhere to activated vascular endothelial cells before migrating on and through the endothelium into the underlying tissues [3,10]. These responses are referred to as adhesion, migration and chemotaxis, respectively [3,10]. Translational studies have revealed that neutrophils from septic patients show supranormal adhesion to in vitro cultured endothelium, yet reduced migration [3,30-36]. Migration and chemotaxis of neutrophils have been quantitatively studied in ex vivo experimental settings in which patients’ blood derived neutrophils were allowed to migrate across micropore membranes in transwells towards a gradient of a chemoattractant (e.g., fMLP, casein or IL-8) for a defined length of time, followed by fixation and microscopic quantification [30-36]. Two early studies addressed the potential relevance of impaired migration of neutrophils in post-surgical patients and found a significantly lower total number of migrated neutrophils from patients who developed sepsis [33,34]. Results

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from neutrophils from controls treated with serum or plasma from septic patients were similar in these studies. Two decades later these observations were confirmed in a cohort of septic patients, which revealed that the degree of impairment was associated with non-survival [35]. Furthermore, neutrophils from breast cancer patients who developed bacterial infection after chemotherapy showed less migration when compared to cells from non-infected breast cancer patients [36]. Finally, Najma et al. found a 72% reduction in neutrophil migration into in situ secondary inflammatory sites in septic patients [37].

Although these studies were informative about the level of impairment of migration of neutrophils from septic patients, detailed dynamics underlying these observations could at that time not be obtained. For this purpose, advances in intra-vital and live cell microscopy and microfluidics now provide tools for a qualitative description of dynamic events [38-41]. These methods can give direct visual evidence of deviations from normal behavior during these events, specifically with regard to how septic neutrophils shape (including polarization and spreading) and move (including direction and speed) in comparison to healthy neutrophils, as will be discussed below.

Summary

Figure 1 shows a schematic representation of in vivo neutrophils under resting conditions, infection and sepsis. Sepsis causes an increase in their circulating numbers (including increased numbers of immature neutrophils and precursors), size and stiffness (either as a direct effect of activation or because of the increase in percentage of larger and stiffer immature neutrophils), and a decrease in migration/chemotactic responses. Septic neutrophils are also prone to produce NETs and show less apoptosis, each to be discerned based on their own specific morphological features.

Whilst most studies focus on one particular feature that changes, some indicate that they are dependent on, or the result of, each other, and, as such, part of an integrated immune response. For example, 1) not only decreased neutrophil deformability, but also larger cell size and adhesion to the endothelium caused microvascular sequestration [28]; 2) the ability of neutrophils to migrate was inversely related to increased adhesion and spreading (and concomitant actin re-arrangements) [41]; 3) larger cell sizes were the result of degranulation and membrane addition, which was associated with inflammatory activation [13]; 4) while immature neutrophils were larger and less granular, they were more resistant to apoptosis and prone to the formation of NETs [17].

Furthermore, an integrated series of changes in neutrophils was proposed in studies describing transcriptional profiles of neutrophils and other leukocytes from septic patients and during endotoxemia [42,43]. Data from these studies featured neutrophils that showed simultaneous upregulation of genes involved in actin cytoskeleton signaling, migration and extravasation, and downregulation of genes participating in apoptosis and antigen presentation. The signature of this ‘genomic storm’ may underlie the changes observed in neutrophil morphology, mechanics and motility during sepsis, which emphasizes the likelihood that measurement of the latter can provide a more complete view on neutrophil phenotype manifestation. In the next section we will discuss novel methodologies and techniques that facilitate the measurement of these changes for clinical use in the near future.

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8 NOVEL METHODS FOR IDENTIFICATION OF NEUTROPHIL

PHENOTYPES

This section describes current methodology and advances in the development of new methods to include features of neutrophil morphology, mechanics and motility in the clinical assessment of sepsis (summarized in Table 1).

Automated Measurement of Neutrophil Morphology

As discussed above, distinct neutrophil morphological features associated with inflammation or sepsis have been well defined for over 100 years in microscopic analysis of peripheral blood smears. Reproducibility and prognostic value of this test for sepsis, however, remains poor because of the manual analysis that requires experienced technicians and is subject to human bias [44]. Additionally, analysis of neutrophils in blood smears does not allow for discrimination between local infection and systemic inflammation (e.g., associated with sepsis), nor between bacterial, viral or sterile inflammation. High-throughput automated hematology analyzers (AHA) and standardized clinical flow cytometry may provide means to determine a number of morphological parameters of many neutrophils at the same time and thus may minimize these limitations.

Automated hematology analyzers

Automated hematology analyzers can measure all cell types in peripheral blood of patients. Based on electrical properties (magnetics, impedance) and scatter and absorption of light, they provide detailed information on leukocyte subpopulations and the collectively named Volume, Conductivity and Scatter (VCS) parameters [45,46]. VCS parameters can be measured in whole blood without the need for isolation of neutrophils, extensive sample handling and consequent risk of inducing activation of neutrophils. Some disadvantages of AHA include false identification of clumps of cells or platelets as large (e.g., immature) neutrophils. The latest generation of AHA minimizes these limitations, measures more parameters, and can even be equipped with lasers for measurement of cell surface markers detected by for example immunofluorescently labeled antibodies.

The performance of AHA analyzers for the diagnosis of sepsis was tested in a range of clinical studies, which specifically focused on VCS parameters [4-6,45-48], immature neutrophil counts [12,49-53], and, more recently, the delta neutrophil index (DNI) (i.e., a reflection of the immature neutrophil population established by measuring the neutrophil differentials for MPO-activity and nuclear lobularity [54-59]). This work showed that septic patients usually present with significantly different VCS parameters (i.c., larger volumes and lower scatter, indicating degranulation), and a larger fraction of immature neutrophils, also reflected by a higher DNI (Table 2). Important findings in these studies were that VCS parameters and DNI could serve as early indicators of sepsis (including neonatal sepsis) [49,51,52-55,58,59], that more aberrant values were associated with severity of disease and mortality [46-48,52,56], and that upon treatment or recovery these values returned to baseline [58,59].

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A. Resting B. Local infection C. Sepsis Inﬂamed Tissue Bone Marrow Bone Marrow Bone Marrow Interstitium

i: Hyperadhesion with potential for

EC damage & Vascular Leak

ii: Sequestration

without EC damage

Figure 1. Schematic representation of neutrophil behavior in vivo. Under resting

conditions A: neutrophils are retained in the bone marrow and circulation, and only have short-term, reversible tethering and rolling interactions with the vascular endothelium. During infection/inflammation

B: the circulating inflammatory mediators

stimulate release of neutrophils and precursors from the bone marrow into the circulation. Upon encountering activated endothelial cells, neutrophils upregulate/ activate adhesion molecules (including integrins) on their cell surface. Neutrophils then engage into coordinated interactions with the endothelium (i.e., the leukocyte adhesion cascade; see reference 29 for detailed description of these events) and ultimately transmigrate into the underlying inflamed tissue. As part of a hypothesis on the role of neutrophils under septic conditons

C: neutrophils can become activated by

high levels of circulating bacteria derived agonists, such as endotoxin, and cytokines

such as Tumor Necrosis Factor (TNF- α) (also

known as the cytokine storm). Earlier, albeit limited, data suggest that such activation could i: promote excessive upregulation of integrins on neutrophil cell surfaces, driving hyperadhesion to the endothelium with potential for collateral endothelial damage (Reviewed in reference 3). However, no direct evidence of structural damage to the endothelium after neutrophil adhesion during sepsis is currently available. Furthermore, ii: re-release of neutrophils into the circulation after periods of sequestration/margination (e.g., due to decreased deformability or size discrepancy between relatively large diameter of neutrophils versus the small diameter of capillaries) without structural damage to the endothelium or other tissues has also been described (Reviewed in reference 82).

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Ta b le 2 . C lin ic al s tu di es u si ng a uto m ate d h em ato lo g y a na ly si s o f n eu tr o ph il m o rp ho lo g y i n s ep si s Ar ti cl e [r ef .] Po p ul at io n Pa ra m et ers C o n tr o l 1 Se p ti c 1 P-va lu e C o rr el at io n An al yz er C el ik e t al . 2 0 12 [4 ] N eo na te s V o lu m e C o nd uc ti vi ty Sc at te r 14 8. 4 (1 1.1 ) 16 1. 5 (9 .1) 12 9 (1 0 .3 ) 17 0 .2 ( 18 .9 ) 15 5. 9 (1 1.9 ) 12 5. 5 (1 1. 8 <0 .0 5 <0 .0 5 <0 .0 5 Ea rl y d ia gn o si s o f s ep si s C o ul te r LH 7 80 M ar d i e t al . 2 0 10 [5 ] A d ul ts V o lu m e C o nd uc ti vi ty Sc at te r 13 9 (6 .6 ) 14 3 (3 .3 ) 14 4 (1 2. 1) 15 9 (1 6. 2) 14 6 (4 .3 ) 13 7 (1 2. 0 ) <0 .0 0 1 0 .0 13 <0 .0 0 1 D ia gn o si s o f s ep si s D is cr im in at io n o f l o ca liz ed in fe ct io n an d s ep si s C o ul te r LH 7 50 Le e et a l. 20 13 [6 ] El d er ly V o lu m e C o nd uc ti vi ty Sc at te r (M A LS ) 14 8 ±1 9. 3 15 0 ± 5. 3 13 4 ± 13 .6 16 5 ±2 4. 4 14 6 ±6 .4 13 0 ± 13 .2 <0 .0 0 1 0 .0 3 0 .6 0 7 D ia gn o si s o f s ep si s D is cr im in at io n o f l o ca liz ed in fe ct io n an d s ep si s C o ul te r D xH 8 0 0 C el ik e t al . 2 0 13 [4 5] N eo na te s V o lu m e C o nd uc ti vi ty Sc at te r 14 8. 4 (1 1.1 ) 16 1. 5 (9 .1) 12 9 (1 0 .3 ) 17 0 .2 ( 18 .9 ) 15 5. 9 (1 1.9 ) 12 5. 5 (1 1. 8 <0 .0 5 <0 .0 5 <0 .0 5 W it h C RP / IL -6 fo r ea rl y d ia gn o si s o f se ps is C o ul te r LH 7 80 C ha ve s e t al . 2 0 0 5 [4 6] A d ul ts V o lu m e C o nd uc ti vi ty Sc at te r 14 3 ±4 .8 14 2 ±2 .6 14 6 ±7 .3 15 6 ± 13 .5 14 1 ± 3. 9 14 0 ± 10 .1 0 .0 0 1 0 .2 33 0 .0 0 2 D ia gn o si s o f a cu te b ac te ri al in fe ct io n C o ul te r LH 7 50 C ha ra fe d d in e et a l. 20 11 [4 8] A d ul ts N D W 19 .8 ( 1.1 ) 25 .1 (3 .6 ) <0 .0 5 D ia gn o si s o f S IR S C o ul te r LH 7 50 Pa rk e t al . 2 0 11 [4 9] A d ul ts V o lu m e Sc at te r IG 15 3 ±1 4. 3 14 1 ± 8. 6 0 .2 ± 0 .13 16 8 ±2 8. 8 13 1 ± 14 .4 2. 4 ±4 .6 <0 .0 5 <0 .0 5 D ia gn o si s o f s ep si s C o ul te r D xH 80 0 Sy sm ex X E 21 0 0 M ak ka r et a l. 20 13 [5 1] N eo na te s I:M 2 I:T 2 53 .1/ 97 .2 93 .8 /9 4. 4 D ia gn o si s o f s ep si s M S 95 Se nt hi ln ay ag am e t al . 2 0 12 [5 2] A d ul ts IG C 2 IG ( % ) 2 86 .3 /> 90 92 .2 /> 90 D ia gn o si s o f b ac te re m ia C o ul te r A ct D iff 5 N ig ro e t al . 2 0 0 5 [5 3] N eo na te s IG ( % ) 2 33 /8 8 Pr ed ic ti o n o f p o si ti ve b lo o d c ul tu re Sy sm ex X E-21 0 0

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Ta b le 2 . ( con ti nue d ) Ar ti cl e [r ef .] Po p ul at io n Pa ra m et ers C o n tr o l 1 Se p ti c 1 P-va lu e C o rr el at io n An al yz er Pa rk e t al . 2 0 11 [5 4] N eo na te s D N I 3 0 ( 0 -0 .1) 2. 8 (0 .5 -5 .3 ) / 16 .9 ( 9. 5-35 .6 ) 4 0 .0 0 3 / <0 .0 0 1 D ia gn o si s o f s ep si s an d p re d ic ti o n o f se ve ri ty Si em en s A D V IA 2 12 0 Li m e t al . 2 0 14 [5 5] A d ul ts D N I 5 48 .2 84 .2 0 .0 0 7 Pr ed ic ti o n o f s ep si s in S BP pa ti en ts Si em en s A d vi a 21 20 N ah m e t al . 2 0 0 8 [5 6] A d ul ts D N I 6 19 .5 65 <0 .0 5 Pr ed ic ti o n o f s ep si s an d o ut co m e Si em en s A d vi a 12 0 Le e et a l. 20 14 [5 7] N eo na te s D N I 7 0 .6 ( 0 .0 -2 .1) 2. 8 (0 .8 0 -5 .5 ) <0 .0 0 1 Pr ed ic ti o n o f t ru e ba ct er em ia Si em en s A d vi a 21 20 K im e t al . 2 0 12 [5 8] A d ul ts D N I 8 1.9 6. 2 0 .0 0 5 D ia gn o si s o f s ep si s Si em en s A d vi a 21 20 Se o k et a l. 20 12 [5 9] N eo na te s D N I 9 0 .0 ( 0 .0 -0 .0 ) 0 .8 ( 0 .0 -1 .7 ) / 3. 4 (1 .5 -5 .3 ) /1 8. 6 (9 .3 -2 4. 7) 0 .0 0 0 1 D ia gn o si s o f s ep si s Si em en s A d vi a 21 20 IG C = Im m at ur e g ran ul o cy te c o un t; IG = Im ma tu re g ra nu lo cy te s; I: M = Im m at ur e : M at ur e ne ut ro ph il ra ti o ; I :T = Im m at ur e ne ut ro ph il : T o ta l n eu tr o ph il ra ti o ; D N I = D el ta N eu tr o ph il In d ex ; M A LS = m ed ia n an gl e lig ht s ca tt er ; N D W = N eu tr o ph il D is tr ib ut io n W id th ; C RP = C -r ea ct iv e Pr o te in ; S IR S = sy st em ic in fla m m at o ry r es p o ns e sy nd ro m e; S BP = s p o nt an eo us b ac te ri al p er it o ni ti s. 1 V C S da ta p re se nt ed a s m ea n (r an ge ) o r m ea n ± st an d ar d d ev ia ti o n. 2 D at a pr es en te d a s se ns it iv it y / sp ec ifi ci ty fo r pr ed ic ti o n o f s ep si s / ba ct er em ia ( % ). 3 D at a pr es en te d a s m ed ia n ( ra ng e) . 4 D at a re pr es en ts s ep si s / se pt ic s ho ck . 5 D N I c ut o ff va lu e 5. 7% (c o nt ro l = < 5 .7 % a nd s ep si s = > 5. 7% ); D at a pr es en te d a s % o cc ur re nc e o f s ep ti c sh o ck a m o ng st S PB p at ie nt s. 6 D N I c ut o ff va lu e 40 % (c o nt ro l = < 40 % a nd s ep si s = >4 0 % ); D at a pr es en te d a s % o cc ur re nc e o f p o si ti ve b lo o d c ul tu re . 7 D N I c o nt ro l = b lo o d c ul tu re c o nt am in at io n; D N I s ep ti c in t hi s st ud y = tr ue b ac te re m ia . D at a pr es en te d a s m ed ia n (r an ge ). 8 D N I c o nt ro l = s ur vi vo r; D N I s ep ti c = no n su rv iv o r; D N I p re se nt ed a s m ed ia n (% ). 9 D at a pr es en te d a s m ed ia n (r an ge ); S ep si s da ta p re se nt ed as S IR S / Se ps is / S ev er e Se ps is .

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Standardized clinical flow cytometry

Multiparameter flow cytometry uses diffraction of light to measure cell size (forward scatter; FSC) and cellular content (i.e., granularity; side scatter; SSC), along with laser based detection of cell surface markers labeled by fluorescently labeled antibodies. Nowadays flow cytometry is broadly used in the diagnosis of cancer, in assessing immune status of HIV patients, or for the monitoring of immunological disease, but the use in diagnosis and monitoring of sepsis remains limited [60-63]. Recently, Roussel et al. applied a standardized clinical flow cytometric analysis of sepsis in 450 patients by analyzing their neutrophils [64,65]. They found that larger, less granular neutrophils and increased numbers of immature neutrophils (identified with immunofluorescent staining of cell surface markers) were associated with sepsis outcome and mortality.

Of note, several studies compared performance of measurement of the same parameters between automated hematology analysers and flow cytometers and found overlapping results [64-66]. Since the newest generations of automated hematology analyzers are able to measure fluorescence equally well as flow cytometers, they can be used for the simultaneous measurement of neutrophil morphological parameters and cell surface markers associated with their activation status (e.g., CD11b, CD64) and neutrophil immaturity (e.g., CD16, CXCR2).

Automated Measurement of Neutrophil Deformability

Sepsis is associated with changes in neutrophil deformability that can be measured with different assays [7,20-28,67,68]. For example, micropipette assays are useful for ex vivo determination of deformability. With this method single neutrophils are aspirated against the tip of glass micropipettes (usually with a lumen diameter of 2-5 μm and coated with plasma), followed by time-lapse microscopic analysis of the neutrophils’ ability to migrate into the lumen. Other approaches for measurement of deformability include the use of microchannels, magnetic twisting cytometry and atomic force microscopy, which are currently being used in experimental settings [67]. Although these methods are promising, their clinical use is not yet feasible, mostly due to complexity of protocols and interpretation. A further detailed summary of developments in these methods and their technical limitations can be found elsewhere [67].

Also, as mentioned above, techniques that measure deformability separate from other variables are still absent. This was illustrated in samples of a small cohort of septic patients of which blood transit time and number of obstructed microchannels were measured in an automated multiple microchannel flow analyzer [68]. Whole blood from control patients passed easily through the microchannels, while increased numbers of rigid leukocytes (predominantly neutrophils) in blood from septic trauma patients caused significant obstruction of the flow, thereby increasing both transit time and number of obstructed channels. These observations are also indicative of changes in migration and chemotaxis, which are discussed in the next paragraph.

Automated Measurement of Neutrophil Migration and Chemotaxis

The activation state of neutrophils in response to septic conditions has an effect on their migration and chemotaxis [5,6,8,9,33-35,68-76]. In particular, neutrophils from septic patients show less

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migration and chemotactic responses when compared to neutrophils from healthy subjects. As mentioned above, quantitative transwell assays are informative about total endpoint migration and chemotaxis, but are not informative about the actual underlying dynamics. For that purpose, parameters such as speed or direction of migration events are now being merged into clinical use through the rapidly evolving and promising field of microfluidics [69]. Microfluidics in this respect refers to methods in which small volumes of fluids (e.g., whole blood or isolated cells in suspension) are applied to easy-to-use lab-on-a-chip microfluidic devices equipped with standardized gradients of chemoattractants. These systems allow for fast assessment of neutrophil migration and chemotaxis with arrayed time-lapse imaging and tracking algorithms [8,9,69-72] (Table 3). For example, Berthier et al. tested their in house designed microfluidics device for the analysis of neutrophils from an infant who presented with severe recurrent bacterial infections due to a primary immunodeficiency disorder [71]. Their assay revealed impaired polarization and chemotaxis in response to fMLP of neutrophils from the patient when compared to neutrophils from its parents and an age-matched control. Using a similar approach, a clinical study in burn patients (who usually present with a sepsis-like systemic inflammatory response syndrome) observed an inverse relationship between neutrophil migration velocity towards a gradient of fMLP and burn size in adults and children [9]. Interestingly, migration velocities were associated with the development of post-burn bacteremia and sepsis, outlining the potential for clinical use of such analytical approaches. Limitations to be dealt with include the necessity of isolation of neutrophils from whole blood and gradient stabilization (such as described in reference 8).

A clinically more feasible approach, in which these limitations were minimized, was reported in a prospective clinical study by Sackmann et al. to diagnose asthma [8]. Whole blood from patients was pipetted into channels of a microfluidic device coated (or ‘primed’) with a neutrophil integrin ligand (e.g., adhesion molecules such as E-selectin or ICAM-1), which resulted in direct capture of neutrophils to the substrate. The unbound cells and other blood components were washed away with laminar flow and the bound neutrophils were next allowed to migrate towards a controlled gradient of fMLP already present in the device. Significantly lower neutrophil migration velocity was predictive for the presence of asthma, with a sensitivity and specificity of 96% and 73% respectively, and it discriminated asthma from allergic rhinitis. This assay required no additional reagents and only 3 μL of whole blood, making it ideally suitable for situations in which only small blood sample volumes can be acquired, such as in neonatal sepsis. Along with migration velocities this assay can provide information on neutrophil polarization (i.e., leading edge formation) and migration directionality through arrayed time-lapse imaging.

Although the direct use in sepsis diagnostics still remains to be investigated, microfluidic devices provide a feasible, fast and cost-limiting aid. Additionally, microfluidic devices that mimic the in vivo circumstances (e.g., with multiple chemokine gradients and substrates with, or closely resembling, endothelial monolayers) are currently under development and could help providing critical insight into neutrophil dynamics and underlying molecular cues in health and disease [73-76].

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Ta b le 3 . S tu di es m ea su ri ng n eu tr o ph il m ig ra ti o n/ ch em o ta xi s v el o ci ty i n r el at io n to i nfl am m at io n a nd s ep si s Ar ti cl e [R ef .] Se tt in g M ea su re m en t C h em o ki n e Su b st ra te C o n tr o l 1 Pa ti en ts 1 P-va lu e D ev ic e Sa ck m an n et a l. 20 14 [5 ] C lin ic al : a st hm a C he m o ta xi s V el o ci ty fM LP ( 10 0 n M) P-se le ct in ( 10 0 μ g/ m L) 1. 6 1. 33 0 .0 0 2 K .O .A .L .A . Bu tl er e t al . 2 0 10 [6 ] C lin ic al : b ur n pa ti en ts C he m o ta xi s V el o ci ty fM LP ( 10 0 n M) Fi br o ne ct in ( 10 0 μ M ) 18 ± 5 9 ±6 <0 .0 1 M ic ro flu id ic d ev ic e Zh o u et a l. 20 0 4 [9 ] Ex p er im en ta l M ig ra ti o n N o ne G la ss 1. 7 2 1. 5 3/3 .6 4 / 6. 3 5 n. s. /< 0 .0 1/ <0 .0 1 Fl o w c el l s ys te m D ui gn an e t al . 1 98 6 [3 3] C lin ic al : p o st s ur gi ca l se ps is C he m o ta xi s C as ei n (2m g/ m L) 8μ m p o re M em br an e 86 .8 ± 1.9 5 73 .4 ± 3. 1 5 <0 .0 2 M o d ifi ed B o yd en ch am b er C hr is to u et a l. 19 79 [3 4] C lin ic al : s ep si s C he m o ta xi s C as ei n (5 m g/ m L) 8μ m p o re M em br an e 12 8. 1 ± 2. 4 5 90 .4 ± 2. 9 5 <0 .0 0 1 M o d ifi ed B o yd en ch am b er Ta ve re s et a l. 20 0 2 [3 5] C lin ic al : s ep si s C he m o ta xi s fM LP ( 10 0 n M) 8μ m p o re M em br an e 93 .4 ± 6. 6 6 51 ± 8. 3 6 <0 .0 1 48 w el l c ha m b er (N eu ro pr o b e) M en d o nç a et a l. 20 0 5 [3 6] C lin ic al : b re as t ca nc er / ba ct er em ia C he m o ta xi s fM LP ( 10 0 n M) 8μ m p o re M em br an e 30 .1 ±8 .3 6 2. 8 ±1 .3 6 <0 .0 0 1 48 w el l c ha m b er (N eu ro pr o b e) A gr aw al e t al . 2 0 0 8 [7 0 ] D ev ic e o pt im iz at io n C he m o ta xi s V el o ci ty fM LP , I L-8 V ar io us 1 7 N o ne M ic ro flu id ic d ev ic e Be rt hie r et a l. 20 10 [ 71 ] In fa nt w it h re cu rr en t in fe ct io ns C he m o ta xi s V el o ci ty fM LP ( 10 0 n M) Fi br o ne ct in 0 .15 8 0 .0 7 8 n = 1 M ic ro flu id ic d ev ic e fM LP = N -f o rm yl m et hi o ni ne -l eu ci ne -p he ny la la nin e; K .O .A .L .A = k it-o n-a-l id -a ss ay . 1 D at a pr es en te d a s m ea n (r an ge ) o r m ea n ± st an d ar d d ev ia ti o n (μ m /m in ). 2 N eu tr o ph ils in H BS S. 3 N eu tr o ph ils in H BS S + fM LP (1 0 0 nM ). 4 N eu tr o ph ils in H BS S + add ed p la sm a pr o te in s. 5 D at a pr es en te d a s m ea n d is ta nc e ± st an d ar d e rr o r o f m ea n (μ m ). 6 D at a pr es en te d a s m ea n n o f e m ig ra te d n eu tr o ph ils ± st an d ar d e rr o r. 7 D at a pr es en te d fo r ch em o ta xi s ve lo ci ty o f h ea lt hy n eu tr o ph ils to w ar d s fM LP g ra d ie nt o n P-se le ct in s ub st ra te ( μm /m in ). 8 D at a pr es en te d a s m ea n v el o ci ty ( μm /s ec ).

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8 CONCLUSIONS AND HYPOTHESIS

Based on our review of the literature we conclude that in sepsis the degree of inflammatory activation drives changes in neutrophil morphology, mechanics and motility that are related to clinical outcome. In particular, sepsis causes an increase in circulating numbers of larger, less granular, more rigid neutrophils that show substantially diminished migration responses. The integration of these changes into a diagnostic algorithm along with traditional biomarkers could facilitate the identification of a clinically relevant hyperactivated septic neutrophil phenotype that can be discriminated from other neutrophil phenotypes associated with local inflammation or resting conditions (Figure 2). Until recently, technical limitations, such as the need for manual analysis of the peripheral blood smear, still make made it difficult to properly evaluate the clinical potential of measuring these changes in neutrophils. In fact, the traditional approach (i.e., assessing neutrophil numbers and differentiation) has proven to be insufficient and there is a need for a more complete identification of neutrophil phenotypes for sepsis diagnosis and clinical management [77]. For that purpose, sophisticated automated hematology cell analysers and bedside microfluidic devices reduce practical limitations and may provide feasible and time and cost-limiting aids.

The next step in validation of different phenotypes is to prospectively compare results obtained by integrated measurements of these features in neutrophils between healthy subjects, patients with local inflammation, and patients with sepsis. There are some challenges that need to be considered before implementation of such an approach into the clinic is feasible. First, it is becoming clear that neutrophils, instead of being one single cell type, are in fact a heterogeneous group of cells with different fates and functions [78]. As such, neutrophils analyzed in peripheral blood are not necessarily representative of the marginating pool in the microvasculature in vivo that may have already depleted their innate immune functions (e.g., degranulation, migration, NETosis). Second, diagnostic accuracy of the methodologies mentioned above needs to be further investigated separately for proper establishment of clear cut-off values for each method to discriminate healthy from septic neutrophils, also taking patient heterogeneity into account. Third, the degree of changes in neutrophil morphology, mechanics and motility during sepsis seems closely related to severity of disease and mortality of sepsis. However, how neutrophils and these changes are exactly involved in sepsis pathophysiology, specifically in the development of endothelial damage and subsequent organ failure, remains to be elucidated. To date there are conflicting data on this latter issue. For example, some in vitro data imply increased adhesion of septic neutrophils to the endothelium and loss of endothelial barrier function after incubation with septic neutrophils [3,79]. In contrast, post mortem evaluation of human tissue after sepsis revealed neutrophils in proximity of endothelium without evidence of endothelial damage or increased accumulation of neutrophils in lung tissue, which was not associated with the extent of damage resulting in acute lung injury (ALI) [80-82]. Identification of the here proposed more complex neutrophil phenotypes may help in providing answers with regard to these questions about the role of neutrophils in sepsis pathophysiology.

In conclusion, we propose that integration of neutrophil morphology, mechanics and motility with these novel methods can lead to more accurate diagnosis, monitoring and prognosis of sepsis.

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Moreover, these analyses may be able to substantially contribute to the basic understanding of sepsis, and in due time unveil new and specific treatment options as well as a means to determine therapeutic effects of new treatments.

Acknowledgments

We acknowledge financial support from Tergooi Hospitals, the Drie Lichten foundation, the Ter Meulen Fund (Royal Netherlands Academy of Arts and Sciences KNAW) and the IPRF Early Investigators Exchange Program Award of the European Society for Pediatric Research (all to RZ).

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8 Local Infection

Healthy

Sepsis

Size

Granularity

Count

Immature:mature

Ratio

Cell-Surface

Molecules

Mature “Segmented”

Neutrophil Immature “Band” Neutrophil

Deformability

Migration

Delta Neutrophil

Index

HIGH

x Y x Y x Y

LOW

Figure 2. Schematic representation of healthy, local inflammatory and septic phenotypes of neutrophils.

Controlled inflammatory conditions drive changes in neutrophils. The excessive inflammatory conditions of sepsis promote the presence of higher numbers of neutrophils, higher numbers of (larger) immature neutrophils and large, less granular neutrophils in peripheral blood. Hypothetically, the average deformability and migration velocities of septic neutrophils will be lower than under localized inflammatory conditions. Novel methodologies can analyze many of these variables at the same time. Multiple morphological features (size, volume, granularity, cell type and maturity) can be quickly assessed from high numbers of cells in small volume blood samples with automated hematology analyzers and flow cytometry. The newest generation of hematology analyzers can even incorporate measurement of critical cell surface molecules through fluorescently labeled antibody based detection similar to flow cytometry. For neutrophil deformability measurements, no clinically feasible technique has been developed yet. Neutrophil migration can be measured at the bedside with microfluidic devices using small volumes of whole blood. Integrated measurement of these variables with these novel methods can facilitate more accurate diagnosis, monitoring and management of sepsis.

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