Giemsa-stained specimens were evaluated by a cytopathologist; the cut-off was 1 CTC/specimen (6 mL). Immunohistochemistry with epithelial (pan-CK) and mesenchymal markers (vimentin) was performed. Results: Morphologically diverse malignant CTCs were found in 12/20 patients in at least one blood specimen. CTCs were positive for both vimentin and pan-CK. More patients were CTC positive after neoadjuvant therapy (6/20 vs. 9/15) and CTCs per/ml increased in most of the CTC-positive patients. After surgery, 8/13 patients with available blood specimens were still CTC positive. In clinical follow-up, 5/9 patients who died were CTC-positive. Conclusions: Detection of CTC by filtration within multimodal treatment protocols of non-metastatic EAC is feasible. The rate of CTC positive findings and the quantity of CTCs changes in the course of multimodal neoadjuvant chemoradiation/chemotherapy and surgery.Background: Isolation of circulating tumor cells (CTC) holds the promise to improve response-prediction and personalization of cancer treatment. In this study, we test a filtration device for CTC isolation in patients with non-metastatic esophageal adenocarcinoma (EAC) within recent multimodal treatment protocols. Methods: Peripheral blood specimens were drawn from EAC patients before and after neoadjuvant chemotherapy (FLOT)/chemoradiation (CROSS) as well as after surgery. Filtration using ScreenCell Esophageal adenocarcinoma (EAC) is one of the most rapidly increasing tumor entities in the Western world. Despite application of multimodal treatment protocols, the overall outcome is still limited, even in non-metastatic disease. Five-year survival rates are about 35% with surgery alone and 45% with neoadjuvant chemoradiation (NeoCRT) plus surgery in the “ChemoRadiotherapy for Oesophageal cancer followed by Surgery Study” (CROSS) trial . More th®, Paris, France) have been developed [There is emerging evidence that there are cells in transit that escape CTC enrichment methods based on epithelial surface antigens due to different, more mesenchymal surface antigens in a course of the epithelial-mesenchymal transition (EMT) ,12. Durieveloped . The aim of this study is to assess the longitudinal presence and gross quantity of CTCs during the time course of combined radio-chemotherapeutic/chemotherapeutic and surgical multimodal curative treatment of non-metastatic EAC.n = 20, time-point 2: n = 15, time-point 3: n = 13). Fifteen patients had neoadjuvant chemotherapy according to the FLOT protocol (PeriCTX). Five patients had neoadjuvant chemoradiation according to the CROSS protocol (NeoCRT). One patient committed suicide during the neoadjuvant treatment. Two patients did not undergo a surgery due to poor physical condition or progressive disease with distant metastasis. Overall, 15 patients underwent curative esophagectomy including regional lymphadenectomy. One patient did not undergo final tumor resection since liver metastases were diagnosed during the operation. One patient underwent surgery outside the country and was lost to follow-up. Furthermore, three blood samples were not processed due to technical difficulties . The majority of the study population was male (18/20) and the average age was 62.1 years (Twenty patients with non-metastatic EAC were included in the study (CTC samples: time-point 1: .1 years .Single CTCs or cluster CTCs were detected in 60% (12/20) of the patient population at a minimum of one time-point in the course of treatment. The healthy volunteer samples did not contain cells that appear like CTCs. The number of CTC-positive (>1 CTC/specimen) patients increased after the neoadjuvant part of treatment. Furthermore, 30% (6/20) of the patients were positive at diagnosis, 60% (9/15) after the neoadjuvant therapy, and 61.5% (08/13) after surgery. Six of 12 CTC-positive patients turned from negative to positive during the therapy. Additionally, with exception of two patients, we observed an increase in the number of CTCs per milliliter blood after neoadjuvant therapy in every CTC-positive patient . Most paOne patient  had veryFurthermore, morphological diversity of the isolated CTCs was observed. The CTCs showed variations in size, nuclear-to-cytoplasm (N/C) ratio, and nucleus morphology. Some patients presented CTC clusters, which consisted of much smaller cells than the single CTCs .The cells identified as CTCs by the May-Grünwald Giemsa staining were analyzed for the presence of epithelial (pan-CK) and mesenchymal (vim) markers. Interestingly, all CTCs were positive for vim and pan-CK. They showed, however, different intensity levels of the staining within one patient . Thus, aTissue analysis was performed on the operative specimens of 15 patients. The patients showed an almost equal distribution between the post-neoadjuvant TNM tumor stages . Only 3/We conducted a one-year and two-year follow-up. In this short follow-up, we observed tumor relapse in 9/18 (50%) patients . There wCTC are thought to be living clones of the primary tumor in transit with the ability to set up metastasis in distant organs. They have been found to be of clinical utility in predicting response to treatment and to sharpen prognosis in several malignancies like breast and prostate cancer ,3,10,15.In some studies, on non-metastatic breast cancer, it was shown that CTC could be found in almost all patients . Under nWe found a variety of CTC types. Small cell clusters and medium to large single CTCs were the main CTC subgroups. Several patients showed CTC clusters, which are currently of high interest due to CTC-granulocyte interaction and the suspected induction of CTC proliferation by white blood cells . The CTC®. It reported a significant correlation of the presence of CTC with shorter relapse-free-survival in patients treated by surgery alone [The largest study on single CTC in patients with esophageal cancer preoperatively investigated the presence of CTC in patients with EAC and squamous cell esophageal carcinoma, using the cell surface epithelial marker-based dependent detection method by CellSearchry alone .® is solely based on antibodies against the epithelial EpCAM antigen. There is, thus, a possibility that a relevant subpopulation of CTCs is left undetected.It is suspected that cells undergoing epithelial-to-mesenchymal transition (EMT), including loss of epithelial markers, can escape detection by epithelial marker-based detection systems. The results of a recent study on 21 patients with esophageal squamous cell carcinoma indicate that most CTCs from esophageal cancer had undergone EMT, since only 8.5% of the CTCs were epithelial-CTCs, 58.9% epithelial-mesenchymal-mixed CTCs, and 32.6% were mesenchymal CTCs . In our ®) was used in the present study and tested for feasibility in detecting CTCs in patients with EAC. We detected the presence of morphologically diverse single CTCs and CTC clusters. Since the patients in this study were in a non-metastatic state, the lowest CTC cut-off of 1 cell per sample was applied. Comparable to the results of observations made in patients treated with surgery alone, 30% of the patients with EAC were pretherapeutically CTC-positive in the present study [In light of the above, an antigen-independent size-based filtration method  results is higher than in methods using cell surface antigens. It is also possible that filtration does isolate a more complete but a less relevant fraction of CTCs from the blood. To reach higher specification of the cells in transit, we used epithelial cell-surface labeling for pan-cytokeratin. Additionally, we analyzed the presence of vimentin to investigate the possible presence of EMT with expression of mesenchymal markers. All cells were positive for both markers, but with diverse intensity. On the other hand, only 23% of the samples of the primary tumor had positive expression of vimentin, which supports the presumption that the CTCs undergo EMT. Since the CTCs were stained directly on the filter after removing the May-Grünwald Giemsa staining, the stain-quality was lower than the control immunofluorescence labeling done directly on the control slides. This may have led to false positive or negative results. One may also argue that CD45 staining of CTC samples is missing to exclude white blood cells as potential false positive targets. We are, however, convinced that CTC identification on bright field microscopy by experienced cytopathologists is equal or even superior to purely marker-dependent CTC definitions. In clinical oncology practice, cancer cells are cytopathologically diagnosed in fluids and smears in bright field microscopy. Additional immunostaining is only performed for characterization of the specific tumor entity. ®) with the size-based filtration method (ScreenCell®) for the detection of CTCs in multimodal treatment of EAC with a planned sample size of 438 patients and a clinical follow-up of a minimum of three years. Over 300 patients have already been enrolled. In summary, the present study proves the feasibility of CTC detection in non-metastatic EAC by an antigen-independent size-based filtration method. The rate of CTC-positive findings and the absolute quantity of CTCs are affected by both neoadjuvant chemotherapeutic or radiochemotherapeutic treatment and by surgery. Morphological diversity with single-cell CTCs and clusters of CTCs, but also frequent co-expression of epithelial and mesenchymal markers, were evident. The present study cannot make a statement about the prognostic significance or possible therapeutic consequences of the observed increase of CTCs after therapy or the role of the different morphological CTC subtypes. These questions are currently being investigated in a translational multicenter study conducted with the therapeutic ESOPEC trial (NCT02509286) . This stThis study was conducted as a prospective study in the Comprehensive Cancer Center Freiburg at the University of Freiburg Medical Center, Germany and was approved by the local Ethics Committee (EK No. 190/15). All patients gave full informed consent for material and data acquisition and for all of the following experiments. Patients with histologically-proven EAC in a non-metastatic stage were consecutively enrolled in this study prior to beginning treatment of the disease. The patients had no previous medical history of cancer. The enrolled patients had locally advanced tumor disease (UICC TNM > cT2 cNx cM0) and qualified for a multimodal therapy concept. The decision for the neoadjuvant treatment (chemotherapy (PeriCTX): FLOT Protocol  vs. chemoradiation (NeoCRT): CROSS protocol (41.4 Gy plus carboplatin/paclitaxel)) was made outside the study by the multidisciplinary tumor board. Additionally, we analyzed blood specimens of 10 healthy volunteers for the presence of CTCs.Blood specimens were sampled at three time-points: before the start of the neoadjuvant therapy (time-point 1 (TP1)), after the neoadjuvant therapy (time-point 2 (TP2)), and after surgical resection of the tumor (time-point 3 (TP3)) . All pat®) using 6 mL of patient’s peripheral blood. The blood samples were processed through two ScreenCell® filtration devices , according to the manufacturer’s instructions (3 mL blood per filtration device). CTC isolation was performed within three hours of the blood draw. The ScreenCell® filtration devices are fitted with microfilters that capture the cells on small metal-rimmed filters via low-pressure vacuum filtration. After CTC-isolation, the filters were stained with a standard May-Grünwald Giemsa staining for CTC identification. Cells considered positive for CTCs were epithelioid cells with enlarged (5–20× filter pore size) irregular, hyperchromatic nuclei with well-defined cytoplasm or smaller epithelioid cells (2–5× filter pore size) with round to oval nuclei with well-defined cytoplasm in small or large clusters. CTCs were counted on each filter and the number of CTCs was divided by three to estimate the number of CTC/mL. No cut-off was chosen for CTC evaluation to maintain a complete picture. Two pathologists with one who was a very experienced cytopathologist (M.B.P) reviewed all CTC specimens. Both were blinded to the diagnosis of the patients, reviewed the cells, and made decisions on bright field without immunofluorescence staining.CTC enrichment was performed by cell size-based filtration  with 0.05% Tween 20 (20 min). For optimal epitope retrieval, a HIER  was performed. For this, the filters were boiled for 20 min using Tris/EDTA Puffer (pH 9), then washed with PBS (4×), and blocked with 2% NGS prior to immunofluorescence staining. This was performed using double immunolabeling with a pan-cytokeratin antibody  and anti-vimentin  applied to the filters overnight at 4 °C. Filters were then washed four times with PBS and incubated with the secondary antibodies anti-mouse Alexa Fluor 488  and anti-rabbit Cy3  for one hour. Afterward, the filters were washed twice with PBS, stained with the standard nuclei-stain DAPI (= Hoechst 33342), and washed again with PBS (2×) and water (1×). We defined a very low cut-off of 1 CTC per blood specimen to classify a sample “CTC-positive.” Prior to the patient sample, staining spiking experiments were performed to develop the staining protocol. Esophageal adenocarcinoma cell lines FLO-1 and SKGT-4 were spiked into human blood samples and filtered with the ScreenCellStandard histological analysis of the tumor from the operative specimen was performed, according to the UICC TNM Classification , includiDue to the sample size, only summary statistics were applied.This is the first study to demonstrate CTCs in EAC after neoadjuvant therapy and surgery. It shows that detection of CTC by filtration in non-metastatic EAC is feasible. The rate of CTC positive findings and the quantity of CTCs changes in the course of multimodal neoadjuvant chemoradiation/chemotherapy and surgery. Future studies with larger sample sizes and multicenter design are currently recruiting and will give an answer regarding a question for the predictive impact of CTCs on treatment decisions for EAC.


The purposes of this study are to investigate whether the Characterizing Alzheimer’s disease Risk Events (CARE) index can accurately predict progression from mild cognitive impairment (MCI) to Alzheimer’s disease (AD) on an individual subject basis, and to investigate whether this model can be generalized to an independent cohort. Using an event-based probabilistic model approach to integrate widely available biomarkers from behavioral data and brain structural and functional imaging, we calculated the CARE index. We then applied the CARE index to identify which MCI individuals from the ADNI dataset progressed to AD during a three-year follow-up period. Subsequently, the CARE index was generalized to the prediction of MCI individuals from an independent Nanjing Aging and Dementia Study (NADS) dataset during the same time period. The CARE index achieved high prediction performance with 80.4% accuracy, 75% sensitivity, 82% specificity, and 0.809 area under the receiver operating characteristic (ROC) curve (AUC) on MCI subjects from the ADNI dataset over three years, and a highly validated prediction performance with 87.5% accuracy, 81% sensitivity, 90% specificity, and 0.861 AUC on MCI subjects from the NADS dataset. In conclusion, the CARE index is highly accurate, sufficiently robust, and generalized for predicting which MCI individuals will develop AD over a three-year period. This suggests that the CARE index can be usefully applied to select individuals with MCI for clinical trials and to identify which individuals will convert from MCI to AD for administration of early disease-modifying treatment. Mild cognitive impairment (MCI) has been conceptualized as a transitional clinical state between normal and Alzheimer’s disease (AD)-type dementia . MCI hasBuilding an effective and accurate prognostic model that predicts the progression from MCI to AD is clinically important. To this end, a large number of studies have combined magnetic resonance imaging (MRI)-based features with positron emission tomography (PET) ; cerebroSignificant progress has been made using machine learning tools. Most of the above-mentioned studies have integrated CSF and PET measures. This approach using multiple technologies  is often not available in clinical practice. In particular, while the prediction performance of these models has been validated extensively in a nested cross-validation loop  and the Recent work has begun to address this limitation. Using cortical thickness regions and subcortical volumes, Westman and colleagues combine Alzheimer’s Disease Neuroimaging Initiative (ADNI) data with the AddNeuroMed dataset  to prediHere, we will address this challenge with our newly developed framework, termed the characterizing AD risk events (CARE) index . The CARχ2 = 0.078, p = 0.78, see The demographic and neuropsychological characteristics of the included groups are listed in Both ADNI and NADS datasets showed that the CARE index differentiated P-MCI subjects from N-MCI subjects at baseline see . The comAs shown in As shown in To validate the generalizability of the CARE index to discriminate P-MCI subjects from N-MCI subjects, we applied the optimal the CARE index threshold (6.54) to discriminate between the P-MCI and N-MCI subjects in the ADNI dataset and the MCI subjects in the NADS dataset. We found that, of the 56 MCI subjects, 48 (85.7%) were predicted correctly. MCI subjects with a CARE index score above the threshold have a high diagnostic odds ratio  and relative risk  in MCI progression relative to non-progression to AD. See .As shown in The changes in CARE index score measured at baseline and the three-year follow-up were negatively correlated with changes in mini-mental state examination (MMSE) scores, and the composite Z scores of episodic memory, information processing speed, and visuospatial function in MCI (p < 0.05). See .The most novel result of this study is that the CARE index allows us to accurately predict MCI-to-AD progression on an individual subject basis across different cohorts.This study showed no differences in the conversion rates in individuals with MCI after a 3-year follow-up between ADNI (26.1%) and NADS (28.6%), which agrees with these findings based on ADNI that showed the conversion rates of 26 to 48% after a 3-year follow-up . This suggests our results are unbiased and convincing.Recently, an excellent study by Young and colleagues used an EBP model to develop a biomarker prognostic model that predicts the MCI-to-AD conversion over three years . They ob. However, they use a nested stratified cross-validation procedure to evaluate the predictive performance in the same dataset [A strength of this work is that the overall predictive performance of the CARE index was not only high but also has a fairly balanced sensitivity/specificity, which has significantly outperformed other existing work thus far. Several studies have developed models based on various combinations of CF, MRI, PET, and CSF biomarkers. They have applied these models to distinguish MCI converters from MCI non-converters over a three-year follow-up period, only attaining accuracies in the range of 0.49–0.75 and AUCs in the range of 0.64–0.76 , 21. The dataset , 29. Usi dataset . Using M dataset . However dataset , 33, res dataset . It is o dataset . Most imAnother principal novelty of this study is that we were able to replicate our results in an independent cohort, which suggests that the CARE index is a robust and accurate model with good generalizability. Recently, a few studies have begun to address the generalization bottleneck; however, their prediction performance could not be transferred from one cohort to other cohorts , 15. AndIn the current study, our findings support the fact that combining different biomarkers can predict the conversion status and cognitive decline. The CARE index associated with conversion likely reflects disease severity, i.e., how close an individual is to a significant clinical transition. The CARE index integrates biomarkers derived from behavior, structural, and resting-sate functional MRI modalities. It has important clinical advantages such as cost-efficiency, noninvasiveness, very high test–retest reliability, strong validity for AD pathological process, and clinical popularity. Most importantly, the excellent predictive performance of CARE index in a multicenter study (ADNI) can be applied in a clinical single-center study (NADS). Therefore, the CARE index can be generalized for use in precisely selecting individuals with MCI for a clinical trial. It can aid the development of new disease-modifying drugs by assigning possible surrogate markers of disease progression, and it can reduce the number of subjects needed to detect a significant drug effect . UltimatSome limitations of this study deserve comment. First, sample sizes of P-MCI and N-MCI subjects in the separate ADNI and NADS cohorts, especially the P-MCI subjects, are relatively small. However, it is important to note that our supplementary analysis combining ADNI and NADS cohorts still showed a very high power in predicting conversion from MCI to AD. Therefore, our results remain convincing. Second, we only used parts of MCI subjects from ADNI cohorts due to our entry criteria, which may affect our results. Finally, this study assessed only the predictive power of the CARE index during a three-year follow-up period. Future studies with longer and multiple follow-up times  are needed to refine and generalize our CARE index estimates. Furthermore, the diffusion tensor imaging (DTI) has been considered as an advanced technology on studying the MCI/AD patients. Therefore, the further studies will corroborate and extend high prediction performance of the CARE index and explore whether combining DTI, brain structural and functional MRI can improve this high prediction performance. Finally, other cognitive measures and non-invasive biomarkers may play crucial roles in improving prediction performance that were not addressed here (such as the CDR–sum of boxes and diffusion tensor imaging measures)In conclusions, the CARE index is sufficiently robust and generalizable for predicting which MCI individuals, across datasets, will develop AD over three years. It can be usefully applied to select individual subjects with MCI for clinical trials and to predict with high sensitivity and specificity for early treatment which individual subjects with MCI will convert to AD in the future.The data for study subjects were obtained from two independent datasets: ADNI and NADS.Baseline data used in this study were consistent with data in our previously published study . The detWe selected a total of 74 MCI subjects with a baseline diagnosis of amnestic MCI, based on the requirements in our previously published study . FinallyThe NADS study recruited 87 subjects with a baseline diagnosis of amnestic MCI status. Written informed consent was obtained from all of the participants, and the study was approved by the responsible Human Participants Ethics Committee of the Affiliated ZhongDa Hospital, Southeast University, Nanjing, China.All amnestic MCI subjects met the diagnostic criteria proposed by Petersen and colleagues  and the In the NADS dataset, all subjects underwent a standardized clinical interview and comprehensive neuropsychological assessments that were performed by neuropsychologists . These assessments included mini-mental state examination (MMSE), Mattis Dementia Rating Scale (MDRS); Auditory Verbal Learning Test–immediate recall (AVLT-IR); Auditory Verbal Learning Test–5-min delayed recall (AVLT-5-min-DR); Auditory Verbal Learning Test–20-min delayed recall (AVLT-20-min-DR); Logical Memory Test–immediate recall (LMT-IR); Logical Memory Test–20-min delayed recall (LMT-20-min-DR); Rey-Osterrieth Complex Figure Test (ROCFT); Rey-Osterrieth Complex Figure Test –20-min delayed recall (ROCFT-20min-DR); Trail-Making Tests A and B (TMT–A and B); Digital Symbol Substitution Test (DSST); Digit Span Test (DST); Stroop Color and Word Test A, B, and C; Verbal Fluency Test (VFT); Semantic Similarity (Similarity) test; and Clock Drawing Test (CDT). These tests were used to evaluate multi-domains of cognitive function, including general cognitive function, episodic memory, information processing speed, executive function, and visuo-spatial function, respectively. Note that all subjects in the NADS dataset had both scores of comprehensive neuropsychological assessment and at least one R-fMRI scan with corresponding anatomical scans at 3-year follow-up to investigate the links between the changes of characterizing AD risk event and the changes of neuropsychological performance. Furthermore, we have standardized the tests in the NADS dataset into the ADNI dataset when the cognitive measure scores obtained across different study datasets may have different testing composition.http://adni.loni.ucla.edu/. The details regarding image acquisition are provided in The ADNI data acquisition process is described at The details regarding image acquisition parameters are provided in Conventional preprocessing steps were conducted using Analysis of Functional NeuroImages (AFNI) software, SPM8, and MATLAB . The preprocessing allows for T1-equilibration (removing the first 15 s of R-fMRI data); slice-acquisition-dependent time shift correction (3dTshift); motion correction (3dvolreg); detrending (3dDetrend); despiking (3dDespike); spatial normalization ; averaging white matter and CSF signal retrieval (3dROIstats) using standard SPM white matter and CSF mask in the MNI space; white matter, CSF signal, and motion effect removal (3dDeconvolve); global signal removal necessity check  [FCI), the posterior cingulate cortex (PCCFCI), and the fusiform gyrus (FUSFCI); two gray matter concentration indices (GMI) from the hippocampus (HIPGMI) and fusiform gyrus (FUSGMI); two CSF biomarkers (Aβ1-42 and p-tau levels); and three cognitive biomarkers . Detailed methods to extract FCI and GMI are provided in According to our previously published studies , 10 welloptimal, is determined by the event-based probabilistic (EBP) model. The Soptimal sequence, in which these biomarker events occur, was well studied in our previously published work [The optimal temporal sequence, Shed work . The mathed work , 17 and optimal sequence of biomarker events with missing data is described in In the case of missing biomarkers, the mathematical detail of the Soptimal; collectively, these events comprise the index for CARE index. Each subject’s CARE index score is defined as that at which the order number had the highest likelihood value in Soptimal  test, and Mann-Whitney U tests were used to compare the differences in demographic data, neuropsychological performance, each individual biomarker feature, and the CARE index between N-MCI and P-MCI subjects. A statistical threshold was set at a p < 0.05.The statistical analyses were conducted with SPSS 22.0 software. The two-sample We evaluated the power of the CARE index and of individual biomarkers to discriminate P-MCI subjects from N-MCI subjects with the use of receiver operating characteristic (ROC) curves . To demoTo compare the stability and generalizability of the CARE index to discriminate between P-MCI and N-MCI subjects with those of individual biomarkers, we also applied the optimal classifier of each biomarker obtained from the ADNI dataset to the NADS dataset. To avoid unbalanced class frequency to lead to discrepancies between sensitivity and specificity , 35, we In addition, to avoid limitations due to the relatively small sample size and the differences in the predictive power of the CARE index between the ADNI and NADS cohorts due to the differences of MCI heterogeneity in general, we performed a supplementary analysis with the combined cohort of ADNI and NADS datasets.SI Methods S.13). The statistical threshold was set at a p < 0.05 (FDR-corrected).We performed a multiple linear regression model analysis to examine the relationships between the changes in CARE index and the changes in cognitive performance or clinical variables at baseline and the three-year follow-up in MCI subjects (See details in Supplementary Information


In this research, we propose a novel concept for a non-destructive evaluation of volatiles emitted from ripening grapes using solid-phase microextraction (SPME). This concept is novel to both the traditional vinifera grapes and the cold-hardy cultivars. Our sample models are cold-hardy varieties in the upper Midwest for which many of the basic multiyear grape flavor and wine style data is needed. Non-destructive sampling included a use of polyvinyl fluoride (PVF) chambers temporarily enclosing and concentrating volatiles emitted by a whole cluster of grapes on a vine and a modified 2 mL glass vial for a vacuum-assisted sampling of volatiles from a single grape berry. We used SPME for either sampling in the field or headspace of crushed grapes in the lab and followed with analyses on gas chromatography-mass spectrometry (GC-MS). We have shown that it is feasible to detect volatile organic compounds (VOCs) emitted in-vivo from single grape berries (39 compounds) and whole clusters (44 compounds). Over 110 VOCs were released to headspace from crushed berries. Spatial (vineyard location) and temporal variations in VOC profiles were observed for all four cultivars. However, these changes were not consistent by growing season, by location, within cultivars, or by ripening stage when analyzed by multivariate analyses such as principal component analysis (PCA) and hierarchical cluster analyses (HCA). Research into aroma compounds present in cold-hardy cultivars is essential to the continued growth of the wine industry in cold climates and diversification of agriculture in the upper Midwestern area of the U.S.  Understanding the development of flavor and aroma compounds in wine grapes is crucial to winemaking. Grape berry development is characterized by two sigmoidal growth periods. The first growth period is berry formation from fruit set to lag phase. This is followed by berry-ripening from veraison to harvest . VeraisoV. vinifera . This is expected since vinifera was cultivated as early as the seventh and fourth millennia BC [V. labruscana Bailey and V. vinifera L.). Levels of linalool, hexanal, (E)-2-hexenal, hexanol, and (Z)-3-hexene-1-ol, nerol in berry skins and pulp were influenced by storage temperatures post-harvest [Interest in research of aroma compounds in wine grapes is high. The prevailing share of published research in wine grapes has been in ennia BC . For exaennia BC ,5. Theseennia BC . The decennia BC . Terroirennia BC  and Cabeennia BC . Aroma c-harvest . Viticul-harvest , and eno-harvest . The bulSince the release of high-quality, cold-hardy, and disease-resistant cultivars from the University of Minnesota, the winemaking industry has grown in cold climates such as the upper Midwest region of the U.S. St. Croix, Frontenac, Marquette, and La Crescent cultivars were developed in 1983, 1996, 2006, and 2002, respectively . Currentcis-3-hexenol to trans-2-hexenal during ripening of Frontenac and Marquette berries grown in Quebec, through the destructive blending of the berries [trans-2-hexenal, 1-hexanol, cis-3-hexenol, hexanoic acid, acetic acid, beta-damascenone, and 1-phenylethanol. Marquette had significantly higher levels of linalool, geraniol, and alpha-citral [E,Z)-2,6 nonadienal, beta-damascenone, ethyl octanoate, and isoamyl acetate were compounds with the highest odor activity values (OAV) in wines [There is a gap in knowledge, especially in aroma research, with these interspecific, cold-hardy hybrid grapes. Previous work showed a constant decrease in the ratio of alpha-linolenic acid degradation products,  berries . Frontena-citral . Continuin wines . The OAVin wines ,19,20. Ein wines .Citrus paradise L.) [Myrtillocactus geometrizans) [Various methods of sample preparation have been used to characterize aromas from grapes and wine. Thermal desorption was used to determine volatiles from Solaris wine . Solid pdise L.) , berry ctrizans) , Shine Mtrizans) , Cabernetrizans) ,26, Zuostrizans) , Muscat trizans) , Nero d’trizans) , Monastrtrizans) , and seltrizans) ,17 and ctrizans) ,32,33,34V. vinifera. There is a need for a better understanding of these new cultivars in order to produce quality cold-climate wines that can compete in the world market. To date, this research is the first report of aroma compounds (1) emitted in-vivo from veraison to harvest using two novel sampling methods and collected by SPME and analyzed by GC-MS from Frontenac, Marquette, St. Croix, and La Crescent grape cultivars.A review of SPME use for in-vivo and in-vitro in whole plant and plant organ analysis is found elsewhere . It is cIn this research, we propose a novel concept for a non-destructive evaluation of volatiles emitted from ripening grapes using SPME. This concept is novel to both the traditional vinifera grapes and the cold-hardy cultivars. Our models are cold-hardy varieties in the upper Midwest for which many of the basic multiyear grape flavor and wine style data is needed. Research into aroma compounds present in cold-hardy cultivars is essential to the continued growth of the wine industry in cold climates and diversification of agriculture in the upper Midwestern area of the U.S. The need for data is confounded by the small resources available to conduct long-term research. If proven feasible, the concept of non-destructive analysis of ripening grapes presents a tantalizing possibility to investigate the effects of different viticulture practices throughout the stages of berry ripening on berry aroma. This, in turn, could be used to develop better quality wines. If volatile compounds emitted in-vivo could be identified as developmental biomarkers, portable target VOC detectors could then be developed. These detectors can give vineyards a real-time gauge to guide them in harvesting for flavor.The main objective was to develop the proof-of-concept for a non-destructive (in-vivo) sampling of volatile compounds from growing and ripening grapes. Specific objectives (1–4) were to (1) develop sampling devices to capture volatiles emitted from a whole cluster and single berry; (2) characterize the volatile compounds emitted in-vivo from four cold-hardy grape cultivars using: (2i) whole cluster analysis, (2ii) single berry analysis; (3) compare volatile compounds emitted in-vivo (objective 1) with crushed berry ; (4) search for preliminary links between volatile compounds detected (objectives 1 and 2) and selected: (4i) microclimates (Iowa and South Dakota), (4ii) the individual cultivars , and (4iii) time stages of berry ripening. The working hypotheses were: (1) aroma compound development from veraison-to-harvest can be detected in-vivo by sampling volatile emissions from ripening grapes (from both a single berry and whole grape cluster) and (2) that the flavor accumulation  can be correlated with berry ripening in all four cultivars. Testing these hypotheses can potentially translate into improving viticulture practices that lead to timing the harvesting for flavor. This research aims at addressing the gap in knowledge for cold-hardy grape cultivars by cataloging VOCs from Frontenac, Marquette, St. Croix and La Crescent emitted in-vivo and whole crushed berries throughout berry ripening.In this research, non-destructive and destructive sampling methods for the detection of VOCs emitted from cold-hardy grapes were explored. Non-destructive sampling included (1) a use of polyvinyl fluoride (PVF) chambers temporarily enclosing and concentrating volatiles emitted by a whole cluster of grapes on a vine , and (2)A total of 124 VOCs were identified across all sampling methods, 79 of these VOCs were verified with analytical standards matching retention times and mass spectral data  target library search with at least 80% mass spectral match. Target libraries included (a) the 6 libraries that are included with the AMDIS program, (b) an onsite (our laboratory) library created from analysis of pure standards (200+ compounds), (c) NIST11 mass spectral library described in Materials and Methods section on data analysis). A full summary of VOCs identified in Frontenac, Marquette, St. Croix, and La Crescent berries from South Dakota and Iowa by each sampling method is provided in data paper  with knoPVF chambers were used in 2012 on Frontenac and Marquette in Iowa and South Dakota. Modified glass vials were used in 2013 on Frontenac, Marquette, St. Croix, and La Crescent in South Dakota. Only St. Croix and La Crescent were sampled by modified glass vials in Iowa, limited by funding. Forty-four of the total 124 grape VOCs emitted in-vivo were detected by whole grape cluster sampling chambers in Frontenac and Marquette cultivars grown in Iowa and South Dakota, monitored from veraison to harvest. 3-Methyl-1-butanol and heptanal were emitted and detected in 2012 Iowa Frontenac grapes at veraison. At harvest, 1,4-butanolide was detected . Nonanal2012 Iowa Marquette did not have a ‘representative’ VOC at veraison, as indicated by HCA, and replicate samples had high variability . By harvest, 1-hexadecanol and methyl ethyl ketone had developed. Similarly, 2012 South Dakota Marquette VOCs emitted at veraison  did not Thirty-nine VOCs emitted in-vivo were also detected by modified glass vial (vacuum assisted) method in Frontenac, Marquette, St. Croix, and La Crescent cultivars grown in Iowa and South Dakota. In-vivo detection of VOCs by modified glass vial did not identify a key representative compound in 2013 South Dakota Frontenac grapes at veraison . At harvVOCs detected by modified glass vial emitted from 2013 Marquette grown in South Dakota generally did not vary during berry development. The variability decreased between the replicate samples, indicated by less spread between the data points as the berries developed. Aromas from berries grown in South Dakota during the 2013 growing season  can be cStatistical analysis of VOCs detected from 2013 St. Croix grown in Iowa at veraison and harvest determined 3 important compounds. These compounds were 3-methyl indole, benzyl alcohol, and acetic acid. Decreased variability between replicate samples was observed as the berries ripened, although no strong associations were noticed between these compounds and berry development. Compounds emitted and detected in 2013 St. Croix from South Dakota  were 1,4VOCs detected by modified glass vial emitted from 2013 La Crescent grown in Iowa were highly variable at veraison. A characteristic compound  was determined to be present at veraison. By harvest, octanal was present but not statistically representative. La Crescent berries from 2013 grown in South Dakota  were hig117 grape VOCs were detected by destructive analysis  in Frontenac, Marquette, St. Croix, and La Crescent cultivars grown in Iowa and South Dakota. The sample matrix included skins, pulp, and seeds. Crushed berry analysis was used in 2012 on Frontenac and Marquette cultivars grown in South Dakota, and all 4 cultivars in 2013. A freezer malfunction in resulted in the loss of Iowa 2012 berries stored for crushed berry analysis. VOCs detected after crushing the berries of Frontenac grapes from the 2013 growing season in Iowa were isovaleraldehyde and isoamyl acetate at veraison. At harvest, VOCs were cyclohexanol and) and 3-methyl-1-butanol, as shown in E)-2-hexenoic acid were most associated with Marquette berries at veraison. By harvest, these compounds shifted to styrene, beta-cyclocitral, and nonanal  from water . This noNon-destructive, sampling of VOCs emitted in-vivo from cold-hardy grapes was conducted using 2 methods. PVF film sampling chambers with custom SPME sampling port was used to monitor whole cluster VOC emissions. Modified glass vials supported SPME sampling of individual berries. For comparison to both non-destructive methods, a random 5 berry sample was collected, crushed, and analyzed under controlled laboratory conditions. Statistical analysis using PCA indicated that sampling by PVF chambers and modified glass vials detected similar VOC emission profiles across all 4 cultivars. There was 1 outlier from the glass vial method, indicating a higher than average concentration of styrene in La Crescent grapes. This data could provide evidence of styrene as a product of 2-phenylethanol synthesis from yeast cells  (p. 309)Berry VOCs were sampled within a 3-month growing period each year for 2 years. VOC profile in 2012, sampled by PVF chambers and crushed berries were similar in profile. Data points from the PCA fall close to the origin throughout the year in 2012, not shown. This indicated that VOCs collected via PVF chambers did not show noticeable changes from veraison to harvest. VOC profile in 2013, sampled by modified glass vials and crushed berries were similar in profile (points near the origin) until the end of August, with the exception of the outlier on 14 August 2013, Differences in microclimate of Iowa and South Dakota plots did not affect VOC emissions from 4 cold-hardy grape cultivars. Little difference in VOC emissions is expected from Marquette and Frontenac because of a shared pedigree. Greater changes in VOC emissions was observed between destructive crushed berry analysis and non-destructive in-vivo analysis methods, but not within the non-destructive methods. In Iowa and South Dakota plots, VOCs emitted from St. Croix and La Crescent cultivars continued to change from veraison through harvest. VOCs emitted in-vivo from Frontenac and Marquette cultivars in South Dakota started to decline 8 days and 3 days before harvest, respectively. More research is warranted in order to make recommendations to viticulturists regarding ideal harvest time for maximum aromas in the cold-hardy grapes. Linking correlations between viticultural practices can enhance the quality of wines for new cold-climate cultivars.Several improvements to the proposed in-vivo sampling are warranted. Addition of internal standard (IS) , for exaA detailed description of Materials and Methods is provided elsewhere . BrieflySampling chambers (~5 L volume) for the non-destructive collection of in-vivo volatiles were made from a PVF film and held firm with clean aluminum wire cage framing. Preconditioned (cleaned) PVF chambers were fitted with custom sampling ports for insertion of SPME needles. Typical sampling time was 30 min. A standard 2 mL glass vials were modified by removing flat bottoms  at a glass shop. The edges of were flared and rounded. A half hole septa was added to the screw top to support the SPME needle. The SPME fiber was placed through the septa prior to sampling. After assembly and placement of the vial apparatus on the individual berry , 5 mL ofBerries were collected from each cultivar on the same day and time of in-vivo sampling. Five berries were collected from clusters adjacent to the cluster tagged for in-vivo sampling . Collected berries were frozen prior to analysis and stored in a −20 °C freezer. Berries collected in South Dakota were also frozen and shipped on ice overnight for analysis in Iowa. Frozen berries were hand-crushed in the lab, placed into 20 mL amber screw top vials  with PTFE/silicone septa. A CTC CombiPal  was used for automated SPME sampling. Briefly, the vials were agitated and heated to 50 °C for 10 min, followed by 30 min agitated headspace sampling using 65 μm PDMS/DVB SPME fiber. The fiber was thermally desorbed under a flow of helium prior to each sample exposure. These sampling parameters were determined, not shown.m/z to 350 m/z. Scans were collected in electron ionization (EI) mode with an ionization energy of 70 eV. MS heated zones for quadrupole and source were 150 °C and 230 °C, respectively. Daily tuning of the MS was performed with perfluorotributylamine (PFTBA) before each analysis.A custom multidimensional GC was used , built on a standard Agilent 6890 platform . System automation and data acquisition software were MultiTrax  and ChemStation . Chromatography was performed on two capillary columns connected in series. The first column was 5% phenyl polysilphenylene-siloxane  with a fixed restrictor pre-column. The second polar column was bonded polyethylene glycol in a Sol-Gel matrix . The midpoint between the two columns was maintained at a constant pressure of 0.39 atm by a pneumatic switch. In this research, all effluent from the first column was directed into the 2nd analytical column, i.e., no heartcutting was performed. The instrument was also equipped with a flame ionization detector (FID). Flow to the FID can also directed at the midpoint, but FID was not utilized in this research. True multidimensional analyses were not performed, i.e., the system was used in full heartcut mode, meaning separation was performed on both columns in series. Effluent from the second polar column was simultaneously directed to a single quadrupole MS  and an olfactometry (sniff) port  via an open spit interface at atmospheric pressure. The sniff port is equipped with a purge flow controller and supplied with humidified air at 0.54 atm. Flow to the MS and sniff port is determined by fixed restrictor columns, 1 part to MS and 3 to sniff port. Olfactometry was not utilized in the research. The GC inlet was operated in splitless mode at 250 °C. GC oven parameters start with an initial temperature of 40 °C, held for 3.0 min, followed by a 7 °C per min ramp to 240 °C, held for 8.43 min. Total run time was 40 min. Carrier gas is ultra-high purity (UHP) helium . Temperature of the sniff port and MS transfer lines were 240 °C and 280 °C, respectively. MS full scan range was set from 34 Identification of compounds was performed using Automated Mass Spectral Deconvolution and Identification System (AMDIS) target library search with at least 80% mass spectral match. Target libraries included (a) the 6 libraries that are included with the AMDIS program, (b) an onsite library created from analysis of pure standards (200+ compounds), (c) NIST11 mass spectral library. Analysis of variance (ANOVA) was performed using XLSTAT 2016.04.33113 . The effects of cultivar, site, sampling time, and sampling methods and their interactions on volatiles emitted were analyzed using ANOVA  followed by post-hoc  test. Multivariate analysis was performed using JMP Pro 12.0.1 .We have shown that is feasible to detect VOCs emitted in-vivo from single grape berries (39 compounds) and whole clusters (44 compounds). Over 110 VOCs were released to headspace from crushed berries. Spatial (vineyard location) and temporal variations in VOC profiles were observed for all four cultivars. However, these changes were not consistent by growing season, by location, within cultivars, by ripening stage when analyzed by multivariate analyses such principal component analysis (PCA) and hierarchical cluster analyses (HCA). Research into aroma compounds present in cold-hardy cultivars is essential to the continued growth of the wine industry in cold climates and diversification of agriculture in the upper Midwestern area of the U.S.


This diagnostic study uses Health and Retirement Survey and linked Medicare data to develop and validate an administrative approach to identifying older adults with fall-related injuries and to quantify the inclusiveness and validity across a spectrum of potential diagnoses of fall-related injuries. International Classification of Diseases, Ninth Revision (ICD-9)–coded injuries in older adults?What are the tradeoffs between inclusiveness and validity when expanding the external mechanism-of-injury codes (E-codes) for fall-related injury to include ICD-9 codes, the acute care algorithm maximized validity and the inclusive algorithm prioritized milder injuries seen in outpatient clinic care, doubling the number of fall-related injuries captured compared with E-codes alone.In this diagnostic study of 13 939 older adults (≥65 years) in the nationally representative Health and Retirement Study, an external reference standard of patient interview (E-codes) was used as evidence that a fall caused the injury. With use of hospital and emergency department ICD-9–coded injuries to the existing method of using only fall-related E-codes.The findings suggest that health care systems can increase inclusion of fall-related injury by adding  National injury surveillance systems use administrative data to collect information about severe fall-related trauma and mortality. Measuring milder injuries in ambulatory clinics would improve comprehensive outcomes measurement across the care spectrum.To assess a flexible set of administrative data–only algorithms for health systems to capture a greater breadth of injuries than traditional fall injury surveillance algorithms and to quantify the algorithm inclusiveness and validity associated with expanding to milder injuries.International Classification of Diseases, Ninth Revision codes that extend FRI identification with administrative data beyond the use of fall-related E-codes was developed: an acute care algorithm , a balanced algorithm , and an inclusive algorithm . Data were collected from January 1, 1998, through December 31, 2012, and statistical analysis was performed from August 1, 2016, to March 1, 2019.In this longitudinal diagnostic study of 13 939 older adults (≥65 years) in the nationally representative Health and Retirement Study, a survey was conducted every 2 years and was linked to hospital, emergency department, postacute skilled nursing home, and outpatient Medicare claims (2000-2012). During each 2-year observation period, participants were considered to have sustained a fall-related injury (FRI) based on a composite reference standard of having either an external cause of injury (E-code) or confirmation by the Health and Retirement Study patient interview. A framework involving 3 algorithms with Validity, measured as the proportion of potential FRI diagnoses confirmed by the reference standard, and inclusiveness, measured as the proportion of reference-standard FRIs captured by the potential FRI diagnoses.Of 13 939 participants, 1672 (42.4%) were male, with a mean (SD) age of 77.56 (7.63) years. Among 50 310 observation periods, 9270 potential FRI diagnoses (18.4%) were identified; these were tested against 8621 reference-standard FRIs (17.1%). Compared with the commonly used method of E-coded–only FRIs , FRI inclusion was increased with use of the study framework of algorithms. With the acute care algorithm , validity was prioritized  over inclusiveness . The balanced algorithm showed a 2-year incidence of 14.6% , inclusion of 65.3% , and validity of 83.2% . With the inclusive algorithm, the number of potential FRIs increased compared with the E-code–only method .International Classification of Diseases, Ninth Revision codes may increase inclusion of FRIs by health care systems compared with E-codes and that these algorithms may be used by health systems to evaluate interventions and quality improvement efforts.The findings suggest that use of algorithms with  On the other hand, including mild injury  would favorably increase the power to detect intervention associations, which depended heavily on the rate of outcomes. In addition, mild injuries are important causes of pain and accelerated disability in older people.12 The inclusion of mild injuries in surveillance systems may reduce the validity of the FRI diagnoses while increasing the total number of FRIs identified because not all mild injuries are fall related.As health care systems develop fall injury reduction programs, monitoring of programs' effectiveness requires surveillance of both severe and moderate injuries . There are important tradeoffs involved in broadening surveillance systems beyond the use of only E-codes. On the one hand, such injuries as hip fracture and head trauma are likely to be FRIs because falls are the most common cause of severe, traumatic injury among older adults.16 We enhanced this work by examining inclusiveness and validity across a spectrum of potential inpatient and outpatient FRI diagnoses.Earlier research involving fall surveillance has extended claims-based diagnoses beyond E-codes without explicitly considering tradeoffs of inclusiveness and validity.STARD) reporting guideline.This longitudinal diagnostic study used Health and Retirement Study (HRS) Core and Exit (postmortem) surveys  and linked Medicare claims from inpatient hospital, ambulatory (emergency department and outpatient clinics), and nursing home claims files from January 1, 1998, through December 31, 2012. We included older (age ≥65 years) HRS respondents who consented to providing their Medicare beneficiary numbers for linking their interviews to fee-for-service Medicare (Parts A and B) claims data. The study was approved by the University of Michigan institutional review board; informed consent was waived because this study was a secondary analysis of deidentified data. Data were collected from January 1, 1998, through December 31, 2012, and statistical analysis was performed from August 1, 2016, to March 1, 2019. The study followed the Standards for Reporting of Diagnostic Accuracy  FRI diagnoses beyond fall-related E-codes, patient-reported FRIs  were added to E-coded FRIs  and external (interview) data validation sources. By adopting this next best reference standard, test properties were calculated and they prioritized the injury codes by validity. Because this ICD-9–based algorithm was intended to measure FRIs in future practice, the primary criterion for prioritizing ICD-9 codes as more valid was higher positive predictive value (proportion of tests events confirmed by the reference standard). Thus, further references to validity in this report were synonymous to positive predictive value. Validity was operationalized as a tradeoff with inclusiveness  as the scope of injuries considered to include milder injuries was broadened. This approach was only possible by considering the reference standard, even if imperfect, as a true measure of FRI.To develop a set of ICD-9 diagnosis codes 800-999) excluding burns, splinters, bites, and fractures associated with metastatic bone disease. The remaining diagnoses were considered as potential FRIs to be tested against the reference standard. Next, we created a matrix of potential FRI categories consisting of 3 injury types by 4 anatomic regions. Injury types were bone fractures, sprains, strains, dislocations, and superficial skin injuries. Regions were the head, neck or trunk, upper limb, and lower limb. Finally, a nonspecific and internal injury category was created  encounters, we identified all injuries . These 2-year observation windows were the unit of analysis for testing whether potential FRI episodes were validated by the reference standard, . The earliest date within the FRI episode was used to determine which observation window to use . The useICD-coded injury diagnoses was calculated by ascertaining the copresence of a reference-standard FRI with the test FRI episode within the same observation window , any emergency department or short hospital stay (<3 days), and outpatient clinic only data.Within each level of health care acuity, injury categories were consolidated to simplify the matrix of injuries and reduce categories with low sample sizes. We screened the least-valid categories  for outlier diagnoses with poor validity . These diagnoses were removed from their original categories and placed into a distinct, generalized category of nonvalidated injuries.The results were used to develop a framework involving 3 algorithms that extend FRI identification with administrative data beyond the use of FRI E-codes: (1) an acute care algorithm identifying the most valid and severe injuries, (2) a balanced algorithm occupying a middle ground in validity and inclusion, and (3) an inclusive algorithm, which maximizes inclusion of FRIs found across the health care system. To develop the framework, we sorted the consolidated injury categories from the most to the least valid within each health care acuity level and sequentially included the categories according to their validity. In this way, we quantified the loss in validity (and gain in inclusiveness) as additional, less-valid FRI categories were added. Specifically, E-coded FRIs were the first category to be included in each algorithm because they were, by definition, the most valid administrative data . Then, beginning with acute care settings, we sequentially added the next most valid of the potential FRI categories to E-coded FRIs, reporting the loss in validity and increase in inclusiveness of the resulting cumulative FRI algorithm at each step.22 validity of the final algorithms was presented among the FRIs less than 6 months before the interviews.These steps were then sequentially repeated for consolidated FRI categories across less acute health care settings until all data were considered in the algorithm framework. To address known recall bias resulting from interviewing patients about FRIs,There were 20 334 participants (aged ≥65 years) in the HRS who participated in 83 769 biennial interviews from 2000 through 2012. Of these, 13 939 individuals allowed HRS to link their survey data with fee-for-service Medicare data, resulting in a final analytic sample of 50 310 two-year observation windows. The sample of 13 939 participants was 42.4% male (1672 participants), with a mean (SD) age of 77.56 (7.63) years.ICD-9–coded potential FRIs . After consolidating injury types, the most valid injury category from higher-acuity care settings  were validated at more than 70%. A post hoc sensitivity analysis , which was a 65.7% increase compared with E-codes only, and was 83.2%  valid and 65.3%  inclusive (a 27% increase compared with E-codes only).The inclusive algorithm, which extends beyond hospitals and emergency department care into outpatient clinic care, revealed an incidence of FRIs of 17.4% , which was a 98.2% increase compared with E-codes only, and was 75.2%  valid and 68.4%  inclusive (a 32.8% increase compared with E-codes only).Validity of all 3 algorithms was improved with use of the UCLA/RAND method (requiring procedure codes as inclusion criteria to identify FRIs exclusively diagnosed in outpatient emergency departments and clinics), with the greatest improvement observed for the inclusive algorithm (from 53.2% to 75.2%)  was not included in any of the 3 final algorithms. Adding these remaining injuries would have identified more cases (19.7% incidence and 70.2% inclusiveness) but would have reduced validity (68.6%) .23In this study, we developed and validated an administrative approach using injury diagnostic data sets from across health settings to identify older adults with FRIs ranging from high to low acuity, improving on existing methods that rely on E-codes for detection of fall-related health care. The result was a flexible framework of algorithms that traded off in validity and inclusiveness, with implications for surveillance and quality improvement measurement. Using only high-acuity FRIs, the acute care algorithm was the most valid but least inclusive, with 88.6% of its test FRIs confirmed by the reference standard and approximately 62.1% of all reference-standard FRIs captured by test FRIs. By examining outpatient clinical care diagnoses, the inclusive algorithm had a 32.8% increase in reference-standard FRIs captured by test FRIs with a decrease in validity (from 90% to 75%). Together, these results suggest that by balancing validity with inclusiveness, FRIs of varying severity can be broadly identified using commonly used administrative databases that go beyond the use of E-codes.27 used FRI algorithms that were limited to only E-coded injuries, patient interview, or medical record review (limited by cost). In comparison with previous algorithms,16 we developed more comprehensive algorithms, including less severe injuries  while removing less valid injury diagnoses. This was achieved by leveraging the thousands of injuries in Medicare data that were validated against E-codes and interviews from the nationally representative HRS—a process not possible in other data sets. The large sample of injuries also allowed us to identify injury categories within health care acuity levels to refine the algorithms. This study was also the first, to our knowledge, to offer its algorithm codes for public use.Previous work30 to reduce fall risk among individuals33and within communities.38 The acute care algorithm identified 43% more FRIs compared with the E-code only approach while maintaining strong validity; thus, it may be a valid tool for increasing statistical power to detect significant changes resulting from an intervention . Because of its broader scope but reduced validity, the balanced algorithm can serve to identify fall outcomes for lower-risk quality improvement initiatives in primary care, such as promoting use of the Medicare annual medical wellness visit or for improving care of older persons who fall.40 The inclusive algorithm was the most comprehensive approach (doubling the number of FRIs compared with E-codes only) and included more superficial injuries. By excluding the least-valid injuries while maintaining most other injuries, it captured the most FRIs of the 3 algorithms and therefore may be the most useful for public health monitoring. For individuals who are more frail, this measure captures even the minor injuries that lead to fear of falling and loss of independence.We propose that this algorithm framework can facilitate assessment of interventions44 and clinical practice improvement efforts to reduce falls49 to work, valid tools to measure their effectiveness will be required. However, although large health care systems are increasingly responsible for monitoring quality and outcomes, they often lack the tools to do so. Medicare also does not have critical population-wide fall surveillance for older adults, although falls are one of the leading causes of death among older adults.50 Instead, present approaches to monitoring fall injuries and their costs,51 including data from the US trauma system,52 focus on fall-related death and more severe injury and ignore falls treated in outpatient clinics, which represent a large proportion of older adult falls. Our algorithms can be considered a step toward addressing these limitations and improve monitoring.Each of these surveillance tools can contribute to ongoing efforts in fall prevention. For population-based efforts53 In addition, triangulating diagnostic codes with both patient report and clinician notes would be a resource-intensive but rigorous way to further develop an administrative algorithm for FRI identification.49 These approaches may not be as broadly generalizable as our present claims-based approach given the intersystem differences in electronic health record use, type, and data availability but may be a step toward more accurate monitoring of falls.As increasing attention is paid to falls in an aging US population, one direction is to capture clinical events in electronic health records. Next steps include developing methods to capture falls reported by patients by telephone, email, or electronic visits either by requiring diagnoses for these alternative means of accessing health care or by using natural language processing to read clinical notes.22 and a modest decline in reporting accuracy 1 year after an FRI. In the present analysis, we used relative ranking of injury types to develop the algorithms. Given that the injury types were randomly distributed in time, the 2-year time frame should not bias our results. Second, use of International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) diagnosis codes became mandatory as of 2015, with expansion of both cause of injury and diagnostic injury codes. We believe that these changes will not increase the validity of reported FRIs because there have been no concomitant changes in the mandates for reporting of falls by clinicians. To facilitate future use of the algorithms, we provided proposed ICD-10 codes corresponding to our ICD-9–based algorithms (eTable 11 in the ICD-10 algorithms. Third, the validity of our algorithms may be biased downward because of unreported FRIs occurring within nursing homes and hospitals. One future improvement would be to identify additional reference-standard FRIs in the minimum data set given standard reporting requirements for falls that are mandatory in nursing facilities.We noted 3 limitations. First, because of the HRS design, we could only examine patient reports of FRIs every 2 years. Instead of being contemporaneous, patient report of fall injuries involved a 2-year recall period. Previous research suggested that accuracy of recall using falls calendars declined after 6 monthsICD codes for injuries. By accounting for variability in the validity but also the contribution to overall inclusiveness when adding ICD codes, this research organized the codes into algorithms that can be tailored to the measurement needs of health systems.The findings suggest that fall injury data should be collected broadly in health systems and for use in public health monitoring by leveraging both the validity of E-codes and


In-stent restenosis (ISR) is still a major cause of failure of endovascular stenting treatment in patients with lower extremity arterial occlusive disease (LEAOD). Sensitive and reliable biomarkers for early diagnosis to predict ISR should be considered. This study was conducted to explore the diagnostic value of microRNA in predicting ISR in patients with LEAOD after endovascular stenting treatment. From March 2014 to July 2016, 208 patients  with LEAOD undergoing interventional treatment were enrolled in this research. Patients were divided into the restenosis and non-restenosis groups according to routine postoperative angiography. Circulating microRNAs expression were detected in 208 participants, including 78 ISR patients, 68 non-ISR patients and 62 healthy volunteers. We selected 6 microRNAs from microarray screening as candidates for further testing via qRT-PCR. A receiver operating characteristic (ROC) curve was generated to assess the diagnostic value of circulating microRNAs in predicting ISR for LEAOD patients. The results showed that circulating microRNA-320a and microRNA-572 in patients with ISR (n = 78) had significantly higher expression levels than it from non-ISR and healthy volunteers. By receiver operating characteristic curve analysis, the sensitivity was 82.1% and the specificity was 63.8% for microRNA-320a; the sensitivity was 69.2% and the specificity was 68.9% for microRNA-572, and the AUC was 0.766 and 0.690 for detection of ISR, respectively. Furthermore, 78 patients with ISR had significantly higher circulating expression levels of microRNA-3937 and microRNA-642a-3p and lower circulating expression levels of microRNA-4669 and microRNA-3138 compared with 68 non-ISR patients and 62 healthy volunteers, but they have no significant difference. We found that differential circulating microRNA expression in patients after stenting with ISR, and the data indicate that circulating microRNA-320a and microRNA-572 have promising value in diagnosing ISR in patients with LEAOD. The use of drug-eluting stents (DESs) has drastically reduced the incidence of in-stent restenosis (ISR), as compared with standard balloon angioplasty (POBA) in patients with superficial femoral artery diseases2. In spite of these advantages, ISR is still an important clinical problem. Therefore, a sensitive and reliable biomarker for early diagnosis to predict ISR in patients with lower extremity arterial occlusive disease after stenting would be ideal.Currently, endovascular stenting treatment, such as self-expanding nitinol stents treatment, has become one of the primary treatment in patients with obstructive atherosclerotic disease and have been shown to be superior to percutaneous transluminal angioplasty (PTA)3. Increasing evidence indicates that microRNAs regulate diverse cellular functions, such as proliferation4 and apoptosis5. Therefore, these microRNAs must have been association with the development of cardiovascular diseases. Zahedi et al. proposed that Dicer expression plays a vital role by reducing smooth muscle cells (SMCs) proliferation in vascular repair after vascular injury by generating anti-proliferative microRNAs, such as microRNA-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation6. Some studies have shown that microRNAs can stably exist in the circulating blood and can be used as a biological marker for the diagnosis of disease7. Circulating miRNA-21, miRNA-143, miRNA-145, and miRNA-100 showed differences between ISR patients groups and non-ISR patients groups, and these miRNAs were closely related to the occurrence of ISR after drug eluting stent implantation in coronary artery disease (CAD) patients9. Yu et al. detected serum microRNA-143 expression by using quantitative real-time polymerase chain reaction (qRT-PCR) and found that microRNA-143 can be a promising tool for predicting the ISR in patients with lower extremity arterial occlusive disease10. The purpose of this study was to investigate the expression of circulating microRNA from microarray screening and qRT-PCR and assessed its diagnostic value in patients after stenting with and without ISR, and to provide evidence for early detection of restenosis after stenting.MicroRNAs (microRNAs) are a kind of small (approximately 22 nucleotides), single-stranded noncoding RNAs, which control various biological functions and play important roles in physiologic and pathologic processes by inducing mRNA degradation and translation interruptionThis study was approved by the Ethical Committee of the Affiliated Changhai Hospital of Second Military Medical University, and we confirmed that all research was performed in accordance with relevant guidelines and regulations. All the participants have signed the informed consent before enrollment.From March 2014 to July 2016, 208 consecutive symptomatic patients  with LEAOD who underwent treatment in the Department of Vascular Surgery of Shanghai Changhai Hospital of Second Military Medical University were enrolled in this study. All patients were diagnosed with LEAOD at different levels based on computed tomography angiography (CTA). The inclusion criteria were as follows: (1) major lesions of femopopliteal arteries; (2) at least one distal run-off below the knees after the intervention. Exclusion criteria were as follows: (1) patients who refused to undergo Doppler ultrasound review; (2) patients who did not take anti-platelet aggregation drugs after surgery; (3) patients who had ISR in two or more locations and received revascularization therapy within 1 month after the diagnosis of ISR; (4) patients who had connective tissue diseases, or malignancy.Blood samples for microRNAs detection were obtained from all patients and were processed within 1 hour of collection by two-step centrifugation. Plasma was transferred to 1-mL nuclease Eppendorf (EP) tubes and refrigerated in an ultra-low-temperature refrigerator at −80 °C.Total RNA was taken out from plasma using the microRNA Neasy Mini Kit  in accordance with the instructions from the manufacturer. The concentration and purity of the RNA solution were detected by NanoDrop ND-2000 Spectrophotometer . The samples absorbance at 260/280 and at 260/230 nm were used for further analyses. All of the purified RNA samples were stored at −80 °C for further processing.The expression of candidate microRNAs was measured in the plasma of every sample by human MicroRNA Microarrays  . The microRNAs samples were labeled by the Complete Labeling and Hyb Kit , and hybridized with the Cy3-tagged RNA. Quantile algorithm (Gene Spring Software 12.6) was used to normalize the raw data. The prediction analysis of microarrays (PAM) was used for microarray analysis.Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was carried out to test the candidate microRNAs acquired on microarray. The RNA sample with high quality was used for reverse transcribed reaction, which was performed by the TaqMan microRNA Reverse Transcription Kit . Then qRT-PCR reaction was carried out by Taqman microRNA assays  in an ABI PRISM 7900HT Sequence Detection System . The total volume of the reaction was 10 μL, including 2 μL cDNA template, 5 μL of Universal Master Mix  and 3 μL water. The primers used in our study were designed in accordance with the sequences of the candidate microRNAs. All reaction mixtures were incubated in a 96-well optical plate at 95 °C template denaturation for 10 min, followed by 40 cycles at 95 °C for 15 seconds and at 60 °C for 1 minute. Each reaction was repeated in triplicate. The microRNAs which showed Cycle threshold (CT) values correspond to the number of cycles required for the amplified product to reach a critical threshold of detection. The circulating levels of microRNAs were analyzed by two investigators who were blinded to the clinical data of patients.Technical success was defined as reestablishment of direct flow to the distal artery with residual stenosis of <30% in the femoral arteries. Clinical success was defined as a subjective perception of improved walking distance, resolution of resting pain, or healing of ischemic ulcer, resulting in at least one category improvement based on the Rutherford classification together with an increase in ankle brachial index (ABI). Patency was determined by duplex ultrasonography in all patients, and contrast-enhanced computed tomography was performed if clinically indicated. Restenosis was defined as >2.4 of peak systolic velocity ratio determined by duplex ultrasonography.SPSS 18.0 statistical software was used for data analysis. The data continuous variables are summarized and presented as mean ± standard deviation (SD) or median. One-way ANOVA was used to assess the differences among the three cohorts. The categorical variables among the three groups were assessed using Chi-square tests. Receiver operating characteristic (ROC) analysis by statistical software was used to evaluate the diagnostic performance of the potential microRNAs. P < 0.05 was considered statistically significant.A total 208 participants, including 78 ISR patients, 68 non-ISR patients and 62 healthy volunteers were enrolled in this study and followed up. The baseline characteristics of the study subjects are compared in Table To observe the differential expression levels of microRNAs among patients with ISR, non- ISR and healthy volunteers, we performed gene expression microarray analysis. The microarray analysis showed significant changes in microRNAs, which were up-regulated or down-regulated in ISR groups compared with non- ISR and healthy volunteers Fig. . All assThe 6 candidate microRNAs were tested using the entire sample set with qRT-PCR, and all of them passed quality control. Among the microRNAs, microRNA-320a and microRNA-572 had a significantly difference between the ISR groups and control groups (P < 0.001). While there was no significant difference in microRNA-3937, microRNA-642a-3p, microRNA-4669 and microRNA-3138 between the ISR groups and control groups. The results are summarized in Table ROC analysis was conducted in the 6 candidate microRNAs between ISR and control groups Table . ROC ana11. However, the incidence of ISR restricts the development of the endovascular techniques and ISR is the most frequent and major problem after stent implantation for treatment of LEAOD12. Although some researchers have attempted to prevent the occurrence of ISR, we still do not have an appropriate treatment for ISR13. It is an urgent problem to solve ISR in the treatment of LEAOD and increasing attention has been directed toward the correlative factors of ISR in LEAOD after stent implantation.The present study is to investigate the prognostic value of microRNA in predicting ISR in patients with LEAOD undergoing peripheral angioplasty with endovascular stenting treatment. Endovascular treatment by stent implantation may be considered a first-line treatment option for LEAOD with significant effective, safety and less trauma14. Gareri C et al. focused on microRNAs, which are involved in the regulation of SMCs migration and proliferation15. In addition, they explored the microRNAs related to the platelet activation and endothelial dysfunction, which can lead to both stent thrombosis and in-stent restenosis. Endothelial-derived microRNAs are involved in the modulation of neointima formation enhancement and endothelial recovery impairment. To date, several miRNAs have been shown to participate in the regulation of angiogenesis in the context of endothelial cells. The previous study reported that miR-16 has been shown to be differentially expressed in endometriosis, in which angiogenesis may be involved in the growth of the endothelium and miR-16 participates in the inhibition of angiogenesis in multiple ways16. Sorrentino et al. found that endothelial miR-16 is remarkably upregulated after vascular injury in hind limb ischemia and shows a negative effect on endothelial repair and remote vascular remodeling at the carotid artery17.The key pathophysiologic phenomenon of restenosis after stenting is due to the generation of neointima, which is essentially caused by the proliferation of VSMCs and subsequent abluminal migrationBlood-based biomarker test is convenient, low cost, fast and effective that may be a promising approach for routine screening. In this study, we found that the levels of circulating microRNAs were significantly altered in ISR patients. In particular, our microRNA screening revealed that patients with ISR had significantly differential expression levels of microRNA-320a, microRNA-572, microRNA-3937, microRNA-4669 and microRNA-3138 and microRNA-642a-3p when compared with control groups. From entire sample set testing with qRT-PCR and ROC analysis, the patients with ISR had significantly higher expression levels of microRNA-320a and microRNA-572, reveling their highest diagnostic accuracy for detection of ISR in patients with LEAOD.8, which promoted SMCs proliferation, migration, and neointima formation19. Also, microRNA-143 was reported to play a vital role in modulating VSMC phenotypes20, and microRNA-143 has proposed to be used for their diagnosis and prognosis for postoperative restenosis10. Circulating microRNA-195 predicts target lesion restenosis, atherothrombotic events and target vessel revascularization after infrainguinal angioplasty with stent implantation in patients with peripheral artery disease21. MicroRNA-133a is associated with a higher incidence of major adverse cardiac events in patients with CAD. The differential modulation of miR-133a release in the coronary circulation may reflect pathophysiological mechanism involved in CAD complications and suggest a novel potential role in the development of in-stent restenosis22. However, in the present study, microRNA-145, microRNA-195, microRNA-133a and microRNA-143 were undetectable in almost half of the patients with LEAOD, which were significantly different from cardiovascular diseases. Therefore, these microRNAs may not be suitable as biomarkers for risk stratification of PAD patients, although they have an important role in pathophysiology of restenosis and could represent interesting therapeutic targets15.In earlier studies, microRNAs are known as vital regulators for many cellular mechanism and some researches have showed that circulating microRNAs are related to ISR in peripheral and coronary artery disease. Plasma microRNA-143 and microRNA-145 were reported to be the potential biomarkers for in-stent restenosis25. More specifically, microRNA -320a plays functional roles in regulating the vascular endothelial cell function via targeting neuropilin 126. It has been reported that microRNA -320a plays an important role in the progression of atherosclerosis and endothelial apoptosis27. It has been proved that the over-expression of microRNA-320a in ischemic cardiomyopathy and aortic stenosis28. Subsequently, microRNA-320a expression is reported to be associated with potential cardiovascular target genes29. The human microRNA-572, in particular, has a length of 20 nucleotides and is located in chromosome 430. Recent studies have found that the level of microRNA-572 in blood is altered in different human diseases, such as postoperative cognitive dysfunction31, multiple sclerosis33, ovarian cancer34 and nasopharyngeal carcinoma35. However, we did not find any different expression of microRNA-572 in cardiovascular diseases from former literatures and it was the first time to find the different levels of microRNA-572 in the patients with LEAOD.Numerous evidences indicated that microRNAs showed evidence of vital role in regulating gene expression, which are involved in the pathological process of cardiovascular diseasesOur study has some limitations. First, the sample size of enrolled patients was not very large and a larger sample with a longer follow-up is needed to confirm the diagnostic role of these microRNAs in patients with LEAOD. Second, many factors, such as lifestyle and dietary factors, may affect the incidence of ISR. Third, a future multicenter, randomized, prospective study should be designed and performed to illustrate the relationship between microRNAs and ISR.We found that differential circulating microRNA expression in patients after stenting with ISR, and the data indicate that circulating microRNA-320a and microRNA-572 have promising value in diagnosing ISR in patients with LEAOD. Larger sample with longer follow-up is needed to confirm the diagnostic role of these microRNAs in patients with LEAOD.


The implantologists frequently prescribe antibiotics, analgesics and anti-inflammatories in dental implant surgery. The aims of this study were to evaluate the attitudes of implantologists in Murcia (Spain) to prescribing antibiotics, analgesics and anti-inflammatories in healthy patients during different implant dentistry procedures, and to see how these are influenced by individual dentist’s academic level, professional experience, and ongoing training (attending courses or reading scientific literature on medication use) This cross-sectional study included a total of 200 implantologists from the Murcia area (Spain), who each completed a two-page questionnaire consisting of 26 questions. The implant procedure in which most dentists (n=97) prescribed antibiotics was multiple implant surgery with flap raising, in which 55.6% of these 97 respondents used a prophylactic antibiotic regime for 7 days after implant placement. All subjects (n=200) prescribed analgesics for eight out of the eleven procedures included in the survey and anti-inflammatories in six. Dentists with higher academic levels or longer professional experience prescribed more antibiotics, but those who underwent continuous training (attending courses or reading scientific literature) reduced antibiotic prescription. Dentists often prescribed antibiotics, analgesics and anti-inflammatories in almost all implant procedures in healthy patients, but ongoing training reduced the frequency of antibiotic prescription in some procedures. Key words:Antibiotics, analgesics, anti-inflammatories, dental implant, oral surgery. In recent years, the prosthetic rehabilitation of missing teeth by means of dental implants has become a common treatment, which has proved very accepTableto patients, mainly due to its high success rates . AlthougMeanwhile, concepts regarding the management of post-operative symptoms in implant dentistry have undergone important modification in recent years in response to the advances in our understanding of the physiopathological bases of pain and inflammation, as well the pharmacodynamics of the analgesics and anti-inflammatories used in their treatment. In this way, the tendency is now to prevent post-operative pain and inflammation through pre-operative drug administration, which together with classic post-operative medication will combat post-surgical symptoms effectively ,9.As for anti-inflammatory treatment, non-steroidal anti-inflammatory drugs (NSAIDS) are the most widely used drugs in the world, with an estimated average usage of 80 Tablets per person per year . For thiIn light of these controversial findings and the lack of consensus – only one scientific paper has studied antibiotics prescribing practices in different dental implant procedures  – and thMaterials and Methods- Study designThis transversal observational study included a convenience sample of 200 dentists registered with the College of Dentists of Murcia (Spain), all with at least one year’s clinical experience in dental implant placement. All participants were volunteers and received no remuneration. The study was conducted during the period June 2015 to February 2016.Inclusion criteria were: dentists registered in Murcia with clinical experience of dental implant placement of at least one year. Exclusion criteria were: subject not a dentist and/or insufficient (less than one year) clinical experience in dental implant placement.- Sample size calculationIn June 2015, there were a total of 1,060 dental professionals registered with the College of Dentists of Murcia (Spain). Having made contact with these individuals by telephone, or E-mail, a total of 430 subjects were found to fulfill the inclusion criteria. To calculate a representative sample size, a power of 80% was required , which determined the sample size as 210. After inviting 210 subjects to take part, 10 refused to participate, leaving a final sample size of 200.- Study questionnaire The 200 respondents completed a two-page questionnaire consisting of 26 questions, 115 in face-to-face interviews, 35 by phone, and 50 by e-mail.et al., . Descriptive statistics were calculated for each variable. The associations between the different qualitative variables were analyzed using Pearson’s chi-squared test. Statistical significance was accepted for p≤0.05.The sample group was made up of 200 dentists (58 men and 142 women) from the Murcia region (Spain), all with experience in dental implant placement of at least one year (mean experience 5.04 ± 3.27 years). Descriptive analysis of the study sample showed that 68% were graduates, 25.5% had completed post-graduate Master’s studies, and 6.5% had graduated as medical doctors. Most of the respondents (77.5%) did not attend ongoing training courses in “the use of antibiotics in implant dentistry,” 81% did not attend (ongoing training) courses in “the use of analgesics in implant dentistry,” and 66.5% did not receive ongoing training in “the use of anti-inflammatories in implant dentistry” .The antibiotic of choice was amoxicillin with clavulanic acid (72.5%) or amoxicillin alone (27.5%). When the patient presented a penicillin allergy, clindamycin was the antibiotic of choice in 100% of prescribing subjects. Paracetamol was the most widely used analgesic (80.5%), while NSAIDS were the most commonly used anti-inflammatories (95.5%) .When the possible influence of academic level, professional experience, and ongoing training (through course attendance and/or reading scientific literature) on the use of antibiotics, analgesics and anti-inflammatories during implant treatment , it was The exponential growth of implant dentistry has been accompanied by increasing indications for the uses of antibiotics, analgesics and anti-inflammatories in dental implant surgery. Antibiotics are administered to prevent post-operative infection; if an implant becomes infected the likelihood that it will be lost is high . So implet al., . Comparing the present results with Abukaraky et al.,  the lattet al., ; similar results were obtained by Wasan et al.,  who foun prescribed analgesics for eight out of the eleven procedures included in the questionnaire, and two even used them at the time of impression taking. All respondents (n=200) prescribed anti-inflammatories for six out of the eleven procedures . The lack of consensus regarding the use of anti-inflammatories and the disparity in pain and swelling management are noTable. Trauma In our study, the use of phone or e-mail allowed to complete the 200 questionnaires, because 85 implantologists could not complete it by face-to-face inteviews. These technologies improve recruitment for studies about attitudes and behaviors, including increasing professionals contacts, maximizing convenience for participants, and emphasizing interpersonal relationships between researchers and participants .One limitation that the present study suffered was the difficulty of comparing the results with other works, due to the scarcity of similar research into approaches to antibiotic prescription and the complete lack of articles relating to analgesic and anti-inflammatory use. In the future, when a consensus regarding antibiotic, analgesic and anti-inflammatory regimens in implant dentistry procedures has been reached, further studies will be necessary to assess whether implantologists are following the guidelines established.In conclusion, the present study showed that implantologists frequently prescribe antibiotics, analgesics and anti-inflammatories in different procedures in healthy patients; practitioners with higher academic qualifications and longer professional experience prescribe more antibiotics, while prescriptions are fewer when the implantologist undergoes ongoing training by means of course attendance and reading scientific literature.


We report the case of a patient with B-cell prolymphocytic leukemia who wassuccessfully treated with the novel humanized monoclonal antibody obinutuzumab.This patient was previously treated with the combination of rituximab andbendamustine and had recurrent infusion reactions. Her treatment with rituximaband bendamustine was discontinued when she developed disease progression after 3cycles of therapy. She was then treated with obinutuzumab 1000 mg on day 1 ofevery cycle and chlorambucil 0.5 mg/kg on days 1 and 15 every 28 days to whichshe had greater tolerability. After 4 cycles of treatment, she had resolution ofher clinical symptoms, massive splenomegaly, and normalization of her whiteblood cell count. CD5 and CD23expression is often weak or absent. CD11c, CD103, CD10, and CD25 are not expressed.1 Tumors demonstrating t must be tested by either conventionalcytogenetics, fluorescence in situ hybridization, or by immunohistochemical stainsfor cyclin D1 to exclude the diagnosis of mantle cell lymphoma.B-cell prolymphocytic leukemia (B-PLL) is a rare mature B-cell neoplasm thattypically occurs in the elderly population. This disease is clinically characterizedby a rapidly rising lymphocyte count, splenomegaly, fever, night sweats, and weightloss. Lymphadenopathy is not generally a prominent feature of this malignancy. Thediagnosis of B-PLL is made on a combination of immunophenotypic and genetic findingsin the peripheral blood and bone marrow. By definition the diagnosis requires thatthe prolymphocytes exceed 55% of all lymphoid cells in the peripheral blood.2 Despite advances in the understanding of tumor biology, optimal treatmentoptions have not yet been identified and there are no randomized studies availablefor clinical reference. Treatment strategies have therefore been fashioned from thatof similar but more common neoplasms including chronic lymphocytic leukemia (CLL)and mantle cell lymphoma. Conventional chemotherapy has been used in the pastincluding combination regimens such as cyclophosphamide, doxorubicin, Oncovin, andprednisolone (CHOP), which has yielded only partial responses. In addition, theseresponses are not durable with relapses usually occurring within 12 months. Anotheroption for selected patients is allogeneic stem cell transplant. This offerscurative potential and there are reports of long-term remissions exceeding 5 years.Allogeneic stem cell transplant, however, is best reserved for the few, younger andfit patients with this disease. It is associated with a high risk of morbidity andmortality. More recently, there have been case reports of treatments with thecombination of the anti-CD20 monoclonal antibody, rituximab, and chemotherapy, whichhave produced excellent responses.2 There are, however, no known reports of the more novel humanized type IIanti-CD20 monoclonal antibody obinutuzumab in the treatment of B-PLL. Obinutuzumabhas been compared head to head with rituximab and has yielded superior results inthe management of CLL in a pivotal phase III trial.3 This drug is approved by the Food Drug Administration (FDA) for first-lineuse in CLL and is a preferred regimen in the elderly.B-PLL frequently follows an aggressive clinical course and has historically beenassociated with a poor prognosis with an estimated median overall survival of 3 years.9/L, respectively. Her white blood cell count was 8.7 ×109/L with 67% lymphocytes and 5% atypical lymphocytes. Theperipheral smear showed abundant prolymphocytes  havebeen reported to have activity in B-PLL.710The development of monoclonal antibodies have had a major impact in altering thenatural history of both malignant and benign immune-mediated diseases. Rituximab isthe first FDA licensed and still the most commonly used anti-CD20 monoclonalantibody in B-cell malignancies.11 Obinutuzumab is a type II anti-CD20 monoclonal antibody that recognizes thesame CD20 epitope as rituximab but binds to it in a different orientation and over alarger surface area, allowing a superior induction of direct cell death, enhancednatural killer cell activation, and antibody-dependent cell cytotoxicity. It is,however, less potent in inducing complement-dependent cytotoxicity.3 The mechanism of action of obinutuzumab, in contrast to that of rituximab andofatumumab, may provide greater efficacy.11 For this reason, obinutuzumab-based treatment may display a superior responsein patients with B-PLL as was evidenced in our case.Functionally anti-CD20 monoclonal antibodies may be classified as either type I ortype II. The type I anti-CD20 monoclonal antibodies, rituximab and ofatumumab, leadto complement-dependent cytotoxicity, stimulation of signaling leading to apoptosis,and antibody-dependent cell-mediated cytotoxicity through the recruitment of immunemediator cells.12 The efficacy and safety data from this study served as the catalyst for usingthis regimen in this case.The FDA authorized the use of obinutuzumab in combination with chlorambucil in 2013for the elderly and frail patient with treatment-naïve CLL. Approval was grantedbased on the results of the phase III CLL-11 study. This 3-arm study compared thecombination of obinutuzumab and chlorambucil versus rituximab and chlorambucilversus chlorambucil alone. The results of this study showed marked superiority ofthe obinutuzumab combination in response rates and survival compared with therituximab-based regimen and chlorambucil alone. The median progression-free survivalwas 26.7 months with obinutuzumab-chlorambucil versus 16.3 months withrituximab-chlorambucil versus 11.1 months with chlorambucil alone. Safety andtolerance to treatment was another key measure shown in this study. Less patientsdied from an adverse event with obinutuzumab-chlorambucil (4%) than in therituximab-chlorambucil and chlorambucil-alone groups .13 This oral alkylating agent was first approved for use in 1957 for thetreatment of CLL. The use of chlorambucil has since largely fallen out of favor inpreference to more novel agents, which have improved response rates. It is stillindicated for use in the elderly and frail patient due to its safety and tolerance.Improved complete response rates and progression-free survival are seen whenchlorambucil is combined with an anti-CD20 monoclonal antibody.The authors of this report also acknowledge that chlorambucil has proven efficacy inthe treatment of CLL and other lymphoproliferative disorders. It may have thereforecontributed significantly to the disease response observed in this case. In asystematic review by Lepretre et al, response rates of 31% to 72% were reported withchlorambucil monotherapy in the treatment of CLL; however, complete responses wereas low as 0% to 10%.In conclusion, the combination of obinutuzumab and chlorambucil may have a promisingrole in the treatment of patients with B-PLL and deserves consideration either inthe frontline setting or in the subset of patients who progress on rituximab-basedregimens. More studies are needed to investigate the efficacy of this regimen inpatients with B-PLL and the optimal length of therapy.