Search

SummaryPre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications1. We currently lack efficient screening tools2, 3and early therapies4, 5to address PE. To investigate the early stages of early onset PE, and identify candidate markers and pathways, we performed spatio-temporal multi-omics profiling of human PE placentae and healthy controls and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-seq approach combined with spatial proteo- and transcriptomics and mechanisticin vitrosignalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts. We found a significant increase in the senescence marker activin A in preeclamptic maternal serum in early gestation, before the development of clinical symptoms, indicating a translation of the placental syndrome to the maternal side. Our work describes a new disease progression, starting with a disturbed transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.

BackgroundTherapeutic management of stage II colon cancer remains difficult regarding the decision whether adjuvant chemotherapy should be administered or not. Low rates of recurrence are opposed to chemotherapy induced toxicity and current clinical features are limited in predicting disease relapse. Predictive biomarkers are urgently needed and we hypothesise that the spatial tissue composition of relapsed and non-relapsed colon cancer stage II patients reveals relevant biomarkers.MethodsThe spatial tissue composition of stage II colon cancer patients was examined by in situ sequencing technology with sub-cellular resolution. A panel of 175 genes was designed investigating specific cancer-associated processes and components of the tumour microenvironment. We identified a tumour gene signature to subclassify tissue into neoplastic and non-neoplastic tissue compartments based on spatial expression patterns generated by in situ sequencing (GTC-tool – Genes-To-Count).ResultsThe GTC-tool automatically identified tissue compartments that were used to quantify gene expression of biological processes upregulated within the neoplastic tissue in comparison to non-neoplastic tissue and within relapsed versus non-relapsed stage II colon patients. Three differentially expressed genes (FGFR2, MMP11 and OTOP2) in the neoplastic tissue compartments of relapsed patients in comparison to non-relapsed patients were identified predicting recurrence in stage II colon cancer.ConclusionsIn depth spatial in situ sequencing revealed novel potential predictive biomarkers for disease relapse in colon cancer stage II patients. Our developed open-access GTC-tool allows to accurately capture the tumour compartment and quantify spatial gene expression in colon cancer tissue.

The early human embryo derived from fusion of an oocyte with a single sperm undergoes growth and differentiation and results in an implantation-ready blastocyst. To initiate implantation, the blastocyst hatches from the zona pellucida, thus making it accessible to external inputs. Our modelling of sperm distribution through the uterus indicates that 200–5000 sperms following natural coitus during mid-luteal phase are in a position of reaching the implantation-stage blastocyst in the maternal uterus. We indeed have ultrastructural evidence of sperm cells intruding into the trophectoderm cells of uterine zona-free blastocysts obtained from rhesus monkeys. The question arises whether the negotiation between additional sperm and azonal blastocyst is a feature of evolution yielding adaptation processes influencing the developmental fate of an individual embryo or a neutral by-product in placental mammals. This process potentially bears implications in pregnancy success, sperm competition, and human health.

BACKGROUND In precision oncology ex vivo drug screening systems have the potential to improve clinical outcomes. Traditionally, cancer drugs are tested on cancer cell line models, but these cannot represent an individual patient and are biologically too distinct. Drug screening systems usually rely on viability assays and correlations to genomic alterations. Beside genomic alterations, the cellular metabolism is significantly altered which may lead to drug resistance. Here we aim to establish a drug screening platform using tumor cells derived directly from the individual patient glial tumor tissue, create patient derived tumor cells (PDCs) and combine the outcomes from standardized viability- and genetic-assays with a new developed metabolomics platform. Materials and METHODS Fresh native tissue from patients harbouring low- and high-grade glioma are collected (n=46). Tumor tissue used for NMR-based metabolomic analyses and targeted sequencing analyses as well as PDC isolation. To preserve the original tumor similarity, tissue is short term cultured for two weeks, and PDCs are seeded and treated with a panel of clinical- and preclinical drugs followed by viability assessment, sequencing and metabolomic profiling. RESULTS Culturing of PDCs is successful in ≥ 85% of patient cases, provided that at least 2 g of tumor tissue is available. The automatized high throughput ex vivo drug response identifies drug candidates, which might become relevant for therapeutic approaches in future. It is possible to distinguish between IDH1-wild-type and IDH1-mutant tumors based on the metabolomic profile, which is confirmed by immunohistochemical staining and molecular analysis of IDH1 R132H-mutation. Strong metabolomic variations have been identified, including GABA, lactate, and myo-inositol levels between tumor and healthy tissue. CONCLUSION Entangling drug screening and genetic assays with metabolomic profiling of glial tumors enriches the information about cellular drug response and paves the way for future clinical studies and better understanding of underlying drug resistance mechanisms in gliomas.

DNA damage induced by ionizing radiation may ultimately lead to cell death or initiate cancer cells development. Today it is difficult to estimate what the actual damage to the human body will be, given the fact that today in the world the number of diagnostic procedures using radiation and iodine contrast media is increasing, and the existence of a number of factors that can affect the radiation dose in vivo. At the same time, development of new methods is required, which can determine in a much shorter time what will be the effect of diagnostic radiation on the DNA molecule. For these purposes we develop two competitive inhouse methods for automated quantification of DNA double strand brakes (DDSB) in peripheral blood lymphocytes: immunofluorescence determining of γH2AX with stained microscopic slides and determining the occurrence of DDSB with the flow cytometry. Our initial results shown that computed tomography (CT) can cause damage in the DNA molecule in the form of DDSB, the existence of linear dependence with the increase in low and high range of CTDI and the number of γH2AX, and that iodine contrast media can increase the occurrence of DNA double strand brakes. Keywords: CT examinations, ICM, immunofluorescence, flow cytometry, DDSB

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Various studies found an association of different renin-angiotensin system (RAS) components with gestational duration and preterm birth, as well as with preeclampsia. Approximately 25% of first-time pregnant women develop a mild to severe hypertension in pregnancy or even preeclampsia. Based on recently published single-cell RNA-sequencing, we hypothesized an alternative RAS function in placenta and furthermore, an implication in hypertensive disorders in pregnancy. Placental RAS expression and localization was analyzed via quantitative polymerase chain reaction and in situ mRNA padlock probes. Tissue was collected from first-trimester elective termination (n=198), from healthy third-trimester controls (n=54), from early-onset preeclamptic (n=54) and age-matched controls (n=29), as well as first-trimester placentae from women with a high uterine artery resistance index (high-risk for preeclampsia, n=9) and controls (n=8). Serum levels of Ang (angiotensin) I to IV from women before and after conception were measured via mass spectrometry (n=10). Placental explants were cultured in 2.5% oxygen with Ang II, candesartan, and leptin. Seahorse XF96 MitoStress assays assessed trophoblast metabolism. Here, we show that maternal angiotensin acts on placental LNPEP (leucine aminopeptidase), that is, angiotensin IV-receptor and fetal angiotensin on placental AGTR1 (angiotensin II receptor type 1). Maternal circulating RAS shifts towards Ang IV in pregnancy. Ang IV decreases trophoblastic mitochondrial respiration and increases placental leptin via placental LNPEP. Lower placental LNPEP in preeclampsia and in first-trimester patients at high-risk for preeclampsia suggests a new alternative route in maternal RAS signaling and may contribute to hypertension and disease in pregnancy. The study shows how hypertensive disorders in pregnancy may be connected metabolic alterations that finally seem to contribute to the multifactorial disease in pregnancy, preeclampsia.

Non-coding natural antisense transcripts (ncNATs) are regulatory RNA sequences that are transcribed in the opposite direction to protein-coding or non-coding transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.

Novel androgen receptor (AR) signaling inhibitors have improved the treatment of castration-resistant prostate cancer (CRPC). Nonetheless, the effect of these drugs is often time-limited and eventually most patients become resistant due to various AR alterations. Although liquid biopsy approaches are powerful tools for early detection of such therapy resistances, most assays investigate only a single resistance mechanism. In combination with the typically low abundance of circulating biomarkers, liquid biopsy assays are therefore informative only in a subset of patients. In this pilot study, we aimed to increase overall sensitivity for tumor-related information by combining three liquid biopsy approaches into a multi-analyte approach. In a cohort of 19 CRPC patients, we (1) enumerated and characterized circulating tumor cells (CTCs) by mRNA-based in situ padlock probe analysis, (2) used RT-qPCR to detect cancer-associated transcripts (e.g., AR and AR-splice variant 7) in lysed whole blood, and (3) conducted shallow whole-genome plasma sequencing to detect AR amplification. Although 44&ndash, 53% of patient samples were informative for each assay, a combination of all three approaches led to improved diagnostic sensitivity, providing tumor-related information in 89% of patients. Additionally, distinct resistance mechanisms co-occurred in two patients, further reinforcing the implementation of multi-analyte liquid biopsy approaches.

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.

BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1–76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Objectives: A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities. Methods: SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH). Results: DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy. Conclusion: SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities.

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)(1). A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell's amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (50%) and needs some further attention. This device is not cleared for the use in patients.

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa),We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC andIn situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) hadPadlock probe technology enables quantification of AR-V7, AR-FL, PSA, and

The highly fine-tuned dynamics of cell cycle gene expression have been intensely studied for several decades. However, some previous observations may be difficult to fully decouple from artifacts induced by traditional cell synchronization procedures. In addition, bulk cell measurements may have disguised intricate details. Here, we address this by sorting and transcriptomic sequencing of single cells progressing through the cell cycle without prior synchronization. Genes and pathways with known cell cycle roles are confirmed, associated regulatory sequence motifs are determined, and we also establish ties between other biological processes and the unsynchronized cell cycle. Importantly, we find the G1 phase to be surprisingly heterogeneous, with transcriptionally distinct early and late time points. We additionally note that mRNAs accumulate to reach maximum total levels at mitosis and find that stable transcripts show reduced cell-to-cell variability, consistent with the transcriptional burst model of gene expression. Our study provides the first detailed transcriptional profiling of an unsynchronized human cell cycle.

BACKGROUND Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays—CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs—using the same blood draw. METHODS EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single–cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.

Although tumor genotyping is still the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot or are often difficult to obtain. To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. The analysis of circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA), circulating RNAs, and exosomes, frequently referred to as liquid biopsy, has recently gained enormous momentum. Due to technological advances, novel circulating tumor biomarkers were shown to have a great potential to improve patient treatment in terms of estimation of prognosis, monitoring treatment response, early detection of resistance mechanisms, identification of actionable targets, and detection of minimal residual disease. However, despite all efforts, liquid biopsies are not yet routinely used mainly due to technological hurdles, lack of analytical and pre-analytical standards and conclusive evidence that patients indeed benefit from such analyses. In this chapter, the different entities with respect to state-of-the-art technologies, potential clinical applications, and their limitations are discussed.

High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique, further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%, p = 0.024) and yielded significantly higher CTC numbers (range: 0&ndash, 15 vs. 0&ndash, 5, p = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.

The need to perform gene expression profiling using next generation sequencing and quantitative real-time PCR (qPCR) on small sample sizes and single cells is rapidly expanding. However, to analyse few molecules, preamplification is required. Here, we studied global and target-specific preamplification using 96 optimised qPCR assays. To evaluate the preamplification strategies, we monitored the reactions in real-time using SYBR Green I detection chemistry followed by melting curve analysis. Next, we compared yield and reproducibility of global preamplification to that of target-specific preamplification by qPCR using the same amount of total RNA. Global preamplification generated 9.3-fold lower yield and 1.6-fold lower reproducibility than target-specific preamplification. However, the performance of global preamplification is sufficient for most downstream applications and offers several advantages over target-specific preamplification. To demonstrate the potential of global preamplification we analysed the expression of 15 genes in 60 single cells. In conclusion, we show that global preamplification simplifies targeted gene expression profiling of small sample sizes by a flexible workflow. We outline the pros and cons for global preamplification compared to target-specific preamplification.

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

The here described method of isothermal whole genome amplification (iWGA) uses a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit) that amplifies minute quantities of DNA by multiple strand displacement upon random hexamer primer binding. Starting from genomic DNA or single cells this amplification yields up to 5 μg of iWGA product with fragment lengths of 10 kb and longer. As this amplification lacks the need of fragmenting DNA, its products are well suited for many downstream applications (e.g. sequencing and DNA profiling). On the contrary, degraded DNA samples are not supported by the nature of the amplification and are not well suited.

Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.

Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 μL. The amplification products obtained from single cells can be forwarded to multiple analyses.

Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH).

This protocol describes the use of a 16plex PCR for the purpose assessing DNA quality after isothermal whole genome amplification (WGA). In short, DNA products, generated by amplification multiple displacement amplification, are forwarded to PCR targeting 15 short tandem repeats (STR) as well as amelogenin generating up to 32 different PCR products. After amplification, the PCR products are separated via capillary electrophoresis and analyzed based on the obtained DNA profiles. Isothermal WGA products of good DNA quality will result in DNA profiles with efficiencies of90 % of the full DNA profile.

This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).

This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).

Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH).

The here described method of isothermal whole genome amplification (iWGA) uses a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit) that amplifies minute quantities of DNA by multiple strand displacement upon random hexamer primer binding. Starting from genomic DNA or single cells this amplification yields up to 5 μg of iWGA product with fragment lengths of 10 kb and longer. As this amplification lacks the need of fragmenting DNA, its products are well suited for many downstream applications (e.g. sequencing and DNA profiling). On the contrary, degraded DNA samples are not supported by the nature of the amplification and are not well suited.

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.

Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 μL. The amplification products obtained from single cells can be forwarded to multiple analyses.

This protocol describes the use of a 16plex PCR for the purpose assessing DNA quality after isothermal whole genome amplification (WGA). In short, DNA products, generated by amplification multiple displacement amplification, are forwarded to PCR targeting 15 short tandem repeats (STR) as well as amelogenin generating up to 32 different PCR products. After amplification, the PCR products are separated via capillary electrophoresis and analyzed based on the obtained DNA profiles. Isothermal WGA products of good DNA quality will result in DNA profiles with efficiencies of >90 % of the full DNA profile.

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.