Somatic hypermutation (SHM) status provides an important prognostic indicator for chronic lymphocytic leukemia (CLL), a very common type of mature B-cell leukemia. Owing to the adverse prognosis associated with an unmutated immunoglobulin heavy chain variable (IGHV) status, SHM testing is performed as a standard of care in CLL. Conventionally, SHM testing has been performed using labor intensive and primarily analog Sanger sequencing method following PCR amplification of the clonal immunoglobulin heavy chain gene rearrangements in CLL cells. In comparison, recent availability of next generation sequencing (NGS) allows more versatile detection and direct identification of clonal immunoglobulin gene rearrangements in neoplastic B-cell populations. The ability to identify specific clonal IGHV signature(s) in both baseline (diagnostic) and post-treatment settings enables unique clinical applications of NGS such as determination of SHM status, minimal residual disease (MRD) monitoring, clonal heterogeneity and B cell receptor IG stereotypy. We provide a review of current practices and recommendations for SHM determination using NGS including examples of difficult cases. The IGH region genes encode the antigen binding variable (V) and isotype-specific constant (C) regions of immunoglobulin (IG) heavy chain proteins and their combinations generate tremendous sequence diversity that is important to effectively identify a variety of antigens . The immn = 38–46), IGHD (n = 23), and IGHJ (n = 6) genes  genes . Only thIGH gene rearrangements in B-cells undergo additional somatic mutations on exposure to antigen as part of the germinal center reaction in lymphoid tissues. These acquired or somatic hypermutations (SHM) are mediated by the enzyme activation induced cytidine deaminase (AICD) and mainly involve nucleotide base changes in the CDR regions. SHM occurs in antigen-activated germinal center B cells and improves the fitness of cells involved during a polyclonal immune response through the process of affinity maturation, which optimizes antigen epitope binding by immunoglobulins . SHM is As a result of SHM, the nucleotide sequences of IGH gene rearrangements in affected B-cells are different from their corresponding germline counterparts. In the setting of CLL, SHM status is used to define distinct prognostic subgroups. An “unmutated” clonal IGH gene rearrangement is defined as having very high nucleotide sequence identity to its closest germline IGHV gene. “Mutated” CLL in contrast is characterized by IGHV sequence deviating by some percentage relative to its germline reference. By convention, the SHM status in CLL is reported on the dominant clonal population recognized at diagnosis and is defined as a deviation of >2% (mutated status) or ≤2% (unmutated status) from the closest germline IGHV reference sequence. As confirmed by several studies , unmutat1) and IgBlast2 have greatly facilitated this analytic process. Despite being in use for many years, this assay is still not uniformly performed in many clinical laboratories due to limitations of labor-intensiveness, technical complexity and limited scalability. While this technique generally works well in cases with a single clonal IGH rearrangement, cases with >1 rearrangement, as seen in up to 10% of cases , offers the ability to unambiguously determine the individual clonal sequences and their relative proportions. The ability to determine specific sequences of each of the clones provides a better understanding of the intra-tumor and inter-tumor heterogeneity and clonal evolution. The ability to determine the specific sequence of the clone in the pre-treatment setting allows detection of measurable minimal residual disease (MRD) following therapy. In recent years, many groups have shared their experiences of using NGS assays for SHM testing in CLL . Below wThe use of NGS for SHM analysis was studied by Minimal residual disease assessment in CLL can be done using NGS, typically using the same clone identified at diagnosis for SHM determination . As compNext generation sequencing based analysis in CLL can also be employed for receptor stereotypy analysis. Receptor stereotypy in CLL is defined as the presence of almost identical BCR immunoglobulin (IG) peptide motifs in unrelated CLL patients. Given the massive diversity of IG rearrangements, the statistical probability of any two CLL patients sharing the same or highly similar clonal IG gene products should be exceedingly small, but surprisingly, one-third of CLL patients can be grouped into stereotyped subsets based on similarities of the IGH CDR3 heavy chain variable region. Receptor stereotypy subset analysis is a further refinement of clinical and prognostic subgroup assessment and improves upon the current binary designation of mutated and unmutated CLL. Recent studies have shown that unrelated CLL patients with near-identical BCR IGH profiles or identical receptor stereotypes are comparable to each other in terms of their clinical characteristics, genetic lesions, epigenetic changes, clinical course, and clinical outcome; for example, the largest stereotyped subset is subset #2 (IGHV3-21/IGLV3-21), which exhibits spliceosome genetic alterations, aggressive clinical course and poor outcome irrespective of SHM status. Thus, receptor stereotypy can be a basis of defining more distinct or homogeneous subgroups beyond SHM analysis. In addition, subsets sharing the same stereotype may potentially be candidates for optimized therapeutic approaches .Relatively unbiased library preparation  and high throughput sequencing of all possible VDJ rearrangements are requirements for accurate assessment of clonality using NGS. These seemingly straightforward goals are, however, challenging to achieve owing to the technical and biologic variability of IGHV rearrangements. Briefly, in comparison to the Sanger sequencing based IGHV SHM analysis, evaluation by NGS is typically performed by amplifying extracted DNA using IGH family specific leader and consensus JH primer PCR master mixes. PCR products are purified using bead-based methods, and the resulting libraries normalized and then pooled for sequencing. Key considerations for NGS-based SHM testing are summarized below.∗01 or V4-34∗04). Practically, this situation does not significantly affect the accurate provision of SHM status in most cases, but requires consideration when interpreting results of FR1 PCR. Conversely, SHM poses a potentially significant technical problem for target enrichment in B-cell IGH analysis. Highly mutated regions in IGH genes may not be recognized during the PCR amplification step due to inefficient consensus primer binding; such clones are thus not represented for analysis.As per the ERIC guidelines,  leader pIn summary, for a well-designed assay it is important to include a relatively comprehensive strategy employing either multiple primer sets binding to different locations for each V or J segment; longer primer designs with anchor points at highly conserved positions; or use of full length cDNA with primers positioned outside the V and J regions, which are less likely to undergo SHM . It is iAs stated, in SHM assays performed by NGS, it is important to perform analysis on full-length reads so that a true representation of each sequence is analyzed. The assay needs unambiguous reads of around 350 base pair (bp) length containing the entire IGHV and junction region, which may be obtained in most cases, although samples with suboptimal DNA quality or degradation may result in failure to achieve optimal read lengths. The commercial LymphoTrack assay  with leader primers yields paired end sequence reads of approximately 300 bp length each (301 × 2 reads + 8 bases for leader primer = 610 bp) on the Illumina Miseq platform, so that the entire IGHV region of a given rearrangement can be sequenced in a single fragment. The final sequence is also bioinformatically evaluated for features of a productive rearrangement . In essence, complete in frame reads from the primer region across to the junctional Trp118 would be considered productive. McClure et al., reported an average of 350 bp read length using Ion Torrent PGM platform and Ion 400-bp kit using FR1 PCR primers . The anaThe NGS platform selection is based on various factors like desired turnaround time, nature and volumes of samples, complexity of genetic variants to be tested, pre-existing platform in the lab and the ability to achieve a relatively simple workflow in the clinical laboratory, to name a few. Sequencing platforms that support longer reads and have lowest possible error rate will be preferred as they will provide an accurate assessment of SHM. Platforms with higher error rate in certain genomic context and shorter read length are likely to yield a false elevation in SHM%. Platforms with higher AQ score cut-offs (AQ30 or better) for sequencing quality are likely to provide more accurate sequencing data. The rate of true and reproducible mutations over background sequencing error-rate needs to be carefully validated. Currently, the most popular platforms that can achieve longer read lengths using “short read” methods include the Illumina MiSeq and Ion Torrent instruments. In general, Illumina sequencers can achieve sufficient bidirectional reads (2 × 300 bp) with a lower sequence error rate compared with Ion Torrent, but both are acceptable for use with appropriate validation. The advent of long read technology approaches  may provide alternative means of interrogating the full length of IGH rearrangements, but these systems are currently not optimally suited for clinical use or have unacceptably high raw sequencing error rates.The commercially available positive  and negative  controls supplied with the assay kit such as Lymphotrack should always be run with every batch. The polyclonal controls will help in checking the performance of all the primers used in the assay. In case all the families are not being represented in the commercially available polyclonal control, in-house polyclonal samples may be pooled and validated for use, to have a better V region family representation . The iniFresh  vs. fixed  specimens: Currently, commercial IGHV SHM assays used as lab developed tests (LDT) are validated for fresh sample types including PB and BM only, and provided the CLL population constitutes at least 10% of the sample. The minimum tumor criteria are essential to avoid any false negative result. Use of FNA or fresh frozen samples is feasible, but requires additional validation. Use of the assay for SHM analysis is currently not recommended for FFPE samples due to degradation or cross-linking of DNA.Current NGS based SHM commercial assays are most often validated for genomic DNA (gDNA) use in majority of clinical labs. A total of 500 ng of input gDNA input is required. The degree of SHM is frequently quantified by comparing the resulting IGHV sequence with the closest consensus germline sequence in the IMGT /V-QUEST (V-QUEry and STandardization) database (see text “footnote 1”). In addition, IMGT provides advanced tools for identifying potential insertion/deletion events in the target sequence. An accurate and detailed sequence analysis of V domains (using IMGT/V-QUEST) and CDR3 regions  can be performed using the online tools developed by IMGT . IMGT/HiAlternatively, the IG sequence analysis tool IgBLAST (see text “footnote 2”) can be used to view matches to the germline V, D, J genes, junction details, IGV framework and complementarity determining regions. IgBLAST can be used to analyze both nucleotide and protein sequences. It can process sequences in batches. IgBLAST can also be used to do simultaneous searches against the germline and local IGH databases to reduce the chance of missing germline V gene matches . Despitehttp://www.vidjil.org/. Antigen Receptor Galaxy  is another open-source web-based tool having capabilities to analyze and visualize the high throughput data of immune repertoire including rearranged V(D)J genes, SHM, and class switch recombination J immune repertoire sequencing data . The algbination .http://bioinformatics.fh-hagenberg.at/immunexplorer/.Schaller et al.,  developehttp://bat.infspire.org/arrest/interrogate/ by Bystry et al. . The analytical core uses “data.table” R package for efficient data handling and can maintain sufficient responsiveness with data generated from thousands of clonotypes and millions of reads. Another favorable aspect of the expanded ARResT toolkit is the ability to upload IGH sequences to query a database (ARResT/AssignSubsets) for common stereotyped BCR IG subsets of prognostic significance.Another web-based, interactive application “ARResT/Interrogate” has been made freely available at y et al. . This apAfter massively parallel sequencing, the output is analyzed by the bioinformatics pipeline. The paired-end read data are combined, demultiplexed, and adaptor trimmed to generate FASTA and FASTQ files. Primer sequences are trimmed, and exactly similar sequences (duplicates) are removed. Each unique sequence is then aligned using either the commercially provided software, IMGT/V-QUEST or IgBLAST. The output from Lymphotrack for example is grouped based on different IGHV-J combinations, and the final data is loaded into MySQL database. Nearly identical sequences differing by ≤2 bases are merged to accommodate for PCR or sequencing errors. The top 10 merged read sequences are then returned for further analysis, but access to all sequencing data is maintained if required.The entire IGHV sequence including the CDR3 is compared with the closest germline reference sequence for calculation. The sequence analyzed must include the conserved Cys 104 and Trp 118 residues, and is checked to ensure that the reading frame is preserved  and that no stop codons are encountered. The percentage identity is calculated using a ratio between the total of IGHV region mutations in the IGHV-D-J rearranged sequence, and the length of the most homologous germline IGHV gene:The 98% cutoff to differentiate the cases into mutated and unmutated groups is arbitrary and was originally selected from earlier CLL studies to exclude the possibility of polymorphic variant sequences. It was chosen by convention to avoid the need to analyze the corresponding native germline sequence in each patient. Although other cutoff values  for homology determination have also been suggested, it is recommended to follow the 98% cut off for the sake of data uniformity and reporting . Recentl4) in 2007 and then later updated in 2011 and 2017 (Consensus recommendations for SHM were initially provided by ERIC (European Research Initiative on CLLand 2017 . These gThe following three cases highlight the application of NGS for IGH SHM analysis.The detailed Lymphotrack output for case 1 can be seen in The detailed output for case 2 can be seen in There is a polyclonal pattern with several dominant clones in the visual display of the Lymphotrack data output . The anaDevelopment of consensus validation guidelines, practice standards and specific quality assurance measures will be required to support increasing adoption of NGS-based SHM testing in clinical laboratories. The NGS analysis will likely result in detection of higher proportion of patients with multiple productive IGHV subclones, as seen by KP, SG, and DV contributed conception and design of the study. SG organized the reference database and wrote the first draft of the manuscript. KP and DV wrote sections of the manuscript, edited the manuscript, and contributed the figures. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite concerted global efforts, only a few targeted therapeutics, such as remdesivir, are available to help prevent or treat this disease [Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that mysteriously emerged in late December 2019 in Wuhan City, China . SARS-Co disease , therefo disease . Convale disease . Althoug disease –12. Alth disease –10. As nPrior to the antibiotic era, serum (plasma minus clotting factors) therapy was widely used to treat a range of infectious diseases such as scarlet fever and pneumococcal pneumonia. In 1890, the physiologists von Behring and Kitasato used blood serum from immunized animals to treat diphtheria and tetanus ; subsequThe convalescent plasma therapeutic approach is based on the principle of passive antibody therapy, a short-term strategy whereby antibodies from the blood of someone who recovered from an infection can be administered to protect or treat another person , 21. EffDespite the high rate of SARS-CoV-2 infection, the relatively low mortality rate provides a rich pool of donors . HoweverConvalescent plasma with neutralizing antibodies is currently being used for investigational purposes in the COVID-19 pandemic, and preliminary results from 2 small studies performed in China are encouraging. A pilot study exploring the feasibility of convalescent plasma transfusion to rescue a group of 10 patients with severe disease showed that 1 dose (200 mL) of convalescent plasma with high neutralizing antibody titers was well tolerated, resulted in disappearance of viremia, and improved clinical symptoms in all patients within days of administration . SimilarAlthough convalescent plasma therapy is considered a relatively safe therapeutic modality, there are some potential risks . One theThe success of convalescent plasma therapy hinges on the availability of plasma with high concentrations of antibodies, which may not be a major limitation for COVID-19 because SARS-CoV-2 has been shown to elicit high neutralizing antibody titers in recently convalesced individuals –9. ApherAlthough large-scale randomized clinical trials will ultimately confirm the safety and efficacy of convalescent plasma therapy for COVID-19, a recent safety study by Joyner and colleagues  providedThe global reach of the COVID-19 pandemic and the desperate need for effective treatments have provided an impetus to develop convalescent plasma therapy into a viable  treatment option particularly for the critically ill. Although its efficacy and safety have not yet been fully proven, convalescent plasma therapy for COVID-19 patients is projected to be a safe and potentially effective therapy for prophylaxis and treatment. However, it is critically important to perform rigorous randomized controlled trials to confirm efficacy and safety and to provide evidence for improved meaningful clinical outcomes. Nevertheless, despite the nuanced challenges, the substantial evidence of benefit with use for prior viral infections offers strong precedent for convalescent plasma as a therapeutic approach. Importantly, efforts should be focused not only on evaluation of the feasibility of plasma treatment for infectious diseases but also ensure that use of convalescent plasma therapy takes place according to ethical and controlled conditions.


Carcharhinus falciformis, previously surveyed with the mtDNA control region in the Atlantic and Indian Oceans. Pool-seq methods were able to recover the entire mitochondrial genome as well as thousands of nuclear markers. This volume of sequence data enabled the detection of population structure between regions of the Atlantic Ocean populations, undetected in the previous study . Our results reinforce the conclusion that sampling the mitochondrial control region alone may fail to detect fine-scale population structure, and additional sampling across the genome may increase resolution for some species. Additionally, this study shows that the costs of analyzing 4,988 loci using pool-seq methods are equivalent to the standard Sanger-sequenced markers and become less expensive when large numbers of individuals (>300) are analyzed.Conservation genetic approaches for elasmobranchs have focused on regions of the mitochondrial genome or a handful of nuclear microsatellites. High-throughput sequencing offers a powerful alternative for examining population structure using many loci distributed across the nuclear and mitochondrial genomes. These single nucleotide polymorphisms are expected to provide finer scale and more accurate population level data; however, there have been few genomic studies applied to elasmobranch species. The desire to apply next-generation sequencing approaches is often tempered by the costs, which can be offset by pooling specimens prior to sequencing (pool-seq). In this study, we assess the utility of pool-seq by applying this method to the same individual silky sharks,  Many elasmobranchs around the globe have experienced devastating population declines due to overfishing in both target and non-target fisheries . These sGenetically distinct populations are isolated management units known as stocks; however, stocks can be defined on a smaller scale than genetic populations through other criteria, such as an exclusive economic zone boundry . ReducedFor the past two decades the standard for examining population structure in elasmobranchs has been a section of the mitochondrial genome, usually the control region (mtCR) . Though High-throughput sequencing is a powerful tool for revealing fine-scale population structure that may be missed by single locus studies . HoweverCarcharhinus falciformis ) is the second most commonly harvested shark on Earth , the North Atlantic , Brazil , and the Red Sea . These sample sizes are slightly lower than A total of 143 silky shark fin clips or muscle sections were sampled from commercial or artisanal fisheries across four geographic regions and are the same samples examined in DNA was extracted using Qiagen DNeasy Blood & Tissue kit , following manufacturer protocols. Extracted DNA quality was assessed visually by gel electrophoresis and imaged using Gel Doc E-Z System . Only DNA aliquots with strong genomic DNA bands were further processed, while degraded or overly digested DNA was discarded. Aliquots of high-quality DNA were quantified using an AccuClear Ultra high sensitivity dsDNA quantitation kit  and a SpectroMax M2 . Libraries were pooled with an equal amount of DNA (ng/µl) contributed per individual to minimize individual contribution bias, totaling 2,000 ng of DNA per library. Numbers of individuals per pool are displayed in Carcharhinus falciformis was used as a reference (GeneBank accession number KF801102). Raw paired-end reads were trimmed with TRIMMOMATIC, mapped to the mitochondrial genome reference BWA , and variants called using the dDocent bioinformatics pipeline, modified for pool-seq .MultiQC was used to assess sequence quality scores, sequence length distributions, duplication levels, and overrepresented sequences . To analhttp://ddocent.com/assembly/). Before assembly reads were trimmed using default settings and then an overlap (OL) assembly was performed, followed by clustering with CD-HIT with a–c parameter of 90% similarity. For mapping using BWA  all match, mismatch, and gap open penalty score parameters were also default settings. Different parameters were tested during optimization but did not improve mapping. Within-pool (K1) and between-pool (K2) minimum locus depth values selected for the de novo assembly did impact the results. dDocent provides graphical outputs to help select these values; however, testing a few different values of each is recommended to fully explore the potential of the data by balancing number of contigs by coverage depth (see ddocent.com/UserGuide for details). Selected values for K1 and K2 were 3 and 3 respectively. Once assembled, sequences were mapped, SNPs were called within the dDocent pipeline using FreeBayes, modified for SNP calling in pools (https://github.com/ekg/freebayes). Any contigs that aligned to the mitochondrial genome were removed from this nuclear dataset. The contigs that aligned specifically to the mitochondrial control region were saved for SNP validation to directly compare the results from this pool-seq approach to those previously reported by The bioinformatics pipeline included dDocent followed by AssessPool. Given that no reference genome was available, a reference was constructed using the dDocent de novo assembly and optimized utilizing the reference optimization steps provided on the dDocent assembly tutorial , and SNPs from these data were compared directly to SNPs called within the control region of the mitochondrial pool-seq data generated here. Concordance of this validation set of SNPs was determined by Mantel test in R . INDELs and multiallelic SNPs remain a challenge for quantification software, so we restricted our analysis to biallelic loci . AssessPFst values were all significant  than any inter-Atlantic comparison (ranging from 0.029 to 0.135). However, all comparisons within the Atlantic still showed significant STF values, the highest being between the North Atlantic and Brazil, and the lowest between Brazil and Gulf of Mexico  revealed 804 variable sites: 681 biallelic and 17 multiallelic SNPs. Because coverage in this dataset was fairly low on average, most of these SNPs did not meet the filter threshold. After further filtering for the highest quality markers, 30 SNPs were selected to calculate allele frequencies. Pairwise nificant . The Redf Mexico .STF values between locations than the mitochondrial data, yet all comparisons were still significant  was observed between Gulf of Mexico and the Red Sea, whereas the lowest (FST = 0.014) was between the North Atlantic and Brazil, which had the highest STF value within the Atlantic for the mitochondrial data.Our nuclear data showed 4,988 variants, of which 3,422 were biallelic SNPs and 151 were multialleleic SNPs. A total of 346 SNPs remained after the same filtering process for the highest quality SNPs was applied as for the mitochondrial genome. Nuclear markers showed lower nificant . The RedST value . The higFST values and should not bias interpretations of population structure , and comparisons between the Red Sea and all three Atlantic populations showed the same relative magnitude between both methods.SNPs called in the mitochondrial control region using the pool-seq protocol were compared with those reported in tructure . Three Structure . Despitehttps://usegalaxy.org/) or CyVerse (https://cyverse.org/). It is also important to note that the choice of pool-seq methodology has many caveats, which are discussed in greater detail in the ‘considerations on pool-seq’ section of the discussion below.The findings for cost analysis indicate that pool-seq reaches a threshold at approximately 300 individuals, after which this approach offers cheaper results than individual Sanger sequences. Furthermore, the cost is only twice as expensive at just over 100 individuals . The pooElasmobranchs are being harvested at unsustainable levels in several commercial fishing industries around the world. A fundamental step in successful management of any species is resolving population boundaries so they can be managed on a genetic stock-by-stock basis. As genetic sequencing technologies advance, there is greater opportunity to detect even small-scale genetic differences between populations. When these differences amount to statically significant allele frequencies at the population level, this indicates limited exchange among distinct stocks.Here, we validate the utility of pool-seq using the same individuals as a previous study  and showOne advantage of this pool-seq approach is that we recover SNPs throughout the entire mitochondrial genome along with thousands of additional nuclear loci that together provide greater statistical power to detect finer scale population structure . The pooIn this case, pool-seq lived up to the promise of increased power to detect fine-scale structure, but does it live up to the promise  of beingAs with any sequencing technique, there are still several factors to consider before deciding if pool-seq is appropriate for a particular study. Multiple reviews have been published on high-throughput and pool-seq approaches demonstrating pros, cons, and considerations with these methods, which are beyond the scope of this study. Interested readers should consult STF from pooled data remains a subject of some debate, and new approaches and bias corrections are being actively developed . Therefore, only those Fst values calculated by PoPoolation2 are reported in the main text for ease of presentation.Pooling assumes individuals are from the interbreeding individuals within a single population of the same species. Therefore, care needs to be taken to avoid cryptic species, combining multiple populations (Wahlund effect), or other unintentional bias when selecting individuals to pool . For wideveloped . To accoeveloped  package eveloped  that explues see , howeverThough pool-seq has been shown to be an affordable and reliable tool for population genomics , projectCetorhinus maximus, Prionace glauca, Acanthocybium solandri, The finding of population structure on the scale of North Atlantic/Gulf of Mexico/Brazil is nearly unprecedented for a pelagic shark. Population structure in globally distributed sharks is typically detected on a scale of ocean basins  are listed separately for the Clarke study because MAC < 3 were filtered out for pool-seq data.Click here for additional data file.10.7717/peerj.10186/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10186/supp-3Supplemental Information 3STF matrices produced among the Popoolation approach and Poolfstat approach shows strong correlation . This indicates, at least for this study, the difference in the analytical approaches does not make an overall difference in this study’s conclusions. Additionally poolfstat is not yet capable of calculating p-values for confidence, so for the sake of this study only FST values calculated from Popoolation are used. This may need to be re-evaluated for studies with more pooled regions examined or larger sample sizes. Regional Abbreviations: GM = Gulf of Mexico, NA = North Atlantic, BR = Brazil, RS = Red Sea.Comparison of Click here for additional data file.10.7717/peerj.10186/supp-4Supplemental Information 4Click here for additional data file.


A 20-year-old man with a reported history of asthma presented to the emergency department in cardiac arrest presumed to be caused by respiratory failure.The patient was discovered to have central airway obstruction and concomitant superior vena cava compression caused by a large mediastinal mass—a condition termed mediastinal mass syndrome. While the patient regained spontaneous circulation after endotracheal intubation, he was challenging to ventilate requiring escalating interventions to maintain adequate ventilation.We describe complications of mediastinal mass syndrome and an approach to resuscitation, including ventilator adjustments, patient repositioning, double-lumen endotracheal tubes, specialty consultation, and extracorporeal life support. Masses of the mediastinum are rare. In the Framingham Heart Study, mediastinal masses were present in 0.9% patients2Compression of the vena cava may result in superior vena cava (SVC) syndrome, which is characterized by intermittent swelling of the face and oropharyngeal structures, facial rubor, and/or venous congestion. Compression of pulmonary arteries may mimic massive pulmonary embolism (PE) causing acute right heart failure or circulatory collapse secondary to severely impaired pulmonary venous return, drastically reducing left ventricular preload. Collectively, this constellation of pathologies is known as mediastinal mass syndrome.6A young man with a reported history of asthma presented to the emergency department (ED) with ongoing cardiopulmonary resuscitation (CPR) after suffering from cardiac arrest on the sidewalk in front of the hospital. Family of the patient reported a two-week history of worsening dyspnea prior to his presentation.Initial examination revealed an apneic patient with distended veins across his upper chest and neck suggestive of obstructive pathology as a potential etiology of arrest, in addition to the history of respiratory distress. Medical providers inserted one 14-gauge angiocatheter in the bilateral second intercostal spaces for chest decompression, but no rush of air was noted. The cardiac monitor showed an initial rhythm of wide-complex pulseless electrical activity. With CPR ongoing, the patient was intubated without complication via direct laryngoscopy and manually ventilated with return of spontaneous circulation (ROSC) after approximately six minutes of CPR. No adjuvant medications were given prior to ROSC as the first four minutes of CPR were provided outside of the ED, and intubation with subsequent ROSC was achieved simultaneously with successful peripheral vascular access.2) device. Initial arterial blood gas (with reference ranges in parentheses) after intubation showed a pH of 6.97 (7.35–7.45); pCO2 of 104 millimeters mercury (mm Hg) (reference range 35–45 mm Hg); oxygen (pO2) of 44 mm Hg (75–100 mm Hg); and bicarbonate (HCO3) of 23 milliequivalents per liter (mEq/L) (22–26 mEq/L). After ROSC, his oxygenation was 95%. The patient was given nebulized albuterol and intravenous (IV) magnesium and methylprednisolone for presumed respiratory arrest secondary to severe asthma.No oropharyngeal or airway abnormalities were noted other than minimal edema of the epiglottis. Endotracheal tube (ETT) placement was verified by auscultation, condensation within the tube, and colorimetric carbon dioxide (CO2O) indicating major airway resistance. The patient’s oxygenation decreased to 82%. However, manual bag ventilation was subsequently performed without significant difficulty and with improvement of oxygenation to 95%. Point-of-care ultrasound demonstrated lung sliding bilaterally and showed a heterogeneous mass overlying the heart and obscuring parasternal views. No pericardial effusion was noted. An electrocardiogram demonstrated sinus tachycardia without obvious findings to suggest pericarditis or acute ischemia. A portable chest radiograph was obtained, which showed the ETT in appropriate position and revealed a large, central opacity obscuring the normal airway anatomy and cardiac silhouette  elevated to 90 centimeters of water (cm Hlhouette .20. Computed tomography (CT) demonstrated a large mass in the anterior mediastinum, with almost complete obliteration of the SVC with significant collateralization, as well as severe stenosis of the airways at the level of the lower one-third of the trachea, carina, and main-stem bronchi  attempting to bypass the obstruction; the ventilator was reconnected after manual bag ventilation and ventilated the patient appropriately with the original setting of positive end-expiratory pressure (PEEP) 5 cm H bronchi .2O, and providers raised the patient to a semi-recumbent position, which allowed the ventilator to oxygenate with PIPs in the 40s. Prior to intensive care unit admission, a cooling catheter was placed in the common femoral vein. Repeat blood gas showed improvement with a pH of 7.33 and downtrending lactate.While the patient was undergoing CT the ventilator again failed secondary to increased PIPs, and manual bagging was reinitiated. PEEP was increased to 10 cm HWhat do we already know about this clinical entity?Mediastinal masses are rare and can become large enough to cause compression of the trachea or heart. This is called “mediastinal mass syndrome (MMS).”What makes this presentation of disease reportable?MMS led to cardiac arrest in a young, otherwise healthy patient who was difficult to ventilate secondary to tracheal compression.What is the major learning point?Simple steps such as patient repositioning and increasing positive end-expiratory pressure may be life saving in cases of MMS.How might this improve emergency medicine practice?Providers should be aware of MMS, the steps to correct resulting central airway obstruction and also consider extracorporeal membrane oxygenation.The patient regained consciousness after cooling and rewarming but demonstrated diffuse myoclonus affecting the diaphragm, preventing extubation, and ultimately required tracheostomy.Tissue sampling of the mass revealed a large B-cell lymphoma, and chemotherapy was started. He subsequently developed massive hemorrhage from his right common carotid artery and jugular vein due to malignant erosion into these vessels. He suffered a subsequent arrest and was resuscitated in the operating room with ligation of the bleeding vessels but had diffuse brain injury on magnetic resonance imaging (MRI). His family agreed that further treatment was unlikely to lead to meaningful recovery, and he died on hospital day 52 after care was withdrawn.In regard to anterior mediastinal masses, the most common etiologies are taught as the “4 Ts”: thymoma, thyroid, teratoma, and “terrible” lymphoma. We describe cardiopulmonary arrest in an otherwise healthy young man due to an undiagnosed mediastinal mass. Although the patient regained spontaneous circulation after his airway was secured by endotracheal intubation, he remained difficult to ventilate due to lower airway compression. Mediastinal mass syndrome can lead to arrest due to compression or obstruction of critical structures, including airway obstruction, as in our patient. Circulatory obstruction leading to obstructive shock is possible due to massive PE, pericardial effusion causing tamponade physiology, direct compression of the heart by the mass, and positional SVC syndrome causing sudden decrease in cardiac venous return.DOPES”: Dislodgement/displacement of the ETT or cuff; Obstruction of the airway or ETT; Pneumothorax; Equipment failure (of the ventilator or tubing); and “Stacking” (referring to auto-PEEP or “breath-stacking).”20, but 30–40 is considered acceptable. In our patient, PIP was markedly elevated indicating high resistance to airflow through the tracheobronchial tree, ETT or ventilator tubing which caused ventilatory failure, as opposed to elevated PIP and plateau pressures, which would have indicated a lung compliance issue. High PIP in the setting of normal plateau pressures indicates obstruction and several causes of obstructive physiology must be evaluated. These include the following: kinked ventilator tubing; twisted ETT or ETT partially occluded by blood, mucus, etc.; patient biting ETT; bronchospasm/asthma; and trachea or large bronchus partial occlusion by mass or mucus plugging. 8In the case of the decompensating ventilated patient, providers should first assess for complications arising from mechanical ventilation in a stepwise fashion to aid in decision-making. One popular mnemonic for this approach is “Our patient required advancement of the ETT past the obstructed segment, PEEP, and repositioning to maintain adequate ventilation. Review of the literature indicates these and several other interventions may alleviate central airway obstruction. Repositioning the patient from supine to a semi-recumbent or upright position may both relieve compression of the central airway and increase venous return to the right heart, increasing preload.Passage of a double lumen or wire-reinforced ETT, or placement of an endotracheal stent via bronchoscopy, can also be performed to relieve airway obstruction. Surgical or radiologic removal/debulking of the offending mass may be necessary in the subacute phase of care.In addition to airway compression, patients with large mediastinal masses are also at high risk of SVC syndrome. The literature advocates for obtaining vascular access in the lower extremities such as a femoral central line in cases where the SVC may be severely stenosed or occluded.15Cardiac arrest from mediastinal mass syndrome is rare and can be difficult to manage. Effective management of airway obstruction and adequate ventilation depend on stenting the airway to relieve obstruction. In the emergent setting, options are limited, and relieving airway obstruction must be accomplished with available tools, namely endotracheal intubation, patient repositioning, and positive end-expiratory pressure. Specialty consultation may be both helpful and necessary, and providers should consider ECLS in the patients who remain unstable. In patients with high suspicion for SVC syndrome providers should also consider obtaining vascular access in the lower extremities as well as potential specialist consultation for endovascular stenting of the SVC.


Erythema multiforme (EM) is an acute, self-limiting hypersensitivity reaction that ranges from a mild cutaneous, exanthematous variant with minimal oral involvement to a progressive fulminating severe variant with extensive mucocutaneous epithelial necrosis (SJS & TEN). Knowing the etiological spectrum is the key for management in EM where sometimes leads to mortality. There are no specific diagnostic tests for EM, diagnosis is mainly clinically supported. Here in we report series of cases of EM with different etiological spectrum. EM is an acute, recurrent, self-limited, inflammatory mucocutaneous disease that manifests on the skin and often on the oral mucosa which occasionally can give rise to systemic upset and possibly compromise life . The skiCase 1: a 24-year-old male patient reported with a complaint of extensive painful ulcers and difficulty in eating since 5 days. Significant history of throat infection and common cold two weeks back for which he took azithromycin and diclofenac sodium prescribed by the local private practitioner, subsequently he developed irregular ulceration and hemorrhagic crust on the lips  EM is a hypersentivity reaction. 2) 80% of the cases have HSV (herpes simplex virus) superimposition. 3) Pathagnomic feature of EM - necrotic tissue tags, blood encrustations, typical “target” lesions. 4) Has inclination towards adults, rare in children and elderly people. 5) No specific diagnostic tests, clinical presentation itself gives the diagnosis. 6) Sometimes, clinical features are superimposed with other etiological features, where the proper diagnosis plays vital role in the management.EM as known by its hypersensitivity reaction, it can be precipitated by any of its etiological agents. Recognition and removal of the aetiological agent is crucial step in the management of EM. Early diagnosis remains essential to promptly initiate appropriate management and proper follow up.


Breast cancer is the most common cancer among women worldwide. Although the five-, ten- and fifteen-year survival rates are good for breast cancer patients diagnosed with early-stage disease, some cancers recur many years after completion of primary therapy. Tumor heterogeneity and clonal evolution may lead to distant metastasis and therapy resistance, which are the main causes of breast cancer-associated deaths. In the clinic today, imaging techniques like mammography and tissue biopsies are used to diagnose breast cancer. Even though these methods are important in primary diagnosis, they have limitations when it comes to longitudinal monitoring of residual disease after treatment, disease progression, therapy responses, and disease recurrence. Over the last few years, there has been an increasing interest in the diagnostic, prognostic, and predictive potential of circulating cancer-derived material acquired through liquid biopsies in breast cancer. Thanks to the development of sensitive devices and platforms, a variety of tumor-derived material, including circulating cancer cells (CTCs), circulating DNA (ctDNA), and biomolecules encapsulated in extracellular vesicles, can now be extracted and analyzed from body fluids. Here we will review the most recent studies on breast cancer, demonstrating the clinical potential and utility of CTCs and ctDNA. We will also review literature illustrating the potential of circulating exosomal RNA and proteins as future biomarkers in breast cancer. Finally, we will discuss some of the advantages and limitations of liquid biopsies and the future perspectives of this field in breast cancer management. Breast cancer is the most common invasive cancer and the leading cause of cancer-related deaths among women . The cliBreast cancer is a heterogeneous group of neoplasms originating from the epithelial cells lining the milk ducts or lobules . CurrentBreast cancer generally presents a high degree of intra-tumor heterogeneity as well as cancer cell plasticity, which may affect the progression, pathological response to treatment, and recurrence ,11. ClonThere are several types of tumor material that can be assessed by liquid biopsies, such as circulating tumor cells (CTCs), cell-free circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) . CTCs re® device [® device (Circulating tumor cells (CTCs) are cancer cells that have been shed from the primary tumor or a metastatic lesion into the bloodstream. Even though millions of cancer cells in some primary tumors are shed every day, only a sub-fraction of the CTCs might have the propensity to form distant metastases ,22,23. I® device . The dev® device . Briefly® device . PreviouSeveral studies have shown that detection of CTCs at the time of diagnosis (baseline) and at different following-up time points during or after completion of therapy can have a prognostic value for early breast cancer patients. This was recently demonstrated by Trapp et al., who conducted a retrospective study, including node-positive or high-risk node-negative primary invasive breast cancer patients from the SUCCESS A trial (NCT02181101) . The maiRANKL) and its receptor RANK play critical roles in breast cancer development, both during initial tumorigenesis and in the formation of secondary tumors in the bone [As illustrated by the above-mentioned studies, increasing evidence indicates that CTC enumeration can improve prognostication of breast cancer. Other studies have aimed to determine whether receptors on the surface of the CTCs can predict response to therapy. Receptor activator of nuclear factor-kappaB ligand  found in circulation is highly fragmented and most likely originates from apoptotic and necrotic cells, but the exact origin and mechanism of release are still being investigated . ImportaOnce total cfDNA is isolated from the blood, it can be measured by fluorescence-based methods or by real-time quantitative PCR (RT-qPCR) . TangvarMeasuring the level of ctDNA in cancer patients is a more specific method than measuring cfDNA, and for this reason, it could have a greater potential as a novel method for oncological applications. ctDNA fluctuations during the course of the disease could be correlated with clinical outcome. It was demonstrated by Kruger et al. that high ctDNA molecule numbers and at least two breast cancer-specific mutations, measured by next-generation sequencing (NGS), correlate with poor clinical outcome in ER-positive, HER2-negative metastatic breast cancer patients . Over th3 undetected because the expected mutation-carrying ctDNA concentration in plasma for these patients can be 0.1% or lower of total cfDNA [PIK3CA mutations in plasma ctDNA from breast cancer patients [PIK3CA encodes the catalytic p110a subunit of PI3-kinase and is mutated in 30–40% of all breast cancer [PIK3CA mutations in metastatic tumor tissue and serum ctDNA [PIK3CA mutated ctDNA dropped in patients that displayed response to therapy. This suggests that ddPCR-mediated detection of ctDNA PIK3CA mutations can be used in early monitoring of therapy response [PIK3CA mutations can be detected in metastatic breast cancer patients [PIK3CA mutations in the primary tumors [PIK3CA mutations were detected in ctDNA in blood samples taken before surgery. CtDNA PIK3CA mutational status was also analyzed after surgery for some of the patients, and 5 out of 10 still had detectable ctDNA PIK3CA mutations. However, further studies have to be undertaken to assess whether detected PIK3CA mutated ctDNA after surgery is an identifier of patients with a higher risk of recurrence [PIK3CA mutations on ctDNA has been approved by the FDA. The therascreen PIK3CA RGQ PCR Kit is a laboratory test able to detect 11 mutations in the PIK3CA gene from patients with advanced or metastatic breast cancer. The presence of PIK3CA mutations is linked with response to treatment with PIQRAY®  [®. Another attractive target for ctDNA mutational analyses is the ESR1 gene encoding estrogen receptor alpha (ERα). ESR1 mutations are associated with resistance to aromatase inhibitors (AIs) that are used as estrogen-deprivation therapy in postmenopausal women [ESR1 mutations can be selected during AI treatment of metastatic HR+ breast cancers, which can be detected in ctDNA [ESR1 mutations Y537S and D538G, which lead to ligand-independent constitutive activation of ERα in blood samples from 541 ER+/HER2− breast cancer patients with metastatic diseases that all had undergone AI therapy in different settings  [ESR1 Y537S and/or D538G mutations in ctDNA in 28.8% of the patients. Importantly, the presence of the ESR1 ctDNA mutations was associated with reduced overall survival. Thus, monitoring ESR1 ctDNA mutations can be important in disease control of metastatic ER+ cancers and potentially give valuable information on which patients will benefit from hormonal therapy.Detection and quantification of predetermined alleles from ctDNA by Sanger sequencing, single and multi-locus PCRs, and RT-qPCR have established methods in clinical oncology ,69. Howeal cfDNA ,71. Advaal cfDNA . Both tepatients ,72. PIK3t cancer . By usinum ctDNA . Moreoveresponse . Severalpatients ,76,77. Iy tumors . In 14 pcurrence . Interespelisib) . Thus thal women ,80. Impoin ctDNA ,81,82. IPIK3CA and ESR1 ctDNA mutations plasma samples from patients with HR+, HER2− metastatic breast cancer enrolled in phase 3 PALOMA-3 trial (NCT01942135) [The above-mentioned studies indicate that digital PCR is a sensitive method that is suited for detecting allele variation in ctDNA from clinical samples. A recent study assessed the agreement between the BEAMing and ddPCR techniques in detecting 1942135) . The aut1942135) ,84,85.The revolution within massively parallel NGS technologies has opened up a new era within personalized medicine. The sensitivity of these methods enables the detection of patient-specific chromosomal structural aberrations, copy number variations, and single nucleotide polymorphisms (SNPs) in both tissue samples and liquid biopsies. Both unbiased WGS and more targeted approaches may be used. Even though proof-of-concept studies have been reported, ctDNA analyses by WGS approaches appear to be somewhat limited by the low abundance of ctDNA fraction in total cfDNA, short ctDNA fragment lengths, as well as by high error rates in the detection of rare allele variants ,87. Therhttps://www.natera.com/oncology/signatera-advanced-cancer-detection). The aim of this technology is to improve the detection of minimal residual disease after surgery as well as earlier detection of disease recurrence. Signatera™ provides each individual patient with a customized blood test tailored to match the clonal mutations found in the individual’s tumor tissue by WES. Coombes et al. validated the clinical performance of Signatera™ on breast cancer patient samples [In 2019, the first custom-built NGS-based ctDNA test, Signatera™, was launched  are generally heterogeneous but can broadly be divided into two classes based on size and mechanisms of biogenesis . These aExosomes isolated from breast cancer patients have been shown to have distinct protein- and RNA contents compared to exosomes derived from healthy donors ,104. In HOTAIR were found in circulating exosomes in breast cancer patients than in healthy individuals [HOTAIR levels were also associated with poor prognosis, and it was suggested as a possible predictor of response to chemotherapy and tamoxifen treatment. Zhong et al. reported that serum exosomal levels of the lncRNA H19 were significantly higher in breast cancer patients than healthy controls [SNHG14 were associated with resistance to trastuzumab [Even though the majority of studies on circulating exosomal RNA molecules have been focused on miRNAs, a few studies have shown that exosomal mRNAs and long noncoding RNAs (lncRNAs) might be used as biomarkers. Rodriguez et al. showed that a set of metastasis- and stemness-related mRNAs were higher expressed in plasma exosomes from breast cancer patients with poor disease outcomes than in those that were isolated from patients with good outcomes . Increasividuals . Exosomacontrols . Finallystuzumab .The protein content of exosomes reflects their mode of biosynthesis, and they are typically enriched with endosomal/lysosomal proteins like tetraspanins , ESCRT complex proteins , Rab proteins, Syndecan-1, and Syntenin-1 ,93. QuanERBB2 gene in the immortalized human mammary luminal epithelial cell line HB4a altered the proteomic landscape of the secreted exosomes [Several reports have shown that the proteome of breast cancer-derived exosomes reflects the subtype of the malignant cells. For instance, it has been shown that ectopic overexpression of the HER2− encoding exosomes . Moreoveexosomes ,132,133.The protein content of circulating exosomes in breast cancer patients can potentially predict the response to therapy. It has been suggested that the HER2 receptor on exosomes might act as a decoy for trastuzumab, a HER2-targeting monoclonal antibody used in the clinic today . By doinIn order to have clinical utility, cancer biomarkers should fulfill a set of analytical and clinical criteria ,138. Hig® platform is the only one licensed by the FDA for the isolation of CTCs and their prognostic enumeration in breast cancer [® platform enriches CTCs based on the expression of the epithelial marker protein EpCAM. In breast cancer, as well as other cancers, several lines of evidence suggest that the most aggressive tumor cells are those that have undergone epithelial-mesenchymal transition (EMT), a process that is associated with downregulation of EpCAM as well as other adhesion molecules [®-based CTC detection both at baseline and during therapy might give vital information about residual disease and recurrence. However, further standardization of the assays addressing all the above-mentioned limitations need to be addressed before CTC enumeration can implemented in the clinic as an independent assay to guide treatment decisions [CTCs might give important information about tumor burden and might also contain molecules reflecting cancer-specific features. An important limitation of CTCs is that they may not fully reflect the biology of the underlying tumor. In addition, cancer cells are not confined to one phenotype and might have epithelial, mesenchymal, or stem-like characteristics . Currentt cancer . Howeverolecules ,143. EMTolecules . Thus, oolecules . Anotherolecules . Finallyolecules . This meecisions ,89.PIK3CA and ESR1 ctDNA mutations in plasma samples from breast cancer patients [Detection of ctDNA that reflects the mutational status and clonal evolution of a tumor holds great promise for future monitoring of cancer progression and drug response in breast cancer. The development of high-sensitivity technologies enabling detection of mutant allele frequencies of <0.01% has, without doubt, revolutionized this field ,64. Howepatients . In contpatients . TherefoIt is generally accepted that secreted exosomes provide a protective compartment for circulating biomolecules. Furthermore, in breast cancer and other cancers, tumor-derived exosomes may contain cargo that can give vital information about disease development and progression. However, as illustrated by the limited number of comprehensive clinical studies on exosomes and breast cancer, the field faces challenges that need to be resolved before being clinically applicable. First of all, further research should be conducted on the selection and packaging mechanisms of cargo in the originating cancer cell to better evaluate whether the exosome content in a given sample is based on specific selection or random incorporation of the secreted cargo. In the latter case, inter- and intraindividual heterogeneity of exosomal content might preclude their implementation as reliable biomarkers. Moreover, sensitive methods for purification of exosomes that excludes contaminants like lipoprotein particles, microvesicles, and protein complexes, are needed . Such meMost studies on liquid biopsies have been focused on a single analyte . This is about to change, and more initiatives are now being taken to develop multiparametric assays that incorporate data from separate platforms analyzing different analytes. Such combinatorial strategies require powerful statistical tools and algorithms that can integrate and standardize data obtained from different platforms. In this regard, PROLIPSY (NCT04556916) is a multinational effort, which is exploring whether a combination of CTCs, ctDNA, and tumor-secreted exosomes might assist in early detection and prognostic profiling of prostate cancer. CancerSEEK is an already commercialized blood-based assay that looks at ctDNA and protein biomarkers in parallel. Cohen et al. conducted a multicenter study, enrolling 1000 patients bearing different types of cancer. Their results showed that they could achieve early-detection rates of 69–98% for five different malignancies , with a false-positive rate of less than 1%. Regarding breast cancer, this test could only detect around 30% of the cases, so further improvements are needed . FinallyCurrently, exhaustive validation in clinical trials is required to boost the clinical utility of liquid biopsies. More studies are needed to improve the test’s accuracy and sensitivity of the tests. Moreover, additional investigations are required to clarify whether liquid biopsy provides a representative sampling of all genetic clones within a tumor or if there is a bias to specific subregions of the tumor. Breast cancer is a heterogeneous disease, but the survival rate is good if the patient is diagnosed before distant metastasis has occurred. In many countries, mammography screening programs are implemented for women above a certain age (50 years in Norway), which indeed has led to the early detection and diagnosis of many breast cancers. Early detection might potentially also lead to overtreatment of some patients. In addition, current diagnostic tools for breast cancer are well-established. Based on this, it is unclear whether liquid biopsies can add additional value in breast cancer diagnosis. In sharp contrast, longitudinal monitoring tools that can detect residual disease after therapy, inform about disease progression and predict cancer recurrence at an early stage are desperately needed. Here, biomarkers acquired through liquid biopsies can make a tremendous impact on the future control of breast cancer.


Leadless pacemakers have an accepted role with demonstrable benefits in adults. In contrast, implant experience and follow-up data in pediatric patients are more limited. Clinical indications and patient candidacy for leadless pacing in pediatrics continue to evolve. We present our experience implanting a leadless pacemaker in a four-year-old for the treatment of high-grade atrioventricular block. Implant considerations in small patients, short-term patient and device follow-up data, and follow-up assessment of the vessel used for implantation are discussed. Historically, despite aggressive immunosuppression, he experienced chronic antibody-mediated rejection and markedly elevated panel reactive antibodies precluded retransplantation candidacy. Long-term treatment has prioritized palliative care and maximization of the quality of life.A four-year-old male  systolic function, and mild left ventricle (LV) dilation with mildly decreased systolic function, all of which remained unchanged from prior imaging. Permanent pacing was indicated and both traditional and nontraditional pacing options were considered. The implications of each approach, including the potential impact on vascular access, the relative expected ease of future endomyocardial biopsies, and postprocedure recovery, were discussed. The parents, transplant team, cardiac surgeons, and electrophysiologist agreed to pursue leadless pacemaker implantation. Given the recent availability of the Micra™ AV pacemaker , previously approved by the Food and Drug Administration, at our institution, this device was selected for its potential benefit in addressing the patient’s specific conduction disorder.Inpatient telemetry captured episodes of second-degree, Mobitz type II atrioventricular (AV) block with symptoms of tiredness and dizziness . The vessels were small but, given their capacity for distension, the decision was made to proceed with slow and serial vessel dilation over an exchange-length stiff wire with a series of dilators. Dilation was systematically performed with 8-, 10-, 12-, 16-, 18-, and 22-Fr dilators. Constant assessments of leg perfusion, skin color, and size were performed during the dilation process. The dilator for the Micra introducer sheath (IS) was used as a final dilator before inserting the complete IS set (27-Fr outer diameter) . The insertion of the IS proceeded smoothly without resistance.Conventional right femoral venous implantation was performed under general anesthesia. Ultrasound-guided venous access was obtained, and a 6-French (Fr) sheath was inserted. Contrast injection through the sheath was completed to assess femoral and iliac vein caliber . As such, an apical septal location was pursued instead. The TV was crossed again more inferiorly and clockwise torque was applied thereafter to direct the DC tip toward the apical septum . The device was deployed with at least two splines exhibiting appropriate fixation. Testing demonstrated acceptable functional values . Transesophageal imaging confirmed the implant location and showed no pericardial effusion or significant tricuspid interference. The anchoring tether was cut and removed with preservation of the device’s position . Following DC withdrawal but before IS removal, a 0-Ethibond purse-string suture  was placed around the skin entry site for closure and hemostasis. The site was covered with a pressure dressing. Postimplantation interrogation showed preserved functional values and reasonable accelerometer-derived signals corresponding to atrial contraction .The Micra™ delivery catheter (DC) was prepped and advanced through the IS. To cross the tricuspid valve (TV), active flexion of the DC was performed with counterclockwise torque. Midseptal implantation was initially attempted but adequate septal orientation of the delivery cup could not be achieved. Furthermore, given the dimensions of the chamber, it was felt that midseptal deployment might potentially obstruct the TV apparatus proximally and the RV outflow and pulmonary valve superiorly A right lower-extremity vascular ultrasound performed two days after implantation showed a near-occlusive thrombus in the common femoral vein. The pressure dressing and skin suture were removed on the fourth day after implantation without any evidence of bleeding or hematoma. Given the luminal thrombus, subcutaneous low-molecular-weight heparin (1 mg/kg every 12 hours) was initiated without bleeding recurrence. A follow-up assessment at six weeks after implantation showed normal device function and a reduction in thrombus size with a normal vessel flow pattern on follow-up lower-extremity scanning. By this point, the patient had returned to normal activities without syncope recurrence.To our knowledge, the present case reports the management of the smallest pediatric patient implanted with a Micra™ leadless pacemaker incorporating VDD mode functionality. The patient’s pacing indication, coupled with avoidance of more invasive implant procedures and complications with leads  and subcutaneous device pockets, made leadless pacing preferable, especially in light of his long-term palliative care strategy.3 In contrast, published implant and follow-up data on the use of leadless pacemakers in pediatric patients have been limited, with fewer than 20 total implants reported. The largest single-center cohort to date included only nine patients .4 A conventional femoral venous approach was generally adopted, although all patients were older than 11 years and more than 30 kg in weight.6 Alternatively, Gallotti et al. and Cortez presented different cases performed using right internal jugular venous access in smaller patients.8 The jugular approach was favored given patient and vessel size considerations as well as the notion of easier device maneuverability across the TV and within the RV from a superior approach. Postimplantation assessment of the vessels used for implantation has not been previously reported.Leadless pacing provides effective single-chamber pacing in adults across multiple pacing indications with high rates of implant success, lower complication rates relative to those seen with traditional transvenous devices, and a low infection risk.Our case demonstrates several important considerations to keep in mind when using the conventional femoral venous approach in very small patients. First, slow and serial vessel dilation should be performed. The IS itself, with its hydrophobic outer surface, advanced very smoothly despite the small vessel used in our case and the femoral vessel accommodated the large IS owing to its inherent distensibility. Catheter flexion and device positioning in the RV cavity were easily accomplished from the inferior approach. The application of counterclockwise torque with flexion was extremely effective in crossing the TV. A midseptal implant location for the Micra™ system may not be an ideal choice in small patients, owing to the smaller RV chamber size and potential risks of inflow and outflow interference in these individuals. In addition, adequate septal orientation of the DC tip may not be achievable at this more proximal location on the septum. Postimplantation development of a near-occlusive thrombus in the CFV, while not entirely unexpected, was unfortunate. Although thrombus formation following vascular trauma related to site dilation and large sheath insertion is presumed to be the mechanism, the extended duration of pressure dressing application, which compressed the vessel and slowed venous flows, may also have contributed. It is encouraging that venous patency was preserved with interval reduction of the luminal clot size on short-term anticoagulation therapy.9 This sensed contraction is then used as a surrogate for atrial depolarization from which ventricular pacing can then be sequentially delivered. Given an anticipated low pacing burden and the sporadic nature of Mobitz II AV block in our patient, we were unable to record significant VDD pacing over the short follow-up period. However, manual testing of atrial sensing demonstrated reasonable signals corresponding to atrial contraction and we would anticipate that synchronous pacing can be effectively applied within programmable parameters.Although we hoped the VDD pacing capability of the Micra™ AV pacemaker would be useful for our patient’s primary pacing indication, its use in young patients may be limited due to its relatively low programmable upper tracking rate (115 bpm). Mechanical atrial contraction is sensed using the device’s accelerometer and an automated, enhanced sensing algorithm.10 Given the smaller sizes of RV chambers in pediatric patients, the Micra™ system’s footprint may be proportionately substantial and abandonment of a battery-depleted Micra™ system may limit space for new device implantation. Retrievability was not considered to be a limiting factor in our case given the device’s anticipated long battery longevity in comparison with the patient’s long-term prognosis.Device retrievability is of particular interest in pediatric patients and has been successfully performed up to four years after implantation.In summary, conventional femoral Micra™ implantation is feasible even in small patients. The lower limits for age and size for safe and effective Micra™ pacemaker implantation have not been established but appear to be younger and smaller, respectively, than previously published. The assessment of implant vessel size and its capacity for dilation and large sheath accommodation must be performed. Vessel and cardiac chamber sizes can pose limitations to implantation in smaller patients. Synchronous pacing expands the potential use of leadless pacing even in pediatric patients, although upper tracking rates may be a limitation.


Non-rainfall moisture  is an important driver of plant litter decomposition in grasslands, where it can contribute significantly to terrestrial carbon cycling. However, we still do not know whether microbial decomposers respond differently to NRM and rain, nor whether this response affects litter decomposition rates. To determine how local moisture regimes influence decomposer communities and their function, we examined fungal communities on standing grass litter at an NRM-dominated site and a rain-dominated site 75 km apart in the hyper-arid Namib Desert using a reciprocal transplant design. Dominant taxa at both sites consisted of both extremophilic and cosmopolitan species. Fungal communities differed between the two moisture regimes with environment having a considerably stronger effect on community composition than did stage of decomposition. Community composition was influenced by the availability of air-derived spores at each site and by specialization of fungi to their home environment; specifically, fungi from the cooler, moister NRM Site performed worse  when moved to the warmer, drier rain-dominated site while Rain Site fungi performed equally well in both environments. Our results contribute to growing literature demonstrating that as climate change alters the frequency, magnitude and type of moisture events in arid ecosystems, litter decomposition rates may be altered and constrained by the composition of existing decomposer communities. Understanding when and how microbial communities are driven by different climate variables and when compositional shifts have functional consequences will help us better predict how carbon cycling will change under changing climate conditions.Decomposition of organic matter is a major source of greenhouse gas emissions in terrestrial systems and plays an essential role in ecosystem carbon and nutrient cycling . Biotic One of the most important climatic factors determining microbial community structure and decomposition rates is moisture availability . While rThere are several reasons why microbial communities could differ in NRM- and rain-dominated environments. First, in many systems fog and dew occur more regularly than rainfall , so orgaWe provide the first assessment of how fungal communities are differentially influenced by NRM and rainfall. Specifically, we addressed the following research questions:(1) How does moisture regime (NRM- versus rain-dominated) drive fungal community composition?(2) Do changes in fungal community composition driven by moisture regime influence rates of litter decomposition?We hypothesized that differences in the air-derived fungal communities (fungi that colonized pre-sterilized tillers) as well as selection for different moisture regimes would lead to different fungal communities under the two regimes. We further hypothesized that some fungi would specialize in a particular environment. Finally, we hypothesized that these differences in community structure would affect decomposition rates, with fungi decomposing litter faster under their native moisture regime than in non-native conditions.1 maintained by the Gobabeb Research and Training Centre2. At the Rain Site, we used a HOBO data logger  and sensors to record temperature and relative humidity (S-THB-M002), rain (Davis S-RGD M002), and leaf wetness (S-LWA-M003). Measurements were recorded once per minute and converted into hourly averages. The weather station at the Rain Site failed two months before the end of the experiment, so we used data from a nearby weather station . An analysis comparing weather data from the two eastern stations (Dieprivier and the Rain Site) for ten months before the failure showed that the two sites had comparable weather regimes. Since solar radiation can accelerate litter decomposition by photodegradation  and has two very different moisture regimes in close proximity to one another: the western region is dominated by fog and dew and receives scant rainfall while the inland eastern areas receive more rainfall but very little non-rainfall moisture . Work waradation  and alteradation , we estiradation .To see whether fungal communities functioned similarly under NRM- versus rain-dominated conditions, we reciprocally transplanted litter between the two sites . HereaftStipagrostis sabulicola at the two sites. S. sabulicola is the dominant grass in the Namib Sand Sea and grows in hummocks on unstable dune faces . We confirmed this in the present study by visually examining tillers for evidence of sand blasting after collection.Despite the fact that this study was conducted in a sand dune system, sandblasting and aeolian-driven mass loss were not substantial mass loss drivers in this study. First, we used only coarse tillers, leaving no attached leafy material that would be shredded or easily removed, even during very high winds . SecondlWe used tillers at two different stages of decay to see whether moisture regime structured communities differently as tillers decomposed. Early-stage tillers were harvested from recently senesced stems that still had inflorescences attached (< 2 months post-senescence), were yellow, had no visible fungal growth, and had a visibly intact cuticle. Late-stage tillers were harvested from upright stems that had been standing for at least one year. These were characterized by coverings of light and dark-pigmented fungi and a cracked cuticle that was considerably more permeable to water . The priTo assess how colonization from air-derived fungi affected litter community assembly, we deployed “bait tillers,” which were autoclaved early-stage tillers collected from the NRM Site. We included bait tillers from only one site because space limitations in our litter racks prevented us from including bait tillers from both sites. Sterilization was confirmed by plating tillers on culture media. Autoclaving is an imperfect means of sterilization because it can alter associated chemistry but since our goal was to identify air-derived fungi, this deployment of standardized bait tillers allowed us to estimate the contribution of air-derived fungal sources  since the only fungi present on autoclaved tillers after one year would have been deposited from the air. Importantly, any confounding effects of autoclaving on litter properties would have been the same for litter deployed at both sites, allowing us to use a standardized litter type to compare air-derived fungal communities at the two sites.We measured C:N ratios in litter on a Thermo Finnigan Flash-1112EA microanalyzer . We used an ANKOM 200 Fiber Analyzer and Daisy Incubator  to measure acid detergent lignin, cellulose, and hemicellulose of live tillers and a subset of early- and late-stage tillers to determine initial litter chemistry at the start of the study. To determine whether mass loss was driven solely by differences in leaching from tillers at the two sites, we measured leaching on a subset of recently senesced tillers collected from the two sites that were identical to those used in our decomposition study. We sealed the cut ends of each tiller with glue, submerged them in ultrapure water at 4°C for 24 h, dried them, and weighed them to quantify mass loss due to leaching.PRJNA685174.Fungal communities were analyzed using molecular methods. We extracted DNA from tillers using a MoBio PowerSoil DNA extraction kit supplemented with 10 min of sonication prior to mechanical lysis. We sequenced the ITS region  using 250-bp paired-end sequencing on the MiSeq Illumina (V2) platform at Michigan State University’s Research Technology Support Facility Genomics Core. We used the same clustering and filtering pipeline used for Namib fungal (ITS) sequences described elsewhere . All seq3. Merged sequences were quality filtered to an expected error threshold of 1.0 fastq_filter . We also removed two OTUs that were identified as S. sabulicola, the host plant.Reads per sample ranged from 1,805 to 52,926 averaging 22,425. We used multiple extraction blanks to check for contamination, eventually removing three OTUs from the dataset. After OTU clustering, we removed any taxa that fit all three of the following criteria: it had more than 100 reads when summed across three blanks, had never been found in any previous sequencing datasets from working in the Namib Desert, and had never been cultured from the Namib in previous studies e.g., . This leS. sabulicola tillers in previous studies in the Namib, which had been identified with the longer ITS 1-4 region (∼600 bp)  .Saccharomyces cerevisiae as a standard to quantify ITS gene copies and normalized this to the dry mass of litter used for the extraction to produce a proxy of fungal biomass reported as log10 ITS copies gram–1.As a proxy of fungal biomass, we used quantitative PCR to count the number of ITS gene copies per sample and normalized it to the tiller’s dry biomass . While tt-test and lm functions and analyzed the reciprocal transplant experiment with Type III ANOVAs using the Anova function in the car package. PERMANOVAs and NMDS ordinations were generated using Bray-Curtis distances calculated using the distance function in the phyloseq package  and more frequent rain events than the NRM Site. The average time between wet events (either rain or NRM events) was 33 hours at the NRM Site and 90 hours at the Rain Site (p = 0.001). Dry periods lasting for more than 100 hours were not uncommon at the Rain Site  during the study period was 9% greater in the vicinity of the Rain Site than the NRM Site, though these were based on estimates from proxy sites and likely overestimate differences between our sites. Both sites had similar bidirectional NW and E wind regimes with seasonal variation in wind direction, suggesting similar source regions of air-derived fungi, though the Rain Site had a stronger southern component , Neostagonospora caricis (OTU8), and Phaeococcomyces mexicanus (OTU30)) together accounted for more than 53.0% of reads, but less than 1.4% at the Rain Site (Aureobasidium melanogenum (OTU3), Phaeococcomyces sp. (OTU17), Alternaria alternata (OTU1)) accounted for 50.8% of Rain Site reads but only 9.2% of NRM Site reads.Fungal communities on native tillers differed between the sites . Site exain Site . LikewisR2 = 0.23, P = 0.008)  , they al= 0.008) ; the 73 = 0.008) , suggest= 0.008) .Aureobasidium melanogenum (OTU3), unknown Pleosporales (OTU104), and Alternaria alternata (OTU1)) made up 31.0% of the Rain Site air-derived reads and 25.8% of the NRM Site air-derived reads. The top three air-derived taxa at the NRM Site  made up 43.7% of NRM Site air-derived reads and 10.2% of Rain Site air-derived reads  and Aureobasidium melanogenum (OTU3)] were highly abundant on both bait tillers and native tillers at both sites.The three most abundant air-derived taxa at the Rain Site . Early-stage tillers moved to the NRM Site had higher fungal biomass than those that remained at the Rain Site  and Aureobasidium melanogenum (OTU3) each represented over 10% of read abundances on native or bait tillers at both sites , Neophaeothecoidea spp. (OTU4), Phaeococcomyces mexicanus (OTU30), and Selenophoma linicola (OTU33). Despite their low abundance at the warmer, drier Rain Site, most of these taxa are closely related to organisms known for their desiccation and thermal tolerance. Neophaeothecoidea is a monotypic genus first isolated from protea plants in South Africa and, while its ecology has not yet been described in detail, it’s confamiliars in Teratosphaeriaceae occupy a wide range of habitats including highly acidic soils  and Phaeococcomyces species (OTU17). Pseudopithomyces is often found on dead plant litter and as a pathogen of some plants , is a congeneric of a fungus with a strong preference for the NRM Site (Phaeococcomyces mexicanus OTU30), suggesting that the traits responsible for thriving under the NRM-dominated versus rain-dominated environment may not be so clearly delineated along taxonomic lines.Two fungi showed much greater abundances at the Rain Site than the NRM Site: e plants  includine plants . The clo regions . InteresOverall, we did not find clear differences in desiccation tolerance or general stress tolerance among the fungi that preferred the NRM Site versus the Rain Site based on our literature searches. While culture-based follow-up studies may provide more information about the desiccation and thermal tolerances of these organisms, biotic interactions may also play a role in determining which taxa dominate at each site. For instance, while some NRM Site fungi may be fully capable of surviving the abiotic conditions of the warmer, drier Rain Site, they may simply be outcompeted by others with even slightly greater dessication tolerance. Similarly, Rain Site fungi appear fully capable of utilizing NRM when given the opportunity , but oveThe fact that both of these sites harbored diverse fungi associated with such wide-ranging niches was surprising, since we might have expected that only specialized fungi could tolerate the radiation and desiccation stressors of the hyperarid Namib Desert. Instead, several of the most abundant taxa were found worldwide in mesic and arid environments. This could be partially explained by considering the traits that support wide distributions. In bacteria, some of the traits that permit growth in an arid, sunny environment (such as pigmentation) correlate with large geographic ranges , since tBy using bait tillers, instead of direct air sampling, to assess the air-derived fungal community, we focused on those fungi that were capable of colonizing senesced grass tillers. These results show that the airborne and litter-associated fungal communities appear to interact with one another, representing a shared pool of dominant fungi. Although we were unable to determine whether air-derived communities at these two sites differed primarily due to differences in local plant diversity at the two sites, long-distance dispersal from wind, or other factors, we did find that the two sites contained a large pool of shared taxa. Cosmopolitan taxa (those that were present at both sites) make up the vast majority of reads on bait tillers at both locations, suggesting that similar air-derived species pools are available to colonize litter at both sites. Overall, litter-associated communities differed between the sites much more than air-derived communities did, suggesting that post-colonization processes play a role in structuring fungal communities differently at the two sites.Fungal communities on some transplanted tillers changed, as would be expected if some fungi performed better in one environment than the other. Fungal communities on early-stage tillers shifted while well-established late-stage tillers retained their initial communities . That we2 respiration  as the sole driver of fungal community differences that we observed, though it was likely the dominant factor. We collected standing dead litter from a sand dune system and suspended it aboveground so any differences in soil type would not be a factor. NRM duration differed by a factor of five to ten between the sites while minimum and maximum temperatures were far more comparable . The Raipiration  and littpiration , even wipiration  found thpiration , so we wpiration . Future S. sabulicola tillers at these same locations, found that the relationship between litter gravimetric moisture content and CO2–C flux did not differ between the two sites, demonstrating that fungi from both sites respire similarly under NRM. Our results showing that Rain Site fungi moved to the NRM Site decompose litter at the same rate as native NRM Site fungi would seem to corroborate this. Taken as a whole, this suggests that optimization for fog and dew by NRM specialists may not reflect any particular ability to utilize NRM so much as their inability to cope with the harsher  conditions associated with the Rain Site.Although we did not attempt to identify specific traits that drove differentiation in fungal communities at the two sites, our results suggest that the ability to tolerate the harsher conditions of the warmer, drier rain-dominated environment is an important driver of fungal differentiation across this gradient. The NRM-to-Rain transition may be difficult because the Rain Site is warmer, experiences less frequent wetting, greater solar irradiance, longer dry periods, and shorter NRM events  Table 1, conditiMicrobial biomass strongly regulates decomposition at regional scales and can predict how decomposition rates respond to changing climatic conditions . Fungal Plant litter decomposition is influenced by many factors, including photodegradation , litter The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/KJ and PJ conceived of the study, conducted fieldwork, and edited the manuscript. SE conducted data analysis and edited the manuscript. JL conducted lab work, data analysis, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rates of nipple-sparing mastectomies have increased over the past decade. In 2017, acellular dermal matrix was used in 56% of breast reconstructive procedures, with complication rates similar to operations without AlloDerm. Although persistent nipple discharge after nipple-sparing mastectomy is a rare event, it has been described in the literature. Other authors have described evaluation and treatment on a case-by-case basis. To the best of our knowledge, this is the first case report to describe a persistent unilateral discharge after multiple operative revisions and to provide an algorithmic approach to workup and treatment.BRCA1 mutation who underwent a prophylactic bilateral nipple-sparing mastectomy with immediate reconstruction using AlloDerm. The year following her operation, the patient underwent two surgical revisions, one for implant rippling and one for asymmetry. Six months after her second revision, she presented to our hospital with a capsular contracture and unilateral clear nipple discharge. Her breast ultrasound showed dilated subareolar ducts and a suspicious mass. Magnetic resonance imaging identified a benign-appearing, rim-enhancing fluid collection. She underwent a third revision. One year later, she returned to our clinic with bloody nipple discharge, erythematous skin changes, and a palpable breast lump. Her surgical biopsy showed a fold in AlloDerm and chronic inflammatory changes. She continued experiencing discharge and opted for nipple excision. During the operation, a lacrimal probe demonstrated a direct connection between the discharging external duct and a seroma associated with an area of unincorporated AlloDerm. The section of unincorporated AlloDerm was excised, and no evidence of malignancy was identified. Ten months later, the patient remained symptom-free and had progressed to placement of final silicone implants.We present a case of a 29-year-old Hispanic woman with To the best of our knowledge, this is the first case report to describe a nongravid patient with persistent unilateral sanguineous nipple discharge after multiple operative revisions. A visible communication between the draining duct and a seroma associated with unincorporated AlloDerm was ultimately identified. We present a clinical algorithm for patients with nipple discharge after nipple-sparing mastectomy. Rates of nipple-sparing mastectomy (NSM) are increasing, and although persistent nipple discharge after NSM is a rare event, this is the first case report, to the best of our knowledge, to describe a nonpregnant patient with persistent unilateral sanguineous nipple discharge after multiple revisional operations. A visible communication between the draining duct and a seroma associated with unincorporated AlloDerm  was intraoperatively identified as the cause of the persistent discharge.BRCA1 deleterious gene mutation who underwent prophylactic bilateral NSM and immediate direct-to-implant subpectoral reconstruction using an AlloDerm sling. Three months from the initial operation, the patient underwent breast reconstruction revision with bilateral implant replacement, bilateral superior and lateral capsulotomies, and bilateral fat grafting due to implant rippling with inadequate upper pole projection. She underwent a second revision due to asymmetry several months later, which included a right inferior capsulorrhaphy, bilateral fat grafting, and right breast mastopexy.Our patient first presented to our hospital as a 29-year-old, nonpregnant, G4P2-0-2-2 Hispanic woman with a Six months later, she noticed left-sided clear, yellow nipple discharge from a single duct. The discharge was reproducible with firm pressure. A grade III capsular contracture was present without erythema or evidence of skin lesions. Her breast ultrasound showed unilateral dilated subareolar ducts and a suspicious 1.2-cm heterogeneous mass. Breast magnetic resonance imaging (MRI) identified a thin, circumferential rim-enhancing fluid collection associated with the left implant, without nipple enhancement, and confirmed the heterogeneous mass to be an area of fat necrosis. She underwent revisional excision of residual subareolar tissue, left breast capsulectomy, implant exchange using AlloDerm, and bilateral breast fat grafting. Pathology showed benign breast ducts and lobules without proliferative changes, atypia, or neoplasm.One year later, she returned to our hospital with complaints of left bloody nipple discharge, erythematous skin changes noncontiguous with the nipple–areola complex (NAC), and a left lateral breast lump. An MRI scan again noted the same benign-appearing, small-volume, peri-implant fluid collection, but now with visible extension to the nipple ducts Fig. . After tOver the next 6 months, she continued to experience left nipple discharge that left half-dollar–sized stains on her clothing, usually following exercise or warm showers. A decision was made to return to the operating room for bilateral nipple excision  and conversion of subpectoral implants to AlloDerm-wrapped prepectoral implants. A lacrimal probe demonstrated a direct connection between the discharging external duct and a seroma associated with an area of unincorporated AlloDerm. No pathologic evidence of malignancy was found in either the excised nipple or other submitted tissues Fig. .Fig. 2LTen months following the removal of unincorporated AlloDerm, the patient remained free of signs or symptoms of a recurrent seroma and had progressed to placement of final silicone implants.Rates of NSM have increased over the past decade, with patients seeking the benefits of this procedure for both therapeutic and prophylactic indications. In 2017, members of the American Society of Plastic Surgeons used acellular dermal matrix in 59,774 of 106,295 breast reconstructive surgeries (56% of procedures) [Despite its widespread use, unincorporated AlloDerm is a rare event. Animal research demonstrates complete integration of the graft at 12 weeks with evidence of vascularization seen on both sides . GabrielPersistent nipple discharge in the setting of a prior NSM in postpartum and nonpregnant patients has been described. Tang and colleagues reviewed 1620 NSM patients and identified 27 women who carried successful term pregnancies following an NSM operation. Records show that 6 (22%) of 27 experienced nipple discharge following delivery; 5 women described the discharge as “milky” and 1 as “clear.” All six women reported that discharge stopped after cessation of either breastfeeding  or cessation of fertility treatments. There was even distribution of indications in this small group, both risk reduction and malignancy. Of the remaining 1593 patients who did not become pregnant after their NSM, 4 reported clear discharge (0.25%), 2 of whom had acellular dermal matrix slings in place. One patient opted for complete nipple excision, which resolved the issue, and the other opted just for excision of the nipple’s discharging orifice. This failed to correct the discharge, and the patient declined further intervention. Mean follow-up was 54 months, and all were without evidence of a new or recurrent malignancy. The authors describe “extensive evaluation” of the early patients, although they do not provide full details. Interestingly, in this group of ten patients who experienced nipple discharge, only one nipple was resected, revealing normal histology [Orzalesi and associates documented the only other case of nipple discharge following NSM identified in the literature. They described a 40-year-old woman who developed serosanguinous nipple discharge 2 years after the procedure. Discharge cytology results were suspicious for malignancy, but galactography suggested benign duct ectasia. The patient underwent a selective duct resection that resolved the discharge and allowed preservation of the NAC. Final pathology confirmed columnar cell metaplasia with fibrocystic mastopathy and necrosis .et al. documented two instances of spontaneous nipple discharge following lumpectomy. Using galactography, they identified the source of discharge as a fistulous connection between native ducts and a postlumpectomy seroma [There is literature to support the idea that nipple discharge after breast surgery can be linked to the presence of a seroma. Ramirez-Hernandez y seroma . Koretz y seroma .BRCA1 mutation, elevated our concern for a missed diagnosis of cancer. We found no evidence of cancer. Our patient had several features in common with the other reported cases: normal appearance of the NAC, increase in discharge volume following activity or warm showers, and a lack of specific imaging features suggestive of malignancy.Our patient first noticed nipple discharge after the third breast operation, approximately 12 months after her initial surgery. She described increases in discharge after activity and warm showers, similar to other patients described in the literature. Despite the lack of malignant enhancement by MRI, the evolution of the discharge character from serous to bloody, and an underlying On the basis of our literature review as well as our experience, we suggest the following algorithm when managing a patient with nipple discharge following NSM Fig. . InitialAlthough the University of New Mexico Institutional Review Board does not require approval of retrospective descriptive studies consisting of three or fewer cases, the patient gave us direct written informed consent to publish our experience and use the accompanying images.Nipple discharge following NSM is a rare event. Our review of the literature did not reveal an underlying malignancy in the limited number of case reports that specifically describe normal appearance of the discharging nipple with or without normal imaging. Our experience demonstrates that ductal communication with a seroma surrounding unincorporated AlloDerm can be a cause of nipple discharge. Furthermore, we suggest a treatment algorithm based on a review of the literature and our experience.