The discovery of VLT links VZV with the other better characterized human and animal neurotropic alphaherpesviruses and provides insights into VZV latency.Varicella-zoster virus (VZV), an alphaherpesvirus, establishes lifelong latent infection in the neurons of >90% humans worldwide, reactivating in one-third to cause shingles, debilitating pain and stroke. How VZV maintains latency remains unclear. Here, using ultra-deep virus-enriched RNA sequencing of latently infected human trigeminal ganglia (TG), we demonstrate the consistent expression of a spliced VZV mRNA, antisense to VZV open reading frame 61 (ORF61). The spliced VZV latency-associated transcript (VLT) is expressed in human TG neurons and encodes a protein with late kinetics in productively infected cells in vitro and in shingles skin lesions. Whereas multiple alternatively spliced VLT isoforms (VLT Varicella-zoster virus (VZV) establishes lifelong infection in the majority of the population, but mechanisms underlying latency remain unclear. Here, the authors use ultra-deep RNA sequencing, enriched for viral RNAs, of latently infected human trigeminal ganglia and identify a spliced, latency-associated VZV mRNA. For the best-studied αHVs, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus and bovine herpesvirus 1 (BHV-1), viral latency has been shown to be accompanied by the expression of a single or restricted set of latency-associated transcripts (LATs)6. These transcripts map antisense to the gene encoding the conserved multifunctional HSV infected cell protein 0 (ICP0), to which varicella-zoster virus (VZV) open reading frame 61 (ORF61) is homologous, which besides inhibiting intrinsic cellular antiviral responses is the major transcriptional transactivator of lytic viral genes required for reactivation of latent HSV10. While the function of the LATs remains uncertain (reviewed in ref. 11), there is mounting evidence from work on HSV-1, HSV-2 and BHV-1 that LATs induce generalized transcriptional and/or translational repression12, and several studies have shown that LAT-encoded miRNAs  or proteins  target viral mRNAs including ICP015.During primary infection, neurotropic alphaherpesviruses (αHVs) gain access to neurons in sensory, cranial and autonomic ganglia to establish a lifelong latent infection from which they can reactivate to cause debilitating disease18. While the absence of a canonical latency transcript in VZV may represent a fundamental difference in the evolution and biology of this virus, it is notable that a putative LAT, antisense to ORF61, has also been described for simian varicella virus (SVV), the varicellovirus most closely related to VZV. However, neither the transcript nor its function in SVV infection have been studied in detail8.The exception has been VZV, a human-restricted αHV and causative agent of varicella and herpes zoster, for which no latency transcript mapping antisense to its ICP0 homologue ORF61 has been described20. By contrast, VZV gene expression during latency remains poorly defined23. This is largely due to the lack of appropriate animal24 and, until recently, in vitro26 models, which accurately mimic VZV pathogenesis26. VZV latency has been extensively studied in cadaveric human trigeminal ganglia (TG), a prominent anatomic site of both HSV-1 and VZV latency, yielding conflicting results as to which VZV transcripts and proteins are expressed27. Whereas viral protein detection by immunohistochemistry (IHC) can largely be attributed to non-specific binding of anti-VZV antibody preparations28, the time interval between death and TG specimen processing  determines the number and quantity of VZV transcripts detected22. Using PCR primers targeting all canonical VZV genes, only the lytic gene ORF63 is occasionally detected in TGs with PMI < 9 h18, whereas multiple viral transcripts of different kinetic classes are detected in human TGs with PMI > 9 h22 and also frequently in animal models30.Like other herpesviruses, lytic VZV infection is characterized by full viral gene expression occurring with temporally linked immediate-early (IE), early (E) and late (L) kinetics to generate infectious virus progenyHere we describe the identification of a VZV latency-associated transcript (VLT), consistently detected in VZV and HSV-1 co-infected human TG that lies antisense to ORF61. Although multiple alternatively spliced transcripts are present during productive infection, the unique VLT isoform that supresses ORF61 gene transcription in co-transfected cells predominates during latency. The discovery of VLT unifies the VZV latent viral transcription programme with that of other better-characterized human and animal neurotropic αHVs while removing longstanding barriers to understanding VZV latency.6. Enrichment for polyadenylated HSV-1 sequences in the same TGs and five additional dually latently infected TGs  selected for and analysed by miRNA sequencing of unenriched RNA libraries  cells to demonstrate detection of all currently annotated VZV and HSV-1 genes at high specificity and sensitivity Figs. , enricheWhile no VZV transcripts could be identified in non-enriched TG RNA samples  isoforms (each VLTly isoform uses a different upstream exon) in latently VZV-infected TGs  TGs expressed VLT and 9 of the 15 (60%) VZVPOS TGs co-expressed ORF63 mRNA at lower levels relative to VLT  and two VZV naive human fetal dorsal root ganglia (DRG) by in situ hybridization (ISH). VLT and ORF63 transcripts were localized to both the neuronal nucleus and cytoplasm of distinct neurons in latently VZV-infected TG but not in uninfected DRG  TG neurons expressed VLT than ORF63 transcript , a broad-spectrum herpesvirus DNA polymerase inhibitor32. Whereas no effect on ORF61 (IE gene) and ORF29 (E gene) transcription was observed, ORF49 (leaky L gene) transcription was markedly reduced in PFA-treated VZV-infected cells , with a start codon and polyadenylation site/stop codon in exons 2 and 5, respectively Fig. . A polyclls Fig. . PFA bloely Fig. , demonstles Fig. , while Iles Fig. . Neither33, we tested whether VLT affects ORF61 expression. ARPE-19 cells were co-transfected with four plasmids, each containing a single coding VZV sequence  under the control of a chicken beta-actin  or human cytomegalovirus promoter (for ORF62). VLT expression significantly reduced ORF61, but not ORF62 and ORF63 transcript levels, in co-transfected cells  whose expression was strictly dependent on de novo virus DNA replication (true late kinetics) in lytically VZV-infected cells in vitro and in shingles skin lesions but was undetectable by IHC during latency.We have identified a unique spliced VZV transcript, VLT, which is consistently expressed in latently VZV-infected human TG neurons. The VLT locus, including splice donor/acceptor sites and pVLT coding sequence, is highly conserved between wild-type and vaccine strains of VZV  that can reactivate to cause disease44. The identification of the VLT, 35 years after VZV latency in human sensory ganglia was first described45, provides a significant advance in ongoing efforts to investigate and understand the mechanisms by which VZV establishes, maintains and reactivates from latency.With the availability of effective vaccines, VZV disease is much reduced in those geographic regions where coverage is highHuman TG were obtained at 6:01 h ± 1:47 h (average ± SD) after death  were cultured on ARPE-19 cells as described49. All cell cultures and virus infections were performed in a humidified CO2 incubator at 37 °C.Human retinal pigmented epithelium ARPE-19 cells [American Type Culture Collection (ATCC) CRL-2302] were maintained in 1:1 Dulbecco’s modified Eagle’s medium –Ham’s F12 (Lonza or Sigma-Aldrich) medium supplemented with 8% heat-inactivated foetal bovine serum  and 0.6 mg/mL L-sodium glutamate . The human MeWo melanoma cell [ATCC HTB-65] was maintained in DMEM supplemented with 8% FBS and 0.6 mg/mL L-sodium glutamate. Culture of VZV pOka strain and isolation of cell-free virus have been described previouslyApproximately one-fifth of snap-frozen human TG was mechanically dispersed and used for DNA isolation, while four-fifths of the same specimen was used for RNA isolation. DNA extraction was performed using the QIAamp DNA Kit (Qiagen) according to the manufacturer’s instructions. For RNA isolation, samples were homogenized in TRIzol (Invitrogen), mixed vigorously with 200 µL chloroform and centrifuged for 15 min at 12,000×g at 4 °C. RNA was isolated from the aqueous phase using the RNeasy Mini Kit, including on-column DNase I treatment (Qiagen). DNA and RNA concentration and integrity were analysed using a Qubit Flourometer (ThermoFisher).2 flask) were infected with HSV-1 strain F at a multiplicity of infection of 1 and harvested in 1 mL TRIzol at 24 hpi. RNA isolated as described above was subjected to additional DNase treatment using the TURBO DNA-free Kit according to the manufacturer’s instructions (Ambion).ARPE-19 cells were infected with VZV strain EMC-1 by co-cultivation VZV-infected or uninfected cells at a 1:8 cell ratio and harvested in 1 mL TRIzol at 72 h post-infection (hpi). Alternatively, semi-confluent ARPE-19 cell layers (75 cm51 that incorporated custom-designed SureSelect RNA bait sets for both VZV and HSV-1 in the same reaction at reduced concentration (1:10). Bait sets for VZV . Here each sample was either enriched for polyadenylated mRNA  or underwent rRNA-depletion using a NEBNext® rRNA Depletion Kit [Human/Mouse/Rat] (New England Biolabs) according to the manufacturer’s instructions. Subsequent to either procedure, captured/remaining RNAs were transcribed to produce cDNA. Fragmentation, cDNA second-strand synthesis, end repair, A-tailing and adapter ligation were performed as described in the enrichment protocol. Hybridization was performed using a modified strategyhttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove low-quality 3’ ends and mapped to the human genome (hg19) using the BBMap software (http://sourceforge.net/projects/bbmap/) with default parameters. Unmapped read pairs were subsequently aligned against VZV Dumas (NC_001348) and HSV-1 strain 17 (NC_001806) reference genomes deposited at Genbank using BBMap allowing for properly paired reads only to be carried forward in the analysis. Extremely high duplication levels (>0.99) were observed in all enriched libraries generated from TG RNA, an expected feature of enrichment strategies against ultra-low abundance transcripts. Note, however, that we are not stating that VLT is a low-abundance transcript within a single cell but rather that VZV latently infected cells are relatively scarce, thus diluting the apparent expression level of VLT. To mitigate this, duplicate reads were removed using the Picard Tools MarkDuplicates software (http://broadinstitute.github.io/picard). Resulting assemblies were visualized using a combination of Circos52, Artemis53, Tablet54, SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) software and custom R scripts making use of the Rsamtools and Gviz software packages.Sequence run data were de-multiplexed using bcl2fastq2 v2.17 under stringent conditions (--barcode mismatches 0) and yielded between ~31,000,000 and 107,700,000 paired-end reads per sample. Individual sequence data sets were trimmed using the TrimGalore software (56) and visualized using scatter plots. High correlation (R2 0.9252–0.9678) between enriched and unenriched transcriptomes were observed for both HSV-1  and VZV (n = 1), as shown in Supplementary Fig. To confirm that enrichment for viral nucleic acids did not bias relative levels of viral gene transcription, transcripts per million counts were generated using FeatureCounts . Sequence reads were adaptor-trimmed using TrimGalore and size selected using BBduk (provided with BBmap) so that only sequence reads between 17 and 26 bases in length were retained. These were mapped against VZV strain Dumas and the HSV-1 strain 17 reference genomes as well as miRDB using ShortStack (--dicermin 18 --mincov 1 --mismatches 2 --foldsize 200). No putative VZV miRNAs could be detected in either latently infected TGs or lytically infected ARPE-19 cells. By contrast, canonical HSV-1 miRNAs could be detected in both latently infected TGs and lytically infected ARPE-19 cells at high abundance.miRNASeq libraries were prepared from 1 µg of total RNA, isolated from human TG and lytically VZV- and HSV-1-infected ARPE-19 cells using the NEBNext18 using 2.0–11.7 µg [6.7 µg ± 0.64 (average ± SEM)] of TG-derived total RNA or 5 µg of cell-culture-derived DNase-free RNA and Superscript III reverse transcriptase (Invitrogen) with oligo(dT)12-18 primers (Invitrogen). Taqman qPCR was performed in triplicate on DNA and cDNA using Taqman 2× PCR Universal Master Mix (Applied Biosystems) with primer/probe pairs specific for VZV ORF62, ORF63 and the VLT exon 2→3, 3→4, A→1, B→1 and C→1 junctions and HSV-1 DNA58 and LAT, , β-actin (Applied Biosystems) and the human single copy gene hydroxymethylbilane synthase   Fig.  and 11 ale Figs.  and 6a.TM RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions using total RNA extracted from VZV pOka-infected MeWo cells. PCR was performed using KOD-Plus-Ver.2 DNA Polymerase (Toyobo Life Science) templated with 5’RACE ready cDNA using Universal Primer A Mix and ORF61R626AS or pOka104778R or 3’RACE ready cDNA using Universal Primer A Mix and ORF61R626AS (Supplementary Table TM RLM-RACE Kit (Ambion) according to the manufacturer’s instructions, using 10 µg of total RNA obtained from VZV EMC-1-infected ARPE-19 cells. Nested PCR was performed using Pfu Ultra II Fusion HS DNA Polymerase (Stratagene) and 40 cycles of first round amplification using 5’RACEout_Fw–VLT2-3_Rv primers, followed by 40 cycles of second round amplification using 5’RACEinn_Fw–VLT2_Rv primers (Supplementary Table TM4-TOPO® TA vector (Thermo Fisher Scientific) after addition of 3’A overhang using AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific) or Takara Ex Taq® (Takara Bio) and used to transform One Shot® TOP10 competent Escherichia coli. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen), amplified by PCR using M13Fw and M13Rv primers  and human zoster skin biopsies were analysed by in situ hybridization (ISH) using the RNAScope 2.0 Red Kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. In brief, deparaffinized 5 µm tissue sections were incubated with probes designed to cover VZV ORF63 and VLT exons 2–5. The probes for the human transcript POLR2A and ubiquitin C were used as positive controls and probes specific for the bacterial transcript DAPB were used as negative controls. All probes were designed and produced by Advanced Cell Diagnostics. FastRed was used as substrate to visualize the ISH signal and stained slides were counterstained with haematoxylin and mounted in Ecomount . In some experiments, TG sections were incubated with DNase I (Qiagen) or RNAse [Ribonuclease A (25 µg/mL)+T1  diluted in 1×TBS-t], after pre-treatment step #3 for 1 h at 40 °C. To determine the ratio of VZV and HSV-1 transcript expressing TG neurons, slides were scanned using the Nanozoomer 2.0 HT (Hamamatsu) and scored in Adobe Photoshop CS6 (Adobe). Twelve TG from distinct donors were analysed for VZV ORF63 transcript and VLT and 10 TG for HSV-1 LAT, with on average 664 neurons/section (range: 420–1561) and 1–2 sections per TG. Two herpes zoster skin biopsies from distinct donors were analysed with 2–3 sections per donor for each staining.To select for VZV and HSV-1 DNA-positive TG tissue areas, DNA was isolated from three consecutive 5 µm FFPE tissue sections using the QIAamp DNA FFPE Tissue Kit and analysed by the respective virus-specific qPCR assays. Subsequently, viral DNA5 cells) were infected with pOka VZV cell-free virus (103 plaque-forming units) with and without PFA (200 µg/mL) for 24 h at 37 °C. Total RNA was isolated from cells using NucleoSpin RNA in combination with the NucleoSpin RNA/DNA buffer set (Macherey-Nagel) according to the manufacturer’s instructions. Binding DNA was first eliminated from the column in 100 µL DNA elution buffer, the column was treated with recombinant DNase I  for 1 h at 37 °C and finally RNA was eluted in 60 µL nuclease-free water. cDNA was synthesized with 10.4 µL of total RNA and anchored oligo(dT)18 primer in a 20 µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics). qPCR following a dissociation curve analysis was performed as described previously25 using SYBR Select Master Mix in a StepOnePlus Real-time PCR system (Thermo Fisher Scientific). The primer sets for β-actin and VZV ORF61 and ORF49 genes were described previously25 and primers for VZV ORF29 (ORF29F2381 and ORF29R2440) and VLT (VLTexon1F and VLTexon2R) are presented in Supplementary Table ARPE-19 cells 2 × 10 cells weORF63) or EcoRI and SalI (ORF61) restriction enzymes and subsequently cloned into pCAGGS-MCS-puro (CAG.Empty) via EcoRI and XhoI sites. The resulting VLT-, ORF61- and ORF63-expressing plasmids were named as follows: CAG.VLT-FLAG, CAG.ORF61, and CAG.ORF63, respectively. The CAG.VLT(ATA)-FLAG plasmid, in which the ATG start codon of VLT ORF was mutated to ATA to prevent pVLT expression, was generated using primer VLT-G3A with a QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s recommendation based on the CAG.VLT-FLAG. All primers used to construct VZV gene expression plasmids are listed in Supplementary Table 61 was generous gift from Dr. Jun-ichi Miyazaki . The pcDNA.ORF62 was a generous gift from Dr. Yasuyuki Gomi .The VLT coding sequence , an anti-IE63 antibody was also generated by Sigma-Aldrich by immunizing a rabbit with GST-IE63 protein as described for anti-IE61 antibody62. Briefly, GST-IE63 protein was expressed in and purified from E. coli BL21 transformed with pGEX-IE63, in which the entire DNA fragment except the first ATG  was cloned into pGEX6P-1 bacterial expression vector . The anti-IE63 antibody was purified using GST-conjugated NHS-activated sepharose for depleting anti-GST antibody and GST-IE63-conjugated NHS-activated sepharose.Anti-VLT protein (pVLT) antibody was generated by Sigma-Aldrich by immunizing a rabbit with a synthetic peptide encoding the first 19 amino acids of pVLT  and DYKDDDDK tag (FLAG tag) . Alexa Fluor 488- and Alexa Fluor 594-conjugated goat-anti-rabbit and -anti-mouse IgG  were used as secondary polyclonal antibodies, respectively. Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. Confocal microscopic analysis were performed as previously described64.The following primary mouse monoclonal antibodies directed to the indicated proteins were used: VZV IE62  and Dr. Stipan Jonjić )65, rabbit anti-pVLT (1:100 dilution) or isotype control antibodies overnight at 4 °C. Sections were subsequently incubated with biotinylated secondary goat-anti-rabbit immunoglobulin (Ig) or goat-anti-mouse Ig and streptavidin-conjugated horseradish peroxidase  for 1 h at room temperature. Signal was visualized using 3-amino-9-ethylcarbazole and counterstained with haematoxylin (Sigma-Aldrich). For immunofluorescent staining, Alexa Fluor 488- and Alexa Fluor 594-conjugated goat-anti-mouse and goat-anti-rabbit antibodies  were used, and sections were mounted with Prolong Diamond antifade mounting medium with 4,6-diamidino-2-phenylindole. Confocal microscopic analysis was performed as described64.Deparaffinized and rehydrated 5 µm FFPE sections of human herpes zoster skin lesions and healthy control skin were subjected to heat-induced antigen retrieval with citrate buffer (pH = 6.0), blocked and incubated with mouse anti-VZV IE63 5 cells/well in an 12-well plate). Culture medium was changed at 16 h post-transfection and cultured for another 48 h. Cells were harvested and aliquoted into two fractions. Total RNA extraction from one fraction of transfectants, cDNA synthesis and relative qPCR were performed as described in 'Determination of kinetic class of VZV transcripts' section using the FavorPrep Blood/Cultured Cell Total RNA Mini Kit (FAVORGEN BIOTECH) instead of NucleoSpin RNA. The primer sets for VZV ORF62 (ORF62F2016 and ORF62R2083) and VLT (VLTexon2F and VLTexon2R-2) are presented in Supplementary Table 66 or DYKDDDDK (FLAG) tag (WAKO) for pVLT detection as described previously62. Uncropped images of immunoblotting are shown in Supplementary Figure Plasmid co-transfection was performed using PEImax  . High potency linear polyethyleneimine was dissolved in water (1 mg/mL), adjusted to pH = 7 with NaOH, filtered through an 0.22 µm filter and stored in aliquots at −20 °C until use. CAG.Empty, CAG.VLT-FLAG or CAG.VLT(ATA)-FLAG (2 µg) with CAG.ORF61, pcDNA.ORF62 and CAG.ORF63 (1 µg) were diluted in knockout DMEM/F12 media (Thermo Fisher Scientific) 50 µL) and PEImax solution (6 µL) were diluted in knockout DMEM/F12 media (50 µL), then both diluent were mixed, left at room temperature for 10–15 min to form polyplexes and transfected into ARPE-19 cell Peer Review File(PDF 202 kb)Description of Additional Supplementary Files(PDF 173 kb)Supplementary Data 1(TXT 1887 kb)Supplementary Data 2(TXT 5481 kb)


To analyze the influence of the Kangaroo Position duration in the initial interactions between mothers and preterm infants. This is an exploratory prospective observational study that analyzed the mother-infant interaction during breastfeeding, before hospital discharge. All eligible newborns, with a gestational age of 28-32 weeks and a birth weight of 1,000-1,800 g from June 11 to September 31, 2014 were included. The films of the interaction were evaluated by the “Mother-Baby Interaction Protocol 0-6 months” tool. The duration of the Kangaroo Position during all the hospitalization period was correlated with the interaction between mother and preterm infant.p=0.03); and the longer the time in the Kangaroo Position, the less the mothers talked to their children . The longer the dyad spent time in the Kangaroo Position, the more the newborns made physical contact attempts with their mothers during breastfeeding (r=0.37;  Longer periods in the Kangaroo Position stimulates the initial exchanges of contact between preterm infant with his mother, which suggests a higher alert status of the newborn and a better availability for interactions with the mother during breastfeeding. It consists of keeping the low-weight newborn skin-to-skin, upright, close to the chest of the parents or other family members. It should be performed in an orientated, safe, and monitored manner by a properly trained health team.Among the benefits of the Kangaroo Method are: lower mortality risk,The first exchanges between mother and child as demonstrated by emotional behavior - vocalizations, looking at the mother, reactivity and readiness to interactIn this context, this study aimed at analyzing the influence of the duration of the Kangaroo Position, on the initial interactions of the preterm infant-mother dyad.This is an observational, prospective, exploratory study that adopted a non-probabilistic sample. Preterm newborns with gestational age between 28 and 32 weeks, and birth weight between 1,000 and 1,800 g, born at Hospital Sofia Feldman between June 11 and September 31, 2014, who remained in a neonatal unit in this institution and progressed to exclusive or mixed breastfeeding were included. The exclusion criteria were newborns with severe neurological impairment, such as intraventricular hemorrhage (grades III and IV), and leukomalacia; Apgar score at 5 minutes lower than seven; mothers who gave up due to discomfort with filming; mother-infant dyads who did not record and/or lost the record of the frequency and duration of the Kangaroo Position during hospital admission; and newborns who did not progress to breastfeeding.This study was carried out at Hospital Sofia Feldman, a philanthropic institution that currently performs, on average, 900 births per month.PHASE 1: A daily active search was performed in order to find newborns that met the inclusion criteria of this study. Soon after reading the medical record, clarification of possible doubts, acceptance of participation in the research and the signing of the informed consent, the mother-child dyad was included in the study, and monitored until hospital discharge. In this approach, a record form of Kangaroo Position, with information about the day, frequency, and duration of the position, was affixed to the incubator and both the team and the mother and the family were instructed on its completion. It is noteworthy that all newborns included in this study went through the three steps described by the Kangaroo Method.PHASE 2: Upon discharge from the Speech-Language Pathologist, when the newborn was fed exclusively orally, the research team approached the mother and completed the questionnaire elaborated for this research, with personal and socioeconomic data , as well as data on gestational history, previous knowledge about the Kangaroo Method, experience with breastfeeding and relationship with the child in the delivery room.PHASE 3: Between the discharge from the Speech-Language Pathologist and hospital discharge, newborns who progressed to exclusive or mixed breastfeeding were filmed for three minutes of mother-infant interaction during breastfeeding. Filming was performed in the hospital bed itself and the camera was placed on a support (tripod), adjusted about 90 cm in height, so that it focused both the son’s and the mother’s responses and obtained the best lateral angle.PHASE 4: Two examiners with experience and performance in Neonatology were previously trained to perform analyses of the initial interaction videos through the Mother-Baby Interaction Protocol 0-6 months (MBIP 0-6).PHASE 5: The videos were filmed using a Samsung camcorder, Model S630. For editing, Windows Movie Maker software version 2014 was used, so that all videos had about the same duration. Audacity software version 2012 was used to soften the high volumes of environmental noise. The initial 20 seconds, during which the researcher was still present guiding the mother about the procedure, were excluded. This was to ease the bias due to the fact that, initially, the camera could inhibit the action of the mothers until they became accustomed to its presence.R.In this study, univariate and bivariate analyses were used. The purpose of the univariate analysis was to characterize the sample using descriptive statistics. The bivariate analysis evaluated the linear correlation from the Pearson or Spearman linear correlation coefficients. The data collected were tabulated in a 2007 Microsoft Office Excel spreadsheet, and to analyze the association/correlation between two variables, the Pearson (r) linear correlation coefficient was used, which gives the degree and direction of the linear association between two variables. If r=0, the variables are not linearly correlated; if r>0, there is a positive linear relationship; if r<0, there is a negative linear relationship. It should be noted that all statistical analyses were performed in freeware This study was approved by the Research Ethics Committee of the Hospital Sofia Feldman (HSF), under protocol no. 668.309.We analyzed 32 videos of initial mother-child interaction. In relation to newborns, 17 (53.0%) were males. r=0.36; p=0.01).When excluding the dyads that did not perform the Kangaroo Position, the minimum frequency of this positioning was 1 time and the maximum was 21 times. Regarding the duration of the Kangaroo Position, the minimum total time was 60 minutes and the maximum was 4,290 minutes (about 70 hours). The hospital stay ranged from 14 to 70 days. Therefore, the longer the hospital stay, the more hours the mothers performed the Kangaroo Position (p>0.05).r=0.37; p=0.03).The results showed that the Kangaroo Position favored the attempts of physical contact of the child with the mother during breastfeeding  reported short skin-to-skin contact with the child. This may have happened due to prematurity, low weight or the need for some immediate ventilatory support. Thus, De Schuymer et al.,r=0.47; p=0.006).When correlating the Kangaroo Position and the verbalization that the mothers performed during breastfeeding, data from this study revealed that the longer the Kangaroo Position, the less the mothers talked as the children . This correlation was positive and statistically significant, probably due to the encouragement of some professionals or even the maternal presence in the neonatal unit. Regarding this, the literature points out that professionals working in Neonatology with early intervention are responsible for supporting the interpretation of children’s signals and teaching mothers how to help their children organize the regulatory activity.,Excluding the dyads that did not perform the Kangaroo Position, the minimum frequency of this positioning was once and the maximum was 21 times. Regarding the duration of the Kangaroo Position, the minimum time in this group was 60 minutes, and the maximum time was 4,290 minutes. Therefore, the longer the hospital stay, the longer the mothers performed this position , in addition to the presence of the camcorder. In spite of these limitations, it can be concluded with the present analysis of the mother’s interaction in the short term, after birth, that the greater time spent on the Kangaroo Position favored the attempts of physical contact of the child with the mother. This suggests a greater availability of the newborn to intensify the relationship with the mother during breastfeeding. The preterm newborn has the ability to go beyond feeding and have the time of breastfeeding as an opportunity to perform the initial contact exchanges.Further research is needed to define the other patterns of preterm newborn interaction in order to allow the development of early intervention strategies. To this end, it is important that professionals involved in Neonatology focus their attention on mother-child relationships and be able to identify and intervene early in the difficulties of interaction during hospitalization. Investing in family-centered care is a promising strategy, in addition to using the Kangaroo Position as facilitator of exchanges between the mother-child dyad. However, it is necessary to move forward with regard to the quality of the Kangaroo Position in both infrastructure and ambience, as well as in terms of guidance and assistance to mothers, in order to encourage the occurrence of skin-to-skin contact with the child. It is worth mentioning that the ideal time for staying in the Kangaroo Position depends on the pleasure and comfort felt by the mother and the newborn, which demands adequate guidance and support from the local team.


The authors of this article would like to request the following replacement to the section ACKNOWLEGEMENTS:We are grateful to Mrs. Jin Xu for her assistance in flow cytometry. This study is supported by the National Natural Science Foundation of China (Grant No. 31270032), the National Basic Research Program (973 Program) (No. 2013CB945604), and SJTU Interdisciplinary Research Grant (YG2012ZD05).


JHK & KK: This work was supported by National Research Foundation of Korea Grant Number NFR-2016-S1A3A2924582, Funder: National Research Foundation of Korea, https://www.nrf.re.kr/eng/main. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.There are errors in the Funding section. The correct funding information is as follows: KK: This work was supported by JSPS KAKENHI Grant Number JP17H02626, Funder: Japan Society for the Promotion of Science,


Endoscopic endonasal surgery to access the anterior skull base has evolved in many centres worldwide and provides a minimally invasive alternative, with better patient experience, to open techniques.We present a basic approach to a midline lesion that is the fundamental starting point for wider access to the skull base.The nuances of this technique illustrated here have been developed in many centres to provide a safe procedure that has a low incidence of complications and excellent potential.The online version of this article (doi:10.1007/s00701-016-2916-z) contains supplementary material, which is available to authorized users. Neurosurgeons should be familiar with the relevant rhinological anatomy of the approach, - navigation is helpful.Major landmarks during the procedure are shown in Figs. MRI scans in 3 planes with and without contrast are performed preoperatively and examined for tumour size and lateral extension. A fine cut CT of the paranasal sinuses is examined for important variations such as non aerated sphenoid sinus, nasal deformity, and pattern of septation.Electromagnetic navigation (Medtronic) is used routinely.Stable anaesthesia with smooth emergence and reversal is essential to reduce coughing and postoperative haemorrhage.The patient is placed supine with the head elevated about 15°. Phenylephrine and lignocaine is used in the nose as a vasoconstrictor.A zero degree endoscope is used by the second surgeon acting as cameraman.If a large CSF leak is likely the thigh is draped for the harvesting of fascia lata.Adrenaline (1:1000) soaked patties are used to achieve mucosal decongestant and vasoconstriction.The septal mucosa is infiltrated with lignocaine 1 % with epinephrine 1/100,000. This will be the donor site for the nasoseptal flap.We routinely remove the right middle turbinate and approach pituitary tumours through the right nostril when possible. The left middle turbinate is lateralised (outfractured) by gentle pressure with an elevator.A nasoseptal flap  is routiWe use the exact technique described by . This reA generous posterior septectomy is performed to expose the vomer. When there is likelihood of intrasellar dissection wider exposure is required for the camera and instruments and this is achieved by a wide posterior ethmoidectomy. The bony opening over the dura extends to the medial carotids. Bony septa are drilled flat mindful that the median septum usually deviates laterally to overlie the carotid artery See Fig. .The dural opening should allow as much intrasellar access laterally as possible. We use a X shaped incision.As soon as the dura is opened tumour usually presents itself through the dural opening unless the target is a small adenoma. Tumour is biopsied using either a ring curette or Deckers rongeurs. The tumour is debulked using ring curettes, staying in the inferior half of the tumour to avoid breaching the diaphragm (or inverted dome of tumour capsule). The capsule is held up with a small neurosurgical patty to allow full exploration of the sella. The medial cavernous sinus is examined with a Doppler probe to ensure we have explored as far lateral as possible.After tumour excision we inject Floseal (Baxter) into the sella. see Fig. . We placIf there has been a persistent low volume csf leak during the procedure we use an additional inlay graft of a dural substitute . If the leak is from a visible defect in the diaphragm we place a small piece of Spongistan (Ferrosan) over the defect. If there has been a high volume csf leak we would use fat in the sella and fascia lata as inlay and onlay grafts.If there is persistent csf rhinorrhoea we would explore the operative site to examine the cause of the leak. We have not used lumbar drains or bed rest.The approach described here is a valuable initial step in approaching the anterior skull base for lesions based in the sella and suprasellar regions. It is flexible and versatile. It can reach from the frontal sinus to the odontoid peg. Modifications are described allowing access to more lateral areas, , 3.This is our standard approach for pituitary adenomas, - secreting or non-secreting – microadenomas and macroadenomas. We are aware that some authors propose not harvesting the nasoseptal flap for small tumours requiring more limited access, or using for example the rescue flap .Lesions such as meningioma and craniopharyngioma and pituitary adenoma that extend beyond the carotid artery laterally will not be accessible by this approach alone and require an extended, combined or cranial approach.The commonest complication of surgery around the sella is csf leak. We feel the best way to avoid this is by routinely using the nasoseptal flap  Our csf We try not to damage residual pituitary tissue to preserve pituitary function. Unless there is an obvious capsule we do not search for one as this can prejudice pituitary function.The carotid artery is at risk during drilling. We only expose the carotid artery if necessary and identify it preoperatively on CT, and intraoperatively using navigation and doppler. In large tumours the carotid artery may be skeletonised by tumour expansion and extra care may be required during tumour excision.Troublesome venous bleeding from sinuses is controlled with head elevation, compression with patties and Floseal (Baxter). The anterior intercavernous sinuses may bleed on opening the dura and can be pre-emptively clipped.Examination of the pituitary dedicated MR and CT should identify abnormal anatomy. Careful endocrine assessment before during and after surgery is essential. A short period of corticosteroid support before formal endocrine assessment shortly after surgery is used. Careful assessment of fluid balance and management of diabetes insipidus is vital.A routine postoperative scan and Goldman visual field tests are arranged for 3 months after surgery to assess residual tumour.Patients are educated regarding csf leaks and given contact. They are advised to mobilise but to avoid blowing their nose, sneezing or coughing or straining if possible. They have an early (4 weeks) rhinological review. We warn patients that they will lose some sense of smell (and taste) temporarily.A team of neurosurgeon and ENT surgeon is recommended.Examination of the MR and CT imaging is mandatoryAnaesthetic conditions should promote minimal blood loss during the approach.Consider a nasoseptal flap in most casesA generous posterior septectomy provides space for instrument manoeuvre.The dural opening should allow wide access to the sella.Explore the lateral walls of the sella and medial cavernous sinus.Use a small Doppler probe to identify the carotid artery as the lateral limit of tumour excisionThe diaphragma sellae can be held out of the operative field using a neurosurgical pattyEnsure the flap is placed on bone with mucosa removed.ESM 1(MOV 264119 kb)Below is the link to the electronic supplementary material.


The use of antiangiogenic therapy in non-small cell lung cancer (NSCLC) requires thorough evaluation of patient characteristics in order to avoid potential safety issues, particularly pulmonary haemorrhage (PH). The aim of this consensus by a panel of experts was to identify important criteria for the selection of patients with NSCLC who would benefit from antiangiogenic therapy.Radiologists and oncologists were selected for the expert panel. The nominal group technique (NGT) and the Delphi questionnaire were used for consensus generation. The NGT consisted of four steps, the result of which was used to set the Delphi questionnaire. A final report was generated based on the opinions of the experts from the panel.An extremely important prerequisite for the evaluation of an antiangiogenic therapeutic approach in patients with NSCLC was thorough clinical and radiological analysis of the relationships between tumour and vascular or anatomical structures (performed in close co-operation by oncologists and radiologists). The panel identified major parameters to be considered before the use of antiangiogenic treatment, collectively agreeing on the relevance of tumour cavitation, vascular infiltration, endobronchial growth and thromboembolism for chest tumour sites, and of the presence of aneurysms, extra-thoracic bleeding, brain metastases or thrombi for extra-thoracic sites. Moreover, a structured report containing information not only on the tumour but also on the general vascular status is essential to guide the treatment choice The experts agreed that tumour localization in the absence of vessel infiltration, cavitation, and the use of antiplatelet therapy are relevant parameters to be assessed, but their presence should not necessarily exclude a patient from receiving antiangiogenic therapy.Close co-operation between oncologists and radiologists in the diagnosis, treatment selection, and assessment of response is essential for ensuring therapeutic appropriateness in the NSCLC setting. It should be noted that neither the use of antiplatelet therapy nor tumour localisation are to be considered as contraindications to antiangiogenic treatment. Tumour angiogenesis is a hallmark of cancer pathogenesis and operates through several mechanisms, typically mediated by pro-angiogenic factors , 2. Vascp = 0.003) [p = 0.003; 6.5 versus 6.1 months, respectively, in the high-dose group, p = 0.03) and is well tolerated in patients with advanced NSCLC [Non-small cell lung cancer (NSCLC) is one of the two major types of lung cancer, accounting for up to 85% of lung cancers and is associated with a 5-year survival rate of 15.9% . Favoura= 0.003)  and in t= 0.003) . In addied NSCLC  and ARIEed NSCLC  the safeed NSCLC –14.Severe pulmonary haemorrhage (PH) is a relatively uncommon but potentially fatal adverse event that occurs preferentially in squamous NSCLC; the incidence of grade ≥3 PH reported during antiangiogenic therapy has been 0.7–1.9% in the phase III and real-world cohort studies, where patients with predominantly squamous cell tumours had been excluded , 15. SinImaging is essential for diagnosis and staging of NSCLC as it helps in determining the size, location and tumour baseline features . Both MuMost importantly, no imaging criteria that can be used for predicting antiangiogenic therapy-associated PH could be clearly identified from the literature. Newer techniques like computed tomography (CT) perfusion imaging, magnetic resonance (MR) perfusion/diffusion imaging or positron emission tomography-computed tomography (PET-CT) do not provide reproducible results across different study centres and are not commonly used in clinical practice. Determining the relevant parameters to be assessed, therefore, is an increasingly important goal for optimal use of antiangiogenic therapy in patients with NSCLC, particularly in light of the development of newer antiangiogenic agents . The aimIn order to evaluate potential risk factors of PH and assess the optimal selection criteria for antiangiogenic therapy in patients with NSCLC, the conflicting scientific and clinical evidence was discussed by a group of six Italian specialists (three oncologists and three radiologists). Members of the expert panel were identified under the following series of criteria , 23: a) The NGT and the Delphi method were used for this study , under tThe NGT was conducted following four steps: 1. Generating ideas: the problem “In your opinion what are the points worthy of study for the clinical-radiological definition of NSCLC?” was presented to the expert panel and the experts responded to it individually and independently by briefly outlining their ideas on paper; 2. Recording ideas: the ideas from the individual members of the panel were recorded, and overlapping ideas were deleted from the list; 3. Discussing ideas: each individual idea was discussed amongst the panel of experts to determine clarity and importance of the idea; 4. Voting on ideas: the discussed ideas were then individually prioritized by the experts. Each expert came up with five ideas they considered most important; the highest priority idea was rated as rank 5, and the least important as rank 1. The final responses from the experts were tallied to get the most favoured group outcomes on the problem. Thus, the NGT was used to gather information about the given question, and the issues that the expert panel considered relevant to answer this question were then used to develop the Delphi questionnaire that was submitted to the expert panel in a second phase. The overall study design is summarised in Fig. The NGT helped in identifying a broad area of important parameters for the evaluation of clinical and radiological features driving the therapeutic management of patients with NSCLC. From an original list of 20 parameters or “ideas”, the NGT led to the identification of six main areas: status of great vessels, status of lung parenchyma, non-morphological evaluation parameters of the tumour, organizational aspects, localisation of the tumour and tumour morphology. The ideas embodied in these broad areas were rated, establishing a limit cut-off consensus percentage of 66%. With these criteria, the expert panel nominated several points as relevant for the choice of antiangiogenic treatment in NSCLC. Central versus peripheral localization of the tumour, ratio of solid to cavitation component of tumours, presence of thromboembolism in great vessels and relationship to non-vascular adjacent thoracic structures were rated, in this order, as important by all the participants (100% consensus). Lung-vessels relationship was rated as important by five out of six participants, as well as the opportunity of a structured report, while presence of thromboembolism in peripheral vessels, use of standardized imaging techniques, general lung status regardless of the tumour presence and pleural effusion were rated, in this order, as important by four out of six participants.Based on the results obtained through the NGT, the Delphi questionnaire was set and answered by the expert panel to generate a consensus report. The results of the Delphi questionnaire are summarised in Table Broad consensus (>80% agreement) was also reached on the following: the importance of information on infiltration into the bronchi and carina; that 1.5 mm or 3 mm thickness is sufficient for evaluating infiltration of vessels by CT scan; that compression of a vascular structure (particularly the aorta) is not a contraindication for antiangiogenic therapy; that endobronchial tumour extension may be a bleeding risk factor; that awareness of the presence of deep vein thrombosis requiring antiplatelet therapy is fundamental when considering antiangiogenic therapy; and that it is useful to have a structured report which contains information on fistulas and tumour margins  was identified as an extremely important prerequisite. Before antiangiogenic treatment is used it is important to examine tumour cavitation, vascular infiltration, endobronchial growth and thromboembolism for chest tumour sites, and of the presence of aneurysms, extra-thoracic bleeding, brain metastases or thrombi for extra-thoracic sites. Overall, based on complete knowledge of relevant parameters, the co-operative efforts of radiologists and oncologists may provide a framework for the selection of patients and safe use of antiangiogenic agents for the treatment of NSCLC in a clinical setting.


Following publication of this article , it cameThis study was also supported by a Project Grant from the Israel Cancer Research Fund (ICRF) and a grant from Israeli Science Foundations (ISF) number (961/15).


Leg fluid accumulation during sedentary behaviours such as sitting can lead to leg edema and associated adverse health consequences. This study investigates the use calf muscle electrical stimulation (ES) to reduce seated leg fluid accumulation. Thirteen non-obese, normotensive men (mean age 51 yr.) with sleep apnea were enrolled in the study. Participants first lay supine for 30 minutes to equalize fluid distribution and then sat for 150 minutes. While seated, participants received either active or sham ES of the calf muscles, according to random assignment. Participants returned one-week later to cross over to the other study condition. Leg fluid was measured continuously while sitting using the bioelectrical impedance method. Fluid accumulation in the leg was reduced by more than 40% using active ES, compared to sham ES . In summary, calf muscle ES is an effective method for reducing accumulation of fluid during long sedentary periods and has potential use as a device for preventing leg edema to treat associated health consequences in at-risk groups and settings. More fluid in the legs is also associated with a greater shift of fluid toward the upper-body when moving from upright to supine posture3, which can increase risks for supine hypertension4, acute decompensation in heart failure5, pulmonary edema6, nocturnal asthma7, and increased sleep apnea severity8. This is particularly problematic in settings that facilitate sedentariness such as the office9 or long-haul flights10, and in high risk populations such as the spinal cord injured11 and individuals with fluid-retaining diseases1. Reducing leg fluid accumulation can serve to prevent the genesis or worsening of these diseases and disorders in high risk populations and settings15.Fluid accumulation in the legs is a known consequence of sedentariness and is associated with edema formation. Development of leg edema is associated with complications including increased risk of leg ulcers, painful swelling, reduced elasticity of the veins and arteries of the legs, and thromboembolic events18. According to the Starling equation, fluid filters from vasculature into the tissue spaces when capillary pressure is increased and/or tissue pressure is decreased19. Fluid continues to shift from the upper to the lower body by displacing fluid that filters into the interstitial spaces of the leg, driven by the enhanced capillary transmural pressure22. Conversely, fluid is reabsorbed into the vasculature when tissue pressure is increased and/or capillary pressure is reduced.During upright sitting or standing, fluid accumulates in the legs by first rapidly filling the capacitance vessels of the lower body23. In addition, improper fit is a common problem, which increases the risk of developing exudative skin lesions24 and deep vein thrombosis25. Alternatively, activation of the skeletal pump in the legs reduces leg fluid by reducing capillary pressure in the legs and increasing tissue pressure26. Walking is one method of activating the skeletal pump, however, it is limited to able-bodied individuals and is less feasible for the elderly or those with mobility limitations. Therefore, in the present study, we experiment with activation of the skeletal pump using electrical stimulation (ES) of the gastrocnemius muscles to prevent leg fluid accumulation while seated.Compression stockings are a commonly prescribed and effective therapy for reducing leg fluid by increasing tissue pressure. However, they are limited by their discomfort and difficulty to apply and remove, contributing to their low adherenceThe overall purpose of this study was to investigate the efficacy of calf muscle ES in preventing leg fluid accumulation while seated for two-and-half hours. The main objectives of this study were first, to determine if calf muscle ES can prevent the accumulation of leg fluid over the seated period; and second, to identify the minimum time period required for active calf muscle ES to reduce seated leg fluid volume. A secondary objective, which was exploratory in nature, was to characterize the temporal changes in leg fluid accumulation with and without calf muscle ES.2. Participants were all normotensive with a mean resting systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate of 117.5 ± 3.2 mm Hg, 80.4 ± 2.6 mm Hg, 93.0 ± 2.5 mm Hg, and 68.3 ± 2.9 beats/min. All participants were free from symptoms of any serious diseases or disorders.As part of a larger study examining the effect of calf muscle ES on fluid shift and sleep apnea, one hundred and thirty-two participants were pre-screened for eligibility and 45 participants moved on to be screened for sleep apnea. From this group, 15 participants were found to have moderate to severe sleep apnea (apnea-hypopnea index = 21.1 ± 4.0 events/hr) and were willing to participate in the full study. One participant was found to have varicose veins and was therefore excluded, and another was lost to follow-up, yielding a final sample size of 13. Weight, height, and blood pressure were measured at a screening visit to confirm participant eligibility. Although sex was not an exclusion criterion, all participants were non-obese men with a mean age of 51 ± 2 years and body mass index (BMI) of 26.5 ± 0.7 kg/mThe study protocol required participants to first lay supine for 30 minutes to equalize fluid distribution and then sit for 150 minutes on two separate days spaced one week apart. On one of the two study days, seated participants received either active or sham ES. Active ES elicited repeated calf muscle contractions in both legs every two seconds, whereas in sham ES the participant could feel the electrical current but it did not elicit calf muscle contractions. The protocols for the sham and active ES conditions were identical in every aspect (e.g. device and electrode type and position), except that in the sham ES condition electrical current was set to a level where the participant could feel the electrical current but a contraction was not elicited. After the first study day, participants returned one-week later to cross over to the other study condition. Leg fluid from ankle to knee was measured in the dominant leg continuously while sitting using the bioelectrical impedance method. Baseline leg fluid volume at the start of sitting did not differ between the sham (1306.3 ± 63.9 ml) and active (1370.8 ± 56.9 ml) ES conditions (P > 0.10). Similarly, baseline calf circumferences measured at the start of sitting were also similar between the sham (38.9 ± 0.8 cm) and active (39.1 ± 0.8 cm) ES conditions (P > 0.10).Leg fluid volume increased significantly over the seated period in both the sham ES condition  and in the active ES condition . However, the interaction effect demonstrated that leg fluid volume increased significantly more in the sham ES condition compared to the active ES condition (P < 0.001). Calf circumference was measured in the dominant leg at the start and end of the seated period. Calf circumference increased significantly over the seated period in the sham ES condition , but was unchanged in the active ES condition . The interaction effect was significant, demonstrating the increase in calf circumference in the sham ES condition was significantly greater than the change during the active ES condition (P < 0.001).The temporal patterns of leg fluid accumulation in the active and sham ES conditions are displayed in Fig. As illustrated in Fig. The difference in leg fluid accumulation between active and sham ES conditions also demonstrated a rise in the variability among subjects Fig.  at the 2To measure the efficacy of the electrical stimulator in removing fluid upon calf muscle contraction, the change in resistance (ΔR) of the leg per contraction was computed. Resistance of tissues is inversely related to fluid volume. Therefore, the sharp rises in resistance per contraction depicted in Fig. The present study reports several key findings: first, electrical stimulation of the calf muscle reduced the accumulation of fluid in the legs by 43% over a two-and-a-half hour (150 min) seated period. Second, during the sham ES condition, leg fluid accumulated according to an exponential decay during the first 25 minutes of sitting, then linearly in the latter period of sitting. Third, variability in the difference in leg fluid accumulation between active and sham ES conditions increased substantially after 20 minutes of sitting. Lastly, over the final 130 minutes of sitting, ΔR per ES contraction was associated with the difference in leg fluid volume between the active and sham ES conditions.27. A comparison of fluid volumes measured by bioelectrical impedance with prior studies is not possible because prior studies have predominantly measured fluid volume in the whole leg (from hip to ankle), while the present study measured fluid volume in the lower leg (from knee to ankle). In terms of calf circumference, wearing compression stockings for one week reduced evening (pre-sleep) calf circumference by 0.5 cm13, one day of ultrafiltration reduced evening calf circumference by 1.0 cm27, and four weeks of walking exercise caused no change in evening calf circumference12. Calf muscle ES performed comparably, reducing calf swelling by 0.7 cm, despite a short duration of use. This warrants further research to examine whether the long-term use (such as 4 weeks) of calf muscle ES can be used to treat sleep apnea by reducing leg fluid accumulation and the consequent fluid shift from the legs to the neck that occurs during sleep.The present study reports that calf muscle ES reduced leg fluid accumulation by 40 ml and reduced calf circumference by 0.7 cm. The clinical relevance for these results can be derived from prior studies investigating interventions to reduce leg fluid prior to sleeping and effectively reduce sleep apnea severity (as measured by the apnea-hypopnea index) by 27% to 36%28. To our knowledge the findings of the present study are the first to demonstrate the immediate capability of calf muscle contraction via electrical stimulation to reduce leg fluid accumulation while seated, compared to a sham control. The experiment by Goddard et al. compared 30 minutes of quiet sitting followed by 30 minutes of calf muscle activation using a plantar reflex–based stimulation device28. These results differed from those reported in present experiment, as Goddard et al. report no accumulation of leg fluid volume over the 30-minute quiet sitting period, and a sustained reduction in leg fluid over the 30-minute period with leg muscle contractions. Differences in the pattern of leg fluid accumulation can likely be attributed to the methodological differences between the studies. Specifically, leg fluid was measured using air plethysmography, only women were studied, and participants sat for 15 minutes prior to beginning leg fluid measurements which means that the majority of the vascular fluid accumulation was not recorded. Furthermore, since each seated period started with 30 minutes of quiet sitting, calf muscle pumping was tested after 45 minutes of quiet sitting. Therefore leg fluid volume was higher when muscle contractions began. As a result, their study investigates the removal of fluid already accumulated in leg, rather than prevention of leg fluid accumulation which was the purpose of present study.Few studies have investigated the immediate effect of calf muscle contraction on leg fluid while uprightet al. measured changes in leg fluid volume using a mercury strain gauge around the largest circumference of the calf while participants were (i) recumbent for 30 minutes, (ii) upright for 10 minutes, (iii) quiet sitting for 15 minutes, then (iv) sitting while using a cycle ergometer for 20 minutes26. They found that calf circumference increased substantially while upright and seated. During cycling, the study demonstrated a strong effect, reducing calf circumference by approximately the same proportion that it increased during quiet sitting . In the present study, measures of calf circumference were also suggestive that active ES had a strong effect in resisting accumulation of fluid in the legs. Active ES of the calf muscle resulted in no significant change in calf circumference over the entire seated period, while sham ES resulted in a 0.79 cm increase in calf circumference. Caution must be used when comparing the efficacy of cycling and calf muscle ES, given the methodological differences between the studies. In particular, Stick et al. evaluated the reduction of leg fluid accumulation during calf muscle exercise after 10 minutes of standing and 15 minutes of quiet sitting. Therefore as previously explained, Stick et al. report the removal of fluid already accumulated, rather than prevention of leg fluid accumulation that was addressed in the current study.Stick 17. However, rapid vascular filling while upright was complete after approximately 5 minutes29; much less than our reported 25 minutes. One explanation for the discrepancy in vascular filling time is differences in body position. In the present study, participants were sitting in a chair, while in the past studies participants were standing at varying degrees head-up tilt. Regardless of the discrepancy in the time point where rapid vascular filling was complete, it is clear that leg fluid accumulated according to two different functions separated at the 25 minute time point. Following the rapid vascular filling, further increase in leg fluid was considered to be driven by enhanced transmural pressure leading to fluid filtration into the interstitial space22. Therefore, the latter portion of sitting was likely a reflection of the accumulation of interstitial fluid.During the first 25 minutes of quiet sitting, fluid accumulated according to an exponential decay function and at a more rapid rate than in the latter portion of quiet sitting Fig. . Past stThe difference in fluid accumulation between the active and sham ES condition describes the reduction in leg fluid accumulation due to active ES while seated. As illustrated in Fig. One plausible explanation for the rise in variability after the first 20 minutes is the type of fluid being removed. In the first 20 minutes, active ES was likely preventing vascular filling while in the latter 130 minutes, active ES had variable effectiveness in preventing fluid filtration into the interstitium. From this, we hypothesized that differences in contraction strength across the subjects might explain the variability in the effectiveness of active ES in preventing interstitial fluid accumulation. As illustrated in Fig. 9; flight attendants or frequent flight travellers susceptible to leg swelling due to a long immobilized period10; individuals with fluid retaining diseases such as renal failure and heart failure1; spinal cord injured11; individuals with sleep apnea caused by nocturnal shift of fluid from the legs to the neck15; and individuals with asthma who experience worsening of asthma symptoms due to nocturnal fluid shift7.The greatest feature of an electrical stimulator for activating the skeletal pump and reducing leg fluid accumulation is its versatility as a therapeutic device. An electrical stimulation system can be developed into a compact, wearable device that can be applied in a wide variety of settings, thereby making it easier to target individuals who are at risk for leg fluid accumulation and its negative consequences. At risk groups include sedentary office workers who experience leg swelling due to long periods sitting30. Ultrasound is a non-invasive method of estimating blood volume in major veins and arteries that could be performed in future studies to detect time-series changes in blood volume31. Our study was also limited in its lack of women participants. While the study was open to women, fewer women responded to the study advertisement and those that did were not eligible for various reasons. The main challenge with enrolling women to participate was the requirement for a sleep apnea diagnosis, which is a more common disorder in men32. To the best of our knowledge, there are no studies investigating differences between the sexes in leg fluid accumulation while upright. However, we have shown in prior studies that fluid shift due to gravity when lying down differed between the sexes. In short, baseline LFV was greater in men compared to women, yet there was no significant difference between the sexes in fluid shift out of the legs when lying down for 90 minutes33. In addition, while the volume of fluid leaving the legs was the same, less fluid accumulates in the thorax and neck of women compared to men, possibly due to fluid sequestration in the abdomen of women33. Since the same amount of fluid shifted out of the lower body in men and women, it is likely that when assuming the upright position the same amount of fluid will shift into the lower body. With respect to between-sex differences in the effectiveness of the calf muscle activation in reducing leg fluid accumulation, to the best of our knowledge this has never been investigated. So while it is unlikely that leg fluid accumulates differently in men compared to women, the effects of calf muscle ES between the sexes cannot be speculated upon, but would be an interesting topic for future study.The main limitation of this study was that vascular and interstitial fluid were not directly measured and inferences made on the transition from vascular to interstitial fluid accumulation are based on the rates of change in leg fluid described by their mathematical models. To confirm these findings, more objective measures should be considered. Indicator dilution methods are an objective measure of fluid compartments, however it is invasive, requiring blood samples for each time measurementIn conclusion, calf muscle ES was effective at reducing the accumulation of fluid in the legs while seated, compared to a sham control. There was no clear time point after which calf muscle ES stopped being effective. While effect size reached a local peak at 20 minutes and then dropped substantially, after continued use the effect size increased substantially to an absolute peak at 130 minutes. Therefore, the longer an individual used calf muscle ES, the more effective it was at preventing leg fluid accumulation. Efficacy of calf muscle ES was consistent in preventing vascular filling in the first 20 minutes seated, but variable in preventing interstitial fluid formation in the latter 130 minutes seated. The variability in preventing interstitial fluid formation was found to be attributed to the amount of fluid ejected from the leg per contraction, where more fluid leaving the leg per contraction was associated with greater prevention of interstitial leg fluid formation. This highlights the importance of maintaining a strong contraction throughout the sedentary period using calf muscle ES. Once validated in a larger sample size, calf muscle ES has potential as a device for preventing leg fluid accumulation and associated health consequences in at-risk groups and settings.2, and blood pressure < 140/90 mmHg. The study was part of a larger study investigating the efficacy of calf muscle electrical stimulation on fluid shift and sleep apnea severity, so all participants had a positive screening for sleep apnea with an apnea-hypopnea index ≥ 10, as determined by a portable sleep monitoring device35. Participants abstained from alcohol and coffee consumption for 24-hours. Participants were excluded if they reported a history of cardiovascular, renal, neurological, or respiratory disorders; reported taking prescribed medication for these disorders; or reported taking over-the-counter medication that might influence fluid retention. Participants were recruited from the community through advertisement.The protocol was approved by the Research Ethics Board of Toronto Rehabilitation Institute and participants provided written informed consent prior to participation. The study was performed in accordance with the approved guidelines and regulations. Criteria for study enrolment were adult men and women with a body mass index < 30 kg/m37. The method is based on Ohm’s law which states that the resistance of tissues to electrical current is directly proportional to its length: R = ρL2/v, where ρ is the resistivity of the fluid, L is the segments length, v is the fluid volume and R is the measured resistance40. This formula has since been adapted to measure impedance of tissues in individual body segments  and calculated according to equation 142.43.Fluid volume was measured in the dominant leg continuously while seated using a bioelectrical impedance measurement system. This method is a non-invasive technique used to estimate fluid volumes of the tissues. The method has been well validated and is highly reproducible with an accuracy within 0.5% compared to reference measures of total body water, repeatability within 0.3%, and test-retest correlation >95%Surface electrodes were placed on the skin at the top and bottom of the lower leg (ankle and knee) as illustrated in Fig. Using a measuring tape, segment length was measured as the distance between the measurement electrodes. Circumferences of the ankle and knee were measured at the level of the measurement electrodes at the start and end of sitting. A line was drawn around the ankle, and knee to ensure measurements were consistent when repeated. Using the same method, calf measurements were also made around the largest part of the calf at the start and end of sitting, for use as an alternative measure of fluid accumulation.44. The stimulation protocol used an asymmetrical biphasic waveform with 40 Hz frequency, 300 μs pulse duration, at the maximally tolerated stimulation amplitude . The duty cycle was 2 seconds on, followed by 2 seconds off. The left and right gastrocnemii were stimulated out of sync, such that when the left gastrocnemius was contracted the right gastrocnemius was relaxed and vice versa. A sham electrical stimulation protocol was also utilized as a control. The sham ES protocol was identical to the active ES protocol described in terms of the current waveform used and electrode type and position. However, the main difference was the amplitude of the electrical current which was set much lower in the sham ES protocol, at a level that elicited the sensation of electrical current travelling through the lower leg, without causing calf muscle contraction.Calf muscle ES was performed using a programmable 4-channel neuromuscular stimulator . Electrical current was delivered transcutaneously to the gastrocnemius muscle groups of both legs using 5 cm by 9 cm StimTrode neurostimulation electrodes  positioned on the calf over the approximate position of the motor pointsLFV is the leg fluid volume (ml), m is the slope of the line (ml/min), b is the y-intercept (ml), t is time (minutes), τ is the time constant (minutes), and LFVend is the expected final value of the leg fluid volume (ml). The model with the best fit was selected to describe each portion of data.Temporal fluid data in the active and sham ES conditions consisted of two distinct portions: an initial portion and a latter portion which differed by their apparent slopes. In addition, the initial portions appeared to increase according to an exponential decay function, whereas the latter portions appeared linear. Similarly, the difference in fluid volume between the active and sham ES conditions also consisted of two distinct portions. Each portion was modeled with both a linear and exponential decay function, described in equations The change in leg fluid volume per contraction was estimated using the change in resistance (ΔR) per contraction. Illustrated in Fig. The study was a randomized, single-blind double crossover protocol. Data collections were completed over two days set one week apart in a quiet, temperature-controlled room following abstention from alcohol and caffeine. Experiments began in the morning between 10 and 11AM for all participants. On the first morning, participants were randomly assigned to sham or active ES. On each morning, surface electrodes were placed on the legs to collect bioelectrical impedance data, as illustrated in Fig. Bioelectrical impedance data was collected continuously at 1.25 kHz using Biopac data acquisition system  and imported into Matlab  for pre-processing. Signals were manually adjusted for motion artifacts based on documented events of movement or seat adjustment occurring during data collection. Signals were then averaged every minute to reduce variability in the signal due to noise or other artifacts.Bioelectrical impedance signals were converted to fluid volumes using Equation Statistical differences in baseline values of leg fluid volume, and calf circumference between study conditions were analysed using the paired t-test . A repeated measures ANOVA was used to compare changes in leg fluid volume, and calf circumference from start to end of sitting, between study conditions. The ANOVA included two factors: timing (start and end) and study condition (sham ES or active ES). The interaction effect tested differences in the change in leg fluid or calf circumference from the start to the end of sitting between active and sham ES conditions. Lastly, the paired t-test  was used to test if the change in leg fluid or calf circumference from start to end of sitting within each study condition was statistically significant.Data distributions were assessed using the Shapiro-Wilk test of normality and considered non-normally distributed if the null hypothesis was rejected at a significance level of p < 0.05. Correlations were performed using Pearson correlations with normally distributed data and Spearman’s rank correlation with non-normally distributed data. A p-value < 0.05 was considered statistically significant. All data are reported as the mean ± SEM.


Fluid therapy is a common and crucial treatment in the emergency department (ED). While fluid responsiveness seems to be a promising method to titrate fluid therapy, the evidence for its value in ED is unclear. We aim to synthesise the existing literature investigating fluid responsiveness in ED.MEDLINE, Embase and the Cochrane library were searched for relevant peer-reviewed studies published from 1946 to present.A total of 249 publications were retrieved of which 22 studies underwent full-text review and eight relevant studies were identified. Only 3 studies addressed clinical outcomes - including 2 randomised controlled trials and one feasibility study. Five articles evaluated the diagnostic accuracy of fluid responsiveness techniques in ED. Due to marked heterogeneity, it was not possible to combine results in a meta-analysis.High quality, adequately powered outcome studies are still lacking, so the place of fluid responsiveness in ED remains undefined. Future studies should have standardisation of patient groups, the target response and the underpinning theoretic concept of fluid responsiveness. The value of a fluid responsiveness based fluid resuscitation protocol needs to be established in a clinical trial. Fluid therapy is a key part of resuscitation of the shocked patient in the Emergency Department (ED). Prompt and effective fluid resuscitation is required to prevent grave outcomes , 2. HoweFluid resuscitation is currently delivered with uncertainty and variability. There is uncertainty about the amount to give and when to stop. There is significant individual variation in the amount of fluid given between patients, especially in the elderly. This variation was evident in recent major clinical trials where the total volume of intravenous (IV) fluid therapy in ED varied from 200 ml to over 10,000 ml –10.Pre-existing medical conditions or acute disease associated myocardial dysfunction alter the response to resuscitation. Critical care studies demonstrate that only half of haemodynamically unstable patients in intensive care units (ICUs) respond to fluid therapy . It is lIn ED, resuscitation remains largely guided by clinical examination, basic monitoring parameters (e.g. blood pressure and heart rate) and biochemical parameters (e.g. blood lactate), which are poor predictors of volume status , 20. WhiThe immediate goal of fluid resuscitation is improving cardiac output (CO) and ultimately improving tissue perfusion. If a fluid bolus does not increase CO, it will not improve tissue perfusion and may be harmful. The haemodynamic response to fluid loading, ‘fluid responsiveness’ has been suggested as a dynamic guide for fluid therapy and a controlled method of resuscitation . TestingOne suggested definition of fluid responsiveness is ‘an increase in a physiologic parameter, preferably cardiac output, within 15 min, superseding twice the error of the measuring technique after a 15-min administration of 6 mL/kg of crystalloids’ . HoweverAlternatively, the preload challenge can be by passive leg raise (PLR)  or positA number of less invasive and noninvasive techniques for haemodynamic monitoring have been developed, which may be appropriate in emergency care. Less invasive methods include trans-oesophageal Doppler, transpulmonary thermodilution, pulse contour and pulse power analysis. Noninvasive methods include end-tidal carbon dioxide, transthoracic Doppler, bioimpedance, plethysmography and bioreactance .Most of the evidence about different fluid responsiveness strategies comes from perioperative and critical care setting, however as anaesthesia and ventilation profoundly affect cardiovascular responses, these data are difficult to apply to the ED. There are no previous systematic reviews on this topic in ED. In this study, we aim to synthesise the existing literature investigating fluid responsiveness in in-hospital emergency care.A systematic review of literature was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .Patient, In adult patients who may need fluid resuscitation in ED,Intervention, Does testing fluid responsivenessComparison, Compared to standard careOutcome, Affect haemodynamic or biochemical parameters, resource utilisation or mortality?We searched the US National Library of Medicine’s MEDLINE database via Ovid interface, Embase and the Cochrane library for relevant peer-reviewed studies published from 1946 to present. A clinical librarian assisted in the literature search.We used the following search terms and subject headings: “emergency”, “fluid/preload or volume responsiveness” and “preload dependency” with appropriate variations where applicable Table . The refCritical appraisal of the studies identified as relevant to the PICO question followed the CONSORT statement for randomized studies and the STARD checklist for diagnostic accuracy studies., 33 Two A total of 249 publications were retrieved of which 22 studies underwent full-text review. Eight relevant studies were identified, with characteristics shown in Table Kuan et al. comparedHou et al.  comparedDuus et al.  evaluateFeissel et al.  evaluateThis is the first review of fluid responsiveness in emergency medicine, and has a number of limitations. This review was not registered in advance, which may increase the risk of reporting bias. Literature search was undertaken by one author (M.H.E.), however, a clinical librarian was involved in the search strategy. We used wide search criteria from three sources and hand searched references to include relevant studies, however we excluded conference abstracts that could not be assessed for methodological quality.While fluid responsiveness is directly relevant to emergency care, we have found very little evidence  on which to base best practice. This small number of studies reflects a relatively new concept to emergency care compared to anaesthesia and critical care - where fluid therapy has evolved over many years , 43. MosThe main sources of heterogeneity were (1) the patient groups, (2) the type of preload challenge, (3) the monitoring method used, and (4) the definition of ‘fluid responsive’.Patients included in the studies had a wide range of underlying conditions of varying severity. In contrast with ICU and peri-operative studies , most paTo give a preload challenge three studies used PLR , 38, 41,Fluid bolus has been used as the criterion method (gold standard) of preload challenge in all but one diagnostic studies –42. The Most of the included studies tested non-invasive monitors to track CO (or one of its surrogates). This is in line with a recent French survey that demonstrated that ED clinicians prefer to use the less invasive and less time-consuming indicators of fluid responsiveness . In our In the diagnostic accuracy studies Table , the varThere was a variety of definitions used to define ‘fluid responsive’. The commonest definition of fluid responsiveness was a 10% increase in SV, cardiac index (CI) or SVI (5 studies) , 41, 42.Previous studies, mostly in ICU, used >0 to 20% change in SV or CO as a threshold for fluid responsiveness . More reThe diagnostic accuracy of different techniques varied greatly Table . This maThe diagnostic accuracy of different fluid responsiveness predictors have been more thoroughly studied in ICU and peri-operative setting. PLR-induced changes in CO has been validated against a ‘gold standard’ fluid bolus in several meta-analyses , 52, 53.In ED patients, spontaneous breathing, arrhythmia and autonomic response to PLR may confound the validity of the test. The test may also be different in ED patients who may be more hypovolaemic in the early stages of treatment, compared to the more fluid replete patients in ICU. The accuracy of the caval index remains questionable in spontaneously breathing patients , and theOne of the three clinical trials was a feasibility study without a primary outcome. The remaining two studies used different primary outcomes – lactate clearance and SOFA score change Table . Both stA secondary outcome for all studies was the amount of fluid given. The fluid responsiveness based protocols triggered more intensive early resuscitation, however the total fluid received by fluid responsiveness and standard care groups seemed to equalise by the end of the first 24 h. So, total fluid volume was the same, but the intervention meant that the fluid was ‘front-loaded’ into the early phase of care. A similar effect has been seen in the recent major protocolised sepsis care trials –10, 56.There are limited data on the usefulness of the fluid responsiveness approach to fluid management. A 2012 Cochrane review showed no mortality benefit from flow-guided haemodynamic approach in the peri-operative setting. This was supported by a recent RCT of a CO-guided haemodynamic algorithm  . There wThe clinical relevance of ‘fluid responsiveness’ in the ED may be similar in principle to other settings. However, fluid management in ED is faced with unique challenges , in addition to limited time, limited resources and sometimes delay in transfer to intensive care. Hence, studies in other settings may not resolve the ED haemodynamic dilemma. As the benefit of fluid administration is often related to early administration, we think that, despite the practical difficulties, studying fluid resuscitation in the ED is fundamental for improving patient outcomes. Fluid responsiveness based resuscitation strategies have not been adequately tested in the ED to know whether or not they influence outcome, but fluid responsiveness remains an attractive concept.Which patient group?Standardised definition of the target patient group is required, and the ‘uncertainty principle’ could be used. It is unreasonable to delay fluid administration for overtly hypovolaemic or septic patients, and a fluid responsiveness test is not needed. It is also unreasonable to give fluid if the clinician thinks that this may be harmful. However, in patients where the clinician is uncertain about the best course of action a test of fluid responsiveness may help. Future research should target the groups where there is clinical uncertainty.What is the target response?It is difficult to interpret a diagnostic test when the target response is ill defined. Current theory suggests that fluid responsiveness is a normal state  , 58. HowWhat outcome should be used?The outcome should be independent of the test and relevant to the patient. Survival (lived/died) is appropriate for large scale interventional clinical trials of a fluid responsiveness based resuscitation protocols. Surrogate outcomes should be carefully assessed for patient relevance. We would suggest that ‘volume of fluid used’ does not pass this test and should not be used as a primary outcome in future studies.When to start and when to stop fluid resuscitation?Fluid responsiveness does not tell us when to start or stop fluid therapy (about 50% of volunteers would have received unnecessary fluid based on a positive response). The human volunteer studies suggest that it may be unwise to use a fluid responsiveness test as a definition of the resuscitated state (an endpoint for resuscitation). A fluid responsiveness test may help the clinician decide if more fluid could help achieve the resuscitated state (by predicting the effect of a fluid bolus), but cannot be used to define when the resuscitated state has been achieved . Future studies should not use fluid responsiveness to determine when to start or stop fluid resuscitation.What is the conceptual framework for fluid responsiveness in ED?Much of the current heterogeneity within the literature seems to be based on different concepts of how a fluid responsiveness test could potentially be used in an ED management protocol. The key concept is that the test is only a predictor of the haemodynamic effect of further fluid resuscitation – the clinician still has to decide whether further resuscitation is required and incorporate the fluid response prediction into the wider picture of the patient’s physiological state. Future studies would have less variation if based around this central concept.We were unable to perform any meta-analysis due to the large variation within the literature. To resolve the current heterogeneity, we would like to suggest some standardisation for future research:One-size-fits-all protocolised ‘fixed goal’ care has been challenged –10 and t


The number of tumour biopsies required for a good representation of tumours has been controversial. An important factor to consider is intra-tumour heterogeneity, which can vary among cancer types and subtypes. Immune cells in particular often display complex infiltrative patterns, however, there is a lack of quantitative understanding of the spatial heterogeneity of immune cells and how this fundamental biological nature of human tumours influences biopsy variability and treatment resistance. We systematically investigate biopsy variability for the lymphocytic infiltrate in 998 breast tumours using a novel virtual biopsy method. Across all breast cancers, we observe a nonlinear increase in concordance between the biopsy and whole-tumour score of lymphocytic infiltrate with increasing number of biopsies, yet little improvement is gained with more than four biopsies. Interestingly, biopsy variability of lymphocytic infiltrate differs considerably among breast cancer subtypes, with the human epidermal growth factor receptor 2-positive (HER2+) subtype having the highest variability. We subsequently identify a quantitative measure of spatial variability that predicts disease-specific survival in HER2+ subtype independent of standard clinical variables . Our study demonstrates how systematic methods provide new insights that can influence future study design based on a quantitative knowledge of tumour heterogeneity. Increasing evidence supports the use of lymphocytic infiltrate to predict prognosis and response to treatment in different cancer types1234577923Tissue microarray (TMA) has played an important role in morphological and molecular analysis of tumour lymphocytic infiltrate31213146161719We therefore proposed a new approach that is fully automated and objective to evaluate biopsy variability of lymphocytic infiltrate across 998 breast cancers. Our aims were: (1) to evaluate the amount of TMA cores required for a reliable estimate of lymphocytic infiltrate; (2) to quantify variability in lymphocytic infiltrate scoring using TMA cores across different breast cancer subtypes; (3) to define the clinical significance of such variability in lymphocytic infiltration.To evaluate if TMA cores can provide a reliable estimate of the degree of lymphocytic infiltration in the whole tumour, we developed a virtual biopsy approach and applied it to 998 primary, untreated breast tumours , Methods37n = 1, cor = 0.87 for n = 2, cor = 0.88 for n = 3), and this correlation reached a plateau at 0.96–0.98 when number of cores n exceeds 15  or whole-tumour section . As expected, we observed an increased correlation between core-based and tumour-based scores of lymphocyte ratio with more cores . This suggests that more cores are required to estimate lymphocytic infiltrate in HER2, possibly due to a higher level of intra-tumour heterogeneity in lymphocytic infiltrate , supporting that the low correlation between core-based and tumour-based scores we observed in HER2 tumours was not due to tumour size but likely reflected a higher level of intra-tumour heterogeneity  0.90 to 0.94) and n = 3 to n = 4 (AUC 0.94 to 0.96), but no difference between n = 2 and n = 3 . In the HER2 subtype, lower accuracy of core-based classification was found in general compared with the scores obtained from other subtypes combined . Again classification based on tumour-based scores was used as gold standard. When all subtypes were combined, we observed an improvement from p = 0.0087, hazard ratio (HR) = 0.32, 95% confidence interval (CI) = ; HER2: p = 0.047, HR = 0.51, CI = ; combined cohorts, High tumour-based lymphocyte ratio scores was associated with favourable disease-specific survival in Luminal A and HER2 subtypes but not Basal and Luminal B , and found that the frequency of skewness being prognostic was 100% upon resampling when more than 48 cores were used . We quantitatively defined variability in core-based lymphocyte ratio by examining the score distribution of 50 randomly sampled cores from each tumour. We used two different variability measures: (1) standard deviation to define deviation from mean, and (2) skewness to measure the degree of asymmetry in a distribution. First, we found that standard deviation was not associated with survival in any subtype. In contrast, high skewness was associated with poor prognosis in the HER2 subtype , multivariate analysis did not converge when analysis was performed on individual cohorts and further division of HER2+ samples into HER2+/ER+ versus HER2+/ER− subtypes was not possible. Therefore, multivariate analysis was performed using all HER2 samples (n = 110) to include node status, tumour size and grade, demonstrating that the ImmunoSkew score is independently prognostic in HER2 breast cancer  and HER2 status, molecular subtypes including PAM50 and IntClust integrative subtypes , we found that patients with high ImmunoSkew score had significantly worst prognosis compared with patients with low ImmunoSkew score, despite having aggressive treatments . Therefore, we expect a substantial improvement in the estimation of immune infiltrate with the use of 2 TMA cores compared with 1 core; the use of 4 cores represents a good trade-off between performance and the amount of tissue required; however, only marginal improvement in performance can be achieved with much more tissue.The use of TMA has contributed to many key discoveries in tumour immune response over the last decade3121314p > 0.05, p = 0.0087, HR = 0.32, 95% CI = ) and HER2 subtypes . Consistent with our findings above, we observed that more cores are needed for the HER2 subtype to gain consistent prognostic value compared with the Luminal A subtype (38 versus 25 cores). Our observation emphasises the need to control how sampling is performed especially for HER2 subtype. This appears to be in contrast to recent studies that estimate TILs from a single TMA core22n = 6: cor = 0.92, AUC = 0.96; other subtypes n = 4: cor = 0.92, AUC = 0.96; Interestingly, significant variation in concordance between core-based and tumour-based lymphocyte ratio was identified according to breast cancer subtypes. We found lower correlation between core-based and tumour-based scores in HER2 compared with other PAM50 intrinsic subtypes . HER2 tup = 0.045, HR = 2.03, CI = ; Validation: p = 0.018, HR = 4.57, CI = ). Multivariate analysis also demonstrated the value of the ImmunoSkew as a prognostic factor independent of other clinical parameters  in HER2 subtype . Combining ImmunoSkew with treatment regimen including chemotherapy and radiotherapy, we found that HER2 patients with high ImmunoSkew had significantly worst prognosis compared with patients with low ImmunoSkew, irrespective of treatments . High ImmunoSkew score reflects a strong regional segregation of immune cells, and its association with poor prognosis may indicate dysfunctional immune infiltrate. Given the increasing amount of evidence that immune infiltrated HER2+ tumours are more sensitive to chemotherapy and other therapies2126Our study to examine biopsy variability led to the discovery of a quantitative measure of spatial heterogeneity in lymphocytic infiltrate, namely the ImmunoSkew score. High ImmunoSkew was found to be associated with poor prognosis in the HER2 subtype  are more elongated. Our image analysis pipelineSegmentation was performed by employing Otsu thresholding followed by distance transform based watershed segmentation to separate the touching objects. Next, an expert pathologist hand annotated a set of 871 primitives (approximately 200 primitives from each of the four classes). For each of these hand annotated primitives, a dictionary of morphological and textural features was computed with an aim to represent each primitive in a high-dimensional feature space. Finally, a multiclass support vector machine (SVM) classifier with Gaussian kernel was trained to model the morphological differences among different tissue primitives. Once trained, the model was used to perform classification of all the primitives identified during stage 1 of the image analysis pipeline.The kernel smoothing was then performed for cells in spatial proximity to cancer cells to remove erroneous erratic classifications. Finally, a multiresolution hierarchical model was employed to provide further improvement in classification accuracy by identifying cancer cell clusters instead of individual cells as identified in the previous steps. The entire image processing pipeline was supplied as an R package CRImage.p < 0.0001). Automated score of lymphocytic infiltrate was defined as the percentage of lymphocytes in all cells by image analysis, and it was found to be highly correlated with pathological score . On average there were 56,815 (mean) cancer cells , 9,730 lymphocytes  and 11,766 other cells  in each tumour.To evaluate the accuracy of our image analysis pipeline, we performed three set of experiments: (1) 10-fold cross validation within the training set (90.1% accuracy)We generated virtual TMA cores from the H&E stained whole-tumour sections using the following computational algorithm.Get the first element of Count the number of cells within the circular region of radius If the total number of cells in the region is greater than Let Since a commonly used size of a TMA core is 0.6 mm, we fixed We derived the following quantitative measures based on image analysis:The ratio of the number of lymphocytes to the total number of cells present in all sections from a single tumour.The ratio of the number of lymphocytes to the total number of cells present in a TMA core.The skewness of the distribution of core-based lymphocyte ratio in a tumour as a measure of spatial heterogeneity in lymphocytic infiltrate. Mathematically,i is the i-th TMA core which varies between 1 to N, and N is the total number of virtual TMA cores.Where The standard deviation of the core-based lymphocyte ratio as another measure of spatial heterogeneity in lymphocytic infiltrate.n cores and benchmarked the average lymphocyte ratio scores of these cores against the tumour-based scores. This was repeated 100 times for n = 1–50 for each tumour. For samples with less than 50 TMA cores, we performed sampling with replacement. To enable fair comparison across tumours during the survival analysis, n TMA cores were randomly sampled from each tumour and the average lymphocyte ratio score was evaluated in survival analysis. This was repeated 50 times for each n, n = 1–50. To ensure the results were comparable, we only used the subset of samples with at least 50 TMA cores.To compare core-based scores with tumour-based scores, we randomly sampled p-value < 0.05 was considered to be statistically significant.Associations between continuous variables were tested by Pearson’s correlation coefficient. Kruskal-Wallis test was used to assess the relationship between continuous and categorical variables. Two-sided paired t-test was used to assess the difference between the two matched continuous variables. To estimate the accuracy of core-based lymphocyte ratio in predicting immune abundance, we used AUC as a measure of accuracy, and random sampling to estimate 95% CIs. Univariate survival analysis was performed with disease specific 10-year survival data using the Kaplan-Meier method, where p-value was calculated using log-rank test. To dichotomise continuous measures including the ImmunoSkew score, we systematically evaluated every hundredth value of each of the above stated measure in the 20–80% range using univariate analysis. A value that best dichotomised survival in the Discovery cohort was selected as the cut-off value and was evaluated in the Validation cohort. Cox proportional hazard model was fitted to the survival data and HR as well as 95% CI was computed to determine the prognostic value. Multivariate analysis was performed using Cox proportional hazard model for predictors found to be statistically significant in univariate analysis. For all statistical tests, In summary, by employing digital image analysis and machine learning, we sought to determine the number of cores needed for an estimate of immune infiltrate in breast tumours. Virtual TMAs were constructed from whole-tumour histology sections and systematically compared. We revealed that HER2+ breast tumours have a higher degree of spatial heterogeneity of lymphocytic infiltrate and therefore require more TMA cores compared with the tumours of other subtypes. This biological nature of HER2 subtype further motivated the discovery of ImmunoSkew, a quantitative measure of intra-tumour heterogeneity of lymphocytic infiltrate that can potentially serve as a prognostic biomarker in HER2 breast cancer based on routinely generated histology samples.How to cite this article: Khan, A. M. and Yuan, Y. Biopsy variability of lymphocytic infiltration in breast cancer subtypes and the ImmunoSkew score. Sci. Rep.6, 36231; doi: 10.1038/srep36231 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.