Differential Immunoexpression of Inhibitory Immune Checkpoint Molecules and Clinicopathological Correlates in Keratoacanthoma, Primary Cutaneous Squamous Cell Carcinoma and Metastases.

Beyond established anti-programmed cell death protein 1/programmed cell death ligand 1 immunotherapy, T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) and its ligand CD155 are promising novel inhibitory immune checkpoint targets in human malignancies. Yet, in cutaneous squamous cell carcinoma, evidence on the collective expression patterns of these inhibitory immune checkpoints is scarce. Complete tumour sections of 36 cutaneous squamous cell carcinoma, 5 cutaneous metastases and 9 keratoacanthomas, a highly-differentiated, squamoproliferative tumour, with disparately benign biologic behaviour, were evaluated by immunohistochemistry for expression of programmed cell death ligand 1 (Tumor Proportion Score, Immune Cell Score), TIGIT, CD155 and CD8+ immune infiltrates. Unlike keratoacanthomas, cutaneous squamous cell carcinoma displayed a strong positive correlation of programmed cell death ligand 1 Tumor Proportion Score and CD115 expression (p < 0.001) with significantly higher programmed cell death ligand 1 Tumor Proportion Score (p < 0.001) and CD155 expression (p < 0.01) in poorly differentiated G3-cutaneous squamous cell carcinoma compared with keratoacanthomas. TIGIT+ infiltrates were significantly increased in programmed cell death ligand 1 Immune Cell Score positive primary tumours (p = 0.05). Yet, a strong positive correlation of TIGIT expression with CD8+ infiltrates was only detected in cutaneous squamous cell carcinoma (p < 0.01), but not keratoacanthomas. Providing a comprehensive overview on the collective landscape of inhibitory immune checkpoint expression, this study reveals associations of novel inhibitory immune checkpoint with CD8+ immune infiltrates and tumour differentiation and highlights the TIGIT/CD155 axis as a potential new target for cutaneous squamous cell carcinoma immunotherapy.

Chemokines and Cytokines in Immunotherapy of Melanoma and Other Tumors: From Biomarkers to Therapeutic Targets.

Chemokines and cytokines represent an emerging field of immunotherapy research. They are responsible for the crosstalk and chemoattraction of immune cells and tumor cells. For instance, CXCL9/10/11 chemoattract effector CD8+ T cells to the tumor microenvironment, making an argument for their promising role as biomarkers for a favorable outcome. The cytokine Interleukin-15 (IL-15) can promote the chemokine expression of CXCR3 ligands but also XCL1, contributing to an important DC-T cell interaction. Recruited cytotoxic T cells can be clonally expanded by IL-2. Delivering or inducing these chemokines and cytokines can result in tumor shrinkage and might synergize with immune checkpoint inhibition. In addition, blocking specific chemokine and cytokine receptors such as CCR2, CCR4 or Il-6R can reduce the recruitment of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) or regulatory T cells (Tregs). Efforts to target these chemokines and cytokines have the potential to personalize cancer immunotherapy further and address patients that are not yet responsive because of immune cell exclusion. Targeting cytokines such as IL-6 and IL-15 is currently being evaluated in clinical trials in combination with immune checkpoint-blocking antibodies for the treatment of metastatic melanoma. The improved overall survival of melanoma patients might outweigh potential risks such as autoimmunity. However, off-target toxicity needs to be elucidated.

Long-term neurocognitive function after whole-brain radiotherapy in patients with melanoma brain metastases in the era of immunotherapy.

BACKGROUND: Whole-brain radiotherapy (WBRT) used to be standard of care for patients suffering from melanoma brain metastases (MBM) and may still be applicable in selected cases. Deterioration of neurocognitive function (NCF) is commonly seen during and after WBRT. Knowledge on long-term effects in melanoma patients is limited due to short survival rates. With the introduction of immune checkpoint inhibitors, patients may experience ongoing disease control, emphasizing the need for paying more attention to potential long-term adverse effects. METHODS: In this single-center study, we identified in a period of 11 years all long-term survivors of MBM who received WBRT at least 1 year prior to inclusion. NCF was assessed by Neuropsychological Assessment Battery (NAB) screening and detailed neurological exam; confounders were documented. RESULTS: Eight patients (median age 55 years) could be identified with a median follow-up of 5.4 years after WBRT. Six patients reported no subjective neurological impairment. NAB screening revealed an average-range score in 5/8 patients. In 3/8 patients a NAB score below average was obtained, correlating with subjective memory deficits in 2 patients. In these patients, limited performance shown in modalities like memory function, attention, and spatial abilities may be considerably attributed to metastasis localization itself. Six out of 8 patients were able to return to their previous work. CONCLUSION: Five of 8 long-term survivors with MBM after WBRT experienced little to no restriction in everyday activities. In 3 out of 8 patients, cognitive decline was primarily explained by localization of the metastases in functionally relevant areas of the brain. The results of our small patient cohort do not support general avoidance of WBRT for treatment of brain metastases. However, long-term studies including pretreatment NCF tests are needed to fully analyze the long-term neurocognitive effects of WBRT.

Evaluation of Psoriasis Area and Severity Index Thresholds as Proxies for Systemic Inflammation on an Individual Patient Level.

BACKGROUND: Psoriasis is a chronic and systemic inflammatory disease with a loss of up to 5 life years, which is thought to be reduced by biologic treatment. Disease severity and eligibility for systemic treatment are often based on the cutaneous psoriasis area and severity index (PASI) with a cut-off of 10 in several European countries. However, it is unclear how well this cut-off reflects systemic inflammation and, consequently, the risk for the development of comorbidity. OBJECTIVES: (1) To assess whether specific PASI thresholds, in particular PASI 10, predict elevated biomarkers of systemic inflammation and cardiovascular risk on an individual patient level. (2) To assess the association of PASI and psoriatic arthritis with biomarkers of systemic inflammation and cardiovascular risk. METHODS: Retrospective cross-sectional study of 72 psoriasis patients without systemic treatment. RESULTS: Overall, 68, 42, and 50% of patients had cardiovascular risk level neutrophil-to-lymphocyte ratio (NLR), C-reactive protein, and elevated platelet-to-lymphocyte ratio (PLR) values, respectively. The respective positive predictive values of PASI 10 were 70, 45, and 70. The performance of the optimal PASI cut-offs according to the Youden index was similarly weak. Subgrouping of patients with a PASI below 10 did not result in a considerably improved reflection of systemic inflammation. PLR was significantly higher in patients with moderate-to-severe compared to mild psoriasis and significantly correlated with PASI in patients with a PASI above 2 (rs = 0.266, n = 64). NLR was significantly higher in patients with psoriatic arthritis. CONCLUSION: Specific PASI thresholds were not well suited to predict elevated biomarkers of systemic inflammation and cardiovascular risk on an individual patient level. Therefore, PASI, and possibly other purely cutaneous measures, may not be ideal as stand-alone parameters to define disease severity and eligibility for systemic treatment. Our results are relevant for the ongoing discussion on the definition of psoriasis severity and eligibility for systemic treatment. Further research addressing the added value of a set of biomarkers of systemic inflammation in the assessment of psoriasis severity would be desirable.

Ablative Fractional Laser-assisted Low-irradiance Photodynamic Therapy for Treatment of Actinic Keratoses in Organ Transplant Recipients: A Prospective, Randomized, Intraindividual Controlled Trial.

Pain and inferior efficacy are major limiting factors of conventional photodynamic therapy for the field treatment of actinic keratoses in immunosuppressed organ transplant recipients. This prospective randomized controlled study evaluates the efficacy and tolerability of ablative fractional laser system pretreatment combined with low-irradiance photodynamic therapy (18.5 mW/cm2) compared with conventional photodynamic therapy (61.67 mW/cm2) in the treatment of actinic keratoses on the face and scalp in organ transplant recipients, using a red light-emitting diode lamp at a total light dose of 37 J/cm2. Low-irradiance photodynamic therapy combined with Er:YAG pretreatment achieved a significantly superior lesion response rate (mean ± standard deviation 77.3 ± 23.6%) compared with conventional photodynamic therapy (61.8 ± 21.4%; p = 0.025) in intra-individual fields at 3 months without negatively impacting pain (p = 0.777) or cosmetic outcome (p = 0.157).

Evaluation of Psoriasis Area and Severity Index as a Proxy for Bio-markers of Systemic Disease under Treatment with Tumour Necrosis Factor-alpha and Interleukin 12/23 Antagonists in Patients with Psoriasis: A Retrospective Cohort Study of 186 Treatment Cycles.

The efficacy of psoriasis treatments is usually evaluated using the Psoriasis Area and Severity Index (PASI). However, there is a lack of systematic statistical assessments of PASI as a proxy for systemic disease in individual patients. Therefore, a retrospective study of 186 treat-ments with adalimumab, etanercept, and ustekinumab for psoriasis (341 patient-years) was performed. While PASI significantly and independently correlated with biomarkers of systemic inflammation (especially neutrophil-to-lymphocyte ratio, C-reactive protein), the strengths were only weak-to-moderate and varied considerably inter-individually. A decrease in PASI indicated a neutrophil-to-lymphocyte ratio decrease and a C-reactive protein decrease or stable low margin C-reactive protein in ≥ 80%. Sensitivity, specificity, and positive predictive value of PASI 0 and PASI 2.75 (optimal Youden Index) for low cardiovascular risk C-reactive protein were 24%, 92%, 85%, and 62%, 61%, 76%, respectively. Performance was similar using absolute thresholds and PASI 100 or PASI 75, and overall worse for low cardiovascular risk neutrophil-to-lympho-cyte ratio and if psoriasis arthritis was present. In conclusion, PASI allows robust low-order estimates of systemic inflammation, but cannot substitute for laboratory biomarkers for more precise assessments.

Scleromyxedema.

Scleromyxedema is a rare, cutaneous deposition disorder from the group of mucinoses, which can affect multiple organs and is virtually always associated with a monoclonal gammopathy. Cutaneous manifestations are usually generalized, 2 to 3 mm sized, dome-shaped or flat-topped, waxy, slightly red to skin-colored papules and sclerodermoid indurations. Neurological, rheumatological, cardiovascular, gastrointestinal, respiratory tract, renal and ophthalmologic manifestations can occur, with decreasing frequency. A serious and potentially lethal complication is the dermato-neuro syndrome which manifests with flu-like prodromes followed by fever, convulsions and coma. Untreated, scleromyxedema usually takes an unpredictable and potentially lethal progressive disease course over several years. According to a widely acknowledged classification by Rongioletti a diagnosis of scleromyxedema can be rendered when (1) generalized, papular and sclerodermoid eruption, (2) a histological triad of mucin deposition, fibroblast proliferation and fibrosis, and (3) monoclonal gammopathy are present, and (4) thyroid disease is absent. Apart from the classic microscopic triad, an interstitial granuloma annulare like pattern was also described. The pathogenesis of scleromyxedema is unknown. A potential role for various, as yet unknown serum factors has been discussed. An unequivocal causal relationship between paraproteinemia and disease manifestations could not be established to date. High dose intravenous immunoglobulins (IVIg) are the first-line treatment of choice according to the most recent European guidelines.

Low irradiance compared with conventional photodynamic therapy in the treatment of actinic keratoses.

BACKGROUND: Photodynamic therapy (PDT) is an effective therapy treating photodamaged areas with multiple actinic keratoses (AK). Still pain during therapy is one of the most challenging obstacles for patients. This retrospective study compares pain and efficacy intra-individual in patients using conventional PDT (c-PDT) compared to a low irradiance PDT protocol (li-PDT) with a reduced irradiance to 25% of c-PDT. METHODS: Thirty-one patients were enrolled into this retrospective analysis treated with li-PDT and c-PDT on comparable fields of actinic damage on the forehead or the cheek. Pain was scored by the patients using a VAS. Moreover, number and time to therapy interruptions were documented. For effectiveness number and grade of AK were counted before and 4 weeks after PDT. RESULTS: Maintaining a total light dose of 37 J/cm2 , a decrease in irradiation in li-PDT patients resulted in significant less pain (VAS score 2.8 vs 7.6) and fewer therapy interruptions compared to treatment with c- PDT (P < 0.0005). No significant difference in treatment outcome was found (P = 0.068). CONCLUSION: Our data shows that li-PDT can reduce pain with at least comparable clinical outcome compared to c-PDT. Therefore, it is an effective and well-tolerated treatment for patients with multiple AK.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease.

BACKGROUND: Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. OBJECTIVE: We sought to determine whether AIBDs develop in Treg cell-deficient scurfy mice. METHODS: Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. RESULTS: Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell-deficient nu/nu mice. CONCLUSION: We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Rituximab as a therapeutic option for patients with advanced melanoma.

Treatment of metastatic melanoma remains challenging, despite a variety of new and promising immunotherapeutic and targeted approaches to therapy. New treatment options are still needed to improve long-term tumour control. We present a case series of seven patients with metastatic melanoma who were treated individually with the anti-CD20 antibody rituximab between July 2014 and July 2015. Two of the patients were treated in an adjuvant setting. All patients had already received a variety of treatments. During an induction phase, the administration of four cycles of weekly rituximab 375 mg/m2 body surface area was planned. After imaging, patients with stable disease continued therapy with rituximab 375 mg/m2 body surface area every 4 weeks up to a maximum of 24 weeks. Two patients experienced grade 2 infusion reactions during the first infusion. Otherwise, treatment was well tolerated and there were no grade 3 or 4 side effects. Staging after the induction phase showed stable disease in five patients, and two patients had progressive disease. Median progression-free survival was 6.3 months (95% CI 4.97-7.53), median overall survival was 14.7 months (95% CI 4.52-24.94), and one patient was still alive in December 2016. In conclusion, rituximab might be a therapeutic option for metastatic melanoma. However, further studies on rituximab among larger patient cohorts are warranted. Evaluation of therapy in an adjuvant setting or in combination with other systemic treatment might, therefore, be of particular interest.

Skin Cancer Classification Using Convolutional Neural Networks: Systematic Review.

BACKGROUND: State-of-the-art classifiers based on convolutional neural networks (CNNs) were shown to classify images of skin cancer on par with dermatologists and could enable lifesaving and fast diagnoses, even outside the hospital via installation of apps on mobile devices. To our knowledge, at present there is no review of the current work in this research area. OBJECTIVE: This study presents the first systematic review of the state-of-the-art research on classifying skin lesions with CNNs. We limit our review to skin lesion classifiers. In particular, methods that apply a CNN only for segmentation or for the classification of dermoscopic patterns are not considered here. Furthermore, this study discusses why the comparability of the presented procedures is very difficult and which challenges must be addressed in the future. METHODS: We searched the Google Scholar, PubMed, Medline, ScienceDirect, and Web of Science databases for systematic reviews and original research articles published in English. Only papers that reported sufficient scientific proceedings are included in this review. RESULTS: We found 13 papers that classified skin lesions using CNNs. In principle, classification methods can be differentiated according to three principles. Approaches that use a CNN already trained by means of another large dataset and then optimize its parameters to the classification of skin lesions are the most common ones used and they display the best performance with the currently available limited datasets. CONCLUSIONS: CNNs display a high performance as state-of-the-art skin lesion classifiers. Unfortunately, it is difficult to compare different classification methods because some approaches use nonpublic datasets for training and/or testing, thereby making reproducibility difficult. Future publications should use publicly available benchmarks and fully disclose methods used for training to allow comparability.

Teledermatology: Comparison of Store-and-Forward Versus Live Interactive Video Conferencing.

A decreasing number of dermatologists and an increasing number of patients in Western countries have led to a relative lack of clinicians providing expert dermatologic care. This, in turn, has prolonged wait times for patients to be examined, putting them at risk. Store-and-forward teledermatology improves patient access to dermatologists through asynchronous consultations, reducing wait times to obtain a consultation. However, live video conferencing as a synchronous service is also frequently used by practitioners because it allows immediate interaction between patient and physician. This raises the question of which of the two approaches is superior in terms of quality of care and convenience. There are pros and cons for each in terms of technical requirements and features. This viewpoint compares the two techniques based on a literature review and a clinical perspective to help dermatologists assess the value of teledermatology and determine which techniques would be valuable in their practice.

A Face-Aging App for Smoking Cessation in a Waiting Room Setting: Pilot Study in an HIV Outpatient Clinic.

BACKGROUND: There is strong evidence for the effectiveness of addressing tobacco use in health care settings. However, few smokers receive cessation advice when visiting a hospital. Implementing smoking cessation technology in outpatient waiting rooms could be an effective strategy for change, with the potential to expose almost all patients visiting a health care provider without preluding physician action needed. OBJECTIVE: The objective of this study was to develop an intervention for smoking cessation that would make use of the time patients spend in a waiting room by passively exposing them to a face-aging, public morphing, tablet-based app, to pilot the intervention in a waiting room of an HIV outpatient clinic, and to measure the perceptions of this intervention among smoking and nonsmoking HIV patients. METHODS: We developed a kiosk version of our 3-dimensional face-aging app Smokerface, which shows the user how their face would look with or without cigarette smoking 1 to 15 years in the future. We placed a tablet with the app running on a table in the middle of the waiting room of our HIV outpatient clinic, connected to a large monitor attached to the opposite wall. A researcher noted all the patients who were using the waiting room. If a patient did not initiate app use within 30 seconds of waiting time, the researcher encouraged him or her to do so. Those using the app were asked to complete a questionnaire. RESULTS: During a 19-day period, 464 patients visited the waiting room, of whom 187 (40.3%) tried the app and 179 (38.6%) completed the questionnaire. Of those who completed the questionnaire, 139 of 176 (79.0%) were men and 84 of 179 (46.9%) were smokers. Of the smokers, 55 of 81 (68%) said the intervention motivated them to quit (men: 45, 68%; women: 10, 67%); 41 (51%) said that it motivated them to discuss quitting with their doctor (men: 32, 49%; women: 9, 60%); and 72 (91%) perceived the intervention as fun (men: 57, 90%; women: 15, 94%). Of the nonsmokers, 92 (98%) said that it motivated them never to take up smoking (men: 72, 99%; women: 20, 95%). Among all patients, 102 (22.0%) watched another patient try the app without trying it themselves; thus, a total of 289 (62.3%) of the 464 patients were exposed to the intervention (average waiting time 21 minutes). CONCLUSIONS: A face-aging app implemented in a waiting room provides a novel opportunity to motivate patients visiting a health care provider to quit smoking, to address quitting at their subsequent appointment and thereby encourage physician-delivered smoking cessation, or not to take up smoking.

Regulatory T cell-derived adenosine induces dendritic cell migration through the Epac-Rap1 pathway.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC.

Routine Laboratory Parameter Dynamics and Laboratory Adverse Events in Psoriasis Patients on Long-term Treatment with Adalimumab, Etanercept, and Ustekinumab.

Only limited data on laboratory parameter dynamics and safety under prolonged biologic treatment in a "real-world" scenario are available for recommendations on screening and monitoring. This study is a retrospective analysis of routine parameter dynamics and laboratory adverse events (LAE) in psoriasis patients on long-term treatment (n = 199) with tumour necrosis factor (TNF)-α-antagonists (adalimumab, etanercept), and the interleukin (IL)12/23-antagonist ustekinumab. Overall, neutrophil (PMN) counts (-11%) and triglycerides (+9%) changed considerably. TNF-α-antagonists and ustekinumab differentially affected lymphocyte counts (+13% and ±0%, respectively). Dynamics were pronounced during the first 180 days of treatment. In 340 treatment-years, 15 Common Terminology Criteria for Adverse Events (CTCAE) III-IV° LAE were recorded (11 involved liver enzymes). They prompted alteration of the biologic regime in only 2 cases. Age, sex, previous systemic treatments, and psoriatic arthritis did not significantly predict LAE. Liver enzyme and triglyceride screening may be warranted in some instances. Our data suggest that unguided monitoring of other routine laboratory parameters is unnecessary under long-term biologic treatment.

High-dose intravenous immunoglobulins for the treatment of dermatological autoimmune diseases.

Based on their immunomodulatory properties, high-dose intravenous immunoglobulins (IVIGs) are successfully used in the treatment of various dermatological autoimmune diseases, in particular pemphigus vulgaris and dermatomyositis. In autoimmune bullous diseases, IVIGs can be used in an adjuvant setting (second- or third-line therapy) once combined immunosuppressive regimens have failed. In dermatomyositis, IVIGs may already be employed as an adjuvant second-line therapy after failure of corticosteroid monotherapy. In scleromyxedema, IVIGs may be considered as first-line treatment, given the lack of effective and safe alternatives. Other potential indications for IVIGs may include severe recalcitrant cases of systemic vasculitis and systemic lupus erythematosus. Toxic epidermal necrolysis may be an indication for high-dose IVIGs if administered early. Common, readily manageable side effects include nausea, headache, fatigue, and febrile infusion reactions. Severe adverse events such as thromboembolic events, anaphylaxis, and acute renal failure are very uncommon. The risk of viral transmission is very low. Potential mechanisms of action include upregulation of inhibitory Fc receptors, reduction of the half-life of endogenous immunoglobulins due to displacement from protective receptor sites, neutralization of autoantibodies by anti-idiotypic antibodies, as well as inhibition of complement activation.

Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification.

Neutrophil extracellular trap (NET) formation is a mechanism of innate immune defence by which neutrophil (polymorphonuclear) granulocytes (PMN) produce net-like structures of decondensed chromatin decorated with antimicrobial peptides for trapping and possibly killing microorganisms. If this process leads to cell death, it is termed NETosis. Alterations of this particular mechanism have been reported to be involved in the pathogenesis of chronic inflammatory diseases including psoriasis and lupus erythematosus. Still, quantification of NETosis poses a considerable challenge. We report and test a refined protocol for morphological NET quantification in healthy human donors that encompasses isolation, stimulation, DNA staining, live imaging and semi-automated offline analysis. The results were highly reproducible and in good agreement with manual counting. The average intra-donor coefficient of variation of NETosis rates to phorbol myristate acetate (PMA) stimulation was low compared to the respective interdonor coefficient of variation (10% vs 82%, n=4, respectively, if experiments were repeated on the same day, and 38% vs 74%, n=6, respectively, if experiments were repeated on average 42±34 days apart). Overall, the interdonor coefficient of variation was 67% (n=10). These findings altogether support the existence of a distinct predisposition of PMN from different donors for undergoing NETosis. Picogreen fluorescence correlated stronger to cell death than to morphological NETosis (r2 =.89, P<.001, n=8, and r2 =.68, P=.012, n=8, respectively). This indicates that cytotoxicity may confound Picogreen fluorescence. Our results and the related protocol may help investigators with the quantification of NETosis and the design of respective basic and translational research studies.