Biopac recorded 3-lead ECG. A camera placed at a 25 cm distance recorded a video stream (100 fps) of a finger illuminated by a laser diode at 639 nm. A chest belt (Polar) monitored respiration. All signals were recorded simultaneously during episodes of spontaneous breathing and paced breathing. Analysis: Post-processing was performed in Matlab to obtain SPG and analyze the SPG, PPG and fiAP mean absolute deviations (MADs) on PWM, plus PAT modulation. Results: Across 2599 beats, the average fiAP MAD with PPG was 0.17 (0–1) and with SPG 0.09 (0–1). PAT derived from ECG–fiAP correlated as follows: 0.65 for ECG–SPG and 0.67 for ECG–PPG. Conclusion: Compared to the clinical NIBP monitor fiAP reference, PWM from an experimental camera-derived non-contact reflective-mode SPG setup resembled fiAP significantly better than PPG from a simultaneously recorded clinical transmission-mode finger clip. For PAT values, no significant difference was found between ECG–SPG and ECG–PPG compared to ECG–fiAP. Objective: The goal was to compare Speckle plethysmography (SPG) and Photoplethysmography (PPG) with non-invasive finger Arterial Pressure (fiAP) regarding Pulse Wave Morphology (PWM) and Pulse Arrival Time (PAT). Methods: Healthy volunteers (n = 8) were connected to a Non-Invasive Blood Pressure (NIBP) monitor providing fiAP pulse wave and PPG from a clinical transmission-mode SpO This work focuses on comparing both Speckle plethysmography (SPG) and Photoplethysmography (PPG) with non-invasive Finger Arterial Pressure (fiAP) regarding Pulse Wave Morphology (PWM) and respiration-induced Pulse Arrival Time (PAT) modulation.SPG is a technology that is derived from Laser Speckle Contrast Imaging (LSCI)  by analysolely depends on blood flow: human blood vessels are flexible, and pressure waves cause the blood vessel walls to expand and contract, thereby compressing the surrounding tissue. This movement will also be perceived by SPG, additionally to the streaming blood cells and should not be neglected. Hence, we studied the influence of blood pressure pulse waves.Although this formed a solid investigation to show that flow modulations correlate well with SPG ,3,4, morThe most accurate way of measuring dynamic blood pressure is invasive (via a catheter) . Blood pUpcoming non-invasive methods use bio-signals, usually PPG, and assess blood pressure through Pulse Wave analysis PWA , Pulse AThis study aims to compare simultaneously recorded SPG and PPG waveform morphologies with the pressure waveform measured by a clinical Finger Arterial Pressure (fiAP) monitor. In addition, this study also compares ECG–fiAP PAT against ECG–PPG and ECG–SPG PAT measurements. With this, we intend to shed light on the possible use of SPG for blood pressure estimation.2 finger clip with output of the near-infrared PPG pulse wave. The reference device used as Non-Invasive Blood Pressure (NIBP) waveform monitor was Finapres Nova . The devA Biopac MP160 was used to record reference ECG, respiration from a chest belt (Polar), the PPG and fiAP signals coming from Finapres NOVA and the common trigger pulses for the laser and camera (for synchronization).A camera  was placed at 25 cm distance to record 100 Laser Speckle Contrast Images per second of a finger being illuminated by a laser diode at a wavelength of 639 nm . The approximated coherence length of the laser diode was 0.9 mm . Camera The study protocol was approved by the institutional ethical committee and device safety board. We recruited 8 healthy volunteers and connected them to the setup as shown in All signals (including synchronization pulses from the microcontroller) were recorded simultaneously  during episodes of spontaneous breathing (3 min) as well as paced breathing  and analyzed for morphology mean absolute deviation (MAD) and PAT modulation . During the experiments, the operator and volunteers wore laser safety glasses. The video sequences were processed to obtain SPG using a standard deviation sliding window (sized 7 × 7 pixels) to scan each video frame, following the equation : SPG  of finger-clip PPG and reflective SPG against a continuous finger Arterial Pressure reference waveform (fiAP). The morphological analysis consisted of the following steps see also : First, MAD values provide an objective absolute calculation of the morphological difference among different signals. It is a convenient method to compare signals coming from different devices with similar morphology.The temporal feature chosen for the secondary analysis was Pulse Arrival Time (PAT). This variable was calculated by measuring the time elapsed between the ECG R-peak and the foot of the fiAP (used as reference), PPG and SPG signals. variations should be comparable .Offset values of PAT are not meaningful because the signals are obtained from different devices, which implies that they have different time delays. However, the PAT t-test for testing the null hypothesis that MAD PPG and MAD SPG are from populations with equal means at the 5% significance level. This would demonstrate whether the difference between MAD values inter-subject is significant for PPG versus SPG. Additionally, we performed two more t-tests to check if the difference in MAD mean values was significantly different between periods of spontaneous versus paced breathing for PPG and SPG.For the morphological analysis, MAD values were calculated beat-to-beat from PPG as well as SPG signals for all subjects. We performed a paired For the temporal analysis, PAT values were calculated beat-to-beat from PPG, SPG and fiAP for all subjects. To analyze the similarity between fiAP and the signals under investigation, intra-subject correlation coefficient was calculated between fiAP and PPG and compared with the correlation coefficient between fiAP and SPG. Finally, an average correlation coefficient was calculated for all subjects.In total, 2599 beats were analyzed . A visual inspection of the SPG, PPG and fiAP morphologies revealed a higher resemblance of the SPG waveform with the fiAP waveform compared to that of PPG, see t-test  to test the null hypothesis that the MAD values from PPG and SPG are from populations with equal means. The t-test showed that the difference in the mean between SPG and PPG MAD is significant (p < 0.0001), rejecting the null hypothesis at the alpha significance level. We performed a paired To check whether the similarity in morphology was not dominantly present for spontaneous or paced breathing, we repeated the morphology analysis discriminating between episodes of spontaneous and paced breathing, see t-tests on MAD PPG and MAD SPG between episodes of spontaneous and paced breathing did not reveal a significant difference, neither for SPG nor for PPG. Both for spontaneous and paced breathing, the remote SPG morphology remained significantly more similar to fiAP than contact PPG.Separate paired t-test results show no significant difference between the PAT correlation coefficients derived from contact PPG and remote SPG when comparing to the fiAP reference: PAT fiAP vs PAT PPG (mean 0.673 ± 0.46) and PAT fiAP vs PAT SPG (mean 0.658 ± 0.47). Average peak-to-foot amplitudes were calculated for SPG and PPG as a surrogate of signal quality between subjects, shown as AC (Alternating Component) PPG (mean 1.25) and AC SPG (mean 0.0084) in Even if we discarded the two subjects with the poorest peak-to-foot values  but have a lower SNR for PPG, whereas SPG excels in reflection mode [PPG PAT has been investigated as a marker for arterial stiffness and BP assessment ,19. Remoion mode . Within the literature, the PPG waveform is often regarded as a proxy of the local arterial blood pressure waveform, and the origin of PPG is mostly explained by the pulsatile volumetric dilatation of the arterial system . In addiIn previous work on SPG , the utiBoth types of vessels could have different impacts on SPG, and this would give a different explanation to SPG’s origin, depending on the location .Another interesting fact is that the applied NIBP fiAP reference device uses an optical probe in the transmission mode (highly resembling PPG) to measure continuous BP in the finger . Neverthremote SPG can be at least as accurate as contact PPG for beat-to-beat interval measurement when comparing it to ECG [In our present study, we discovered no significant difference between contact PPG and remote SPG for PAT measurement. Nevertheless, in a previous study, we found that t to ECG . PAT froMovement artifacts and their suppression mechanisms were not investigated during this study. Yet, it is known that both PPG and SPG are sensitive to movement artifacts , especiatransmissive PPG device and an experimental camera-derived non-contact reflective SPG setup, to the fiAP waveform from a clinical NIBP monitoring reference device. Both pulse wave morphologies and PAT modulations were compared. In this work, we compared a standard contact (finger clip) experimental setup, using a laser with a coherence length of 0.9 mm) showed a significantly higher resemblance to fiAP regarding waveform morphology than PPG from a clinical transmission-mode finger clip. A correlation between PAT derived from SPG was found similar to PAT from PPG when compared to fiAP with the present setup. In contrast to the expected behavior, our findings reveal that the contactless reflection mode SPG (from an same photons, seems a logical next step to investigate how to harvest the best of both modalities in order to advance the field of peripheral circulation monitoring.The realization of an improved probe that would combine SPG and PPG in the same device, deriving both signals from the


Background: Psycho-oncological support (PO) is an effective measure to reduce distress and improve the quality of life in patients with cancer. Currently, there are only a few studies investigating the (expressed) wish for PO. The aim of this study was to evaluate the number of patients who request PO and to identify predictors for the wish for PO. Methods: Data from 3063 cancer patients who had been diagnosed and treated at a Comprehensive Cancer Center between 2011 and 2019 were analyzed retrospectively. Potential predictors for the wish for PO were identified using logistic regression. As a novelty, a Back Propagation Neural Network (BPNN) was applied to establish a prediction model for the wish for PO. Results: In total, 1752 patients (57.19%) had a distress score above the cut-off and 14.59% expressed the wish for PO. Patients’ requests for pastoral care (OR = 13.1) and social services support (OR = 5.4) were the strongest predictors of the wish for PO. Patients of the female sex or who had a current psychiatric diagnosis, opioid treatment and malignant neoplasms of the skin and the hematopoietic system also predicted the wish for PO, while malignant neoplasms of digestive organs and older age negatively predicted the wish for PO. These nine significant predictors were used as input variables for the BPNN model. BPNN computations indicated that a three-layer network with eight neurons in the hidden layer is the most precise prediction model. Discussion: Our results suggest that the identification of predictors for the wish for PO might foster PO referrals and help cancer patients reduce barriers to expressing their wish for PO. Furthermore, the final BPNN prediction model demonstrates a high level of discrimination and might be easily implemented in the hospital information system. Cancer patients have to endure a high burden of distress . DistresUsually, PO includes a broad range of services, such as cost-free counseling centers close to home and external or internal psychiatric respectively psychological consultations . There aIn a larger context, PO represents various aspects of social support, which can be divided into social-emotional (relationships rich in affection), instrumental , problem-oriented (focused on the resolution of a specific problem) and daily support ,21. PercAs well as these barriers to PO for patients with cancer, there have been some studies about factors, which predict the wish for PO. For instance, low emotional wellbeing, lack of social support, younger age, higher level of education, emotional burden unrelated to the disease, lung and breast cancer , currenthus, they may not engage in support services even if those are offered. Dee et al. highlighted a growing proportion of cancer patients in the U.S. who experience financial worries and distress due to healthcare costs [Finally, different healthcare systems across the globe may have some influence on the wish for PO. Although there have been many improvements in providing PO for patients with cancer during the last decades , financire costs . On the Our study aimed (1) to evaluate the number of patients who wish for PO and (2) to identify predictors for the wish for PO in a large patient population with all types of cancer. As a novelty, this study also aimed (3) to build a prediction model for the wish for PO in patients with cancer based on the Back Propagation Neural Network (BPNN). Previous research is based on conventional statistical methods, such as logistic regression analysis. However, these statistical procedures cannot fully represent the multifactorial and intertwined causes for the wish of PO in patients with cancer, and thus, tend to be inaccurate. On the other hand, BPNN is based on the Artificial Neural Network (ANN), which simulates the structure and function of the human brain. Being an adaptive, self-learning and self-organizing system, ANN is able to identify complex non-linear relationships between variables . MoreoveIn this study, we retrospectively included 3063 patients with an initial diagnosis of cancer who were diagnosed and treated at the Comprehensive Cancer Center Zurich (CCCZ) at the University Hospital Zurich. Data were collected from the case files of cancer in- and outpatients between 2011 and 2019. The procedure of selecting the study sample is depicted in The data in our study represent all types of cancer. The proportions of the type of cancer are illustrated in Distress, as defined in the introduction, was assessed with the distress thermometer (DT), which is a commonly used screening tool. Cancer nurses are requested to administer the DT repeatedly during inpatient treatment and outpatient visits in patients with cancer . The DT p-value) was set at p < 0.05. We used IBM SPSS Statistics, version 27  for statistical analysis and Matlab  for BPNN computations. Mean scores, standard deviation and relative and absolute distributions were calculated to describe the prevalence of the wish for PO, distress scores and patient characteristics. An unpaired t test was conducted to compare differences between the number of patients who wish for PO and distress scores. To identify factors that predict the wish for PO, we performed a binary logistic regression analysis with the wish for PO (yes/no) as the dependent variable. In the first step, we analyzed associations of sociodemographics see also  and otheIn a BPNN, information migrates from input layers through hidden layers to output layers. The information in this network only moves in one direction. Hence it is called a feedforward network. To minimize differences between the final calculated output and the target output from the training data, iterations are made based on the error rate obtained in the previous run. By reducing error rates, the BPNN model will become more reliable and generalizable. In this way, BPNN is a standard method of training and adaptively controlling artificial neural networks ,38.p < 0.001). Regarding sex differences, women wished more often for PO and were more frequently distressed than men.The absolute and relative distribution of the wish for PO and the associated distress scores are illustrated in X2 = 1864.462, p < 0.001). The model explained 36.0% of the variance (Nagelkerke R2) and correctly classified 82.2% of cases, indicating high model quality. The model had a sensitivity of 37.8% and a specificity of 98%.In the current study, the BPNN consisted of one input layer, one hidden layer and one output layer. Prior studies already demonstrated that this typical three-layer BPNN is a reasonable compromise between model accuracy and network complexity . Figure Matlab randomly divided the data into a training sample (70%), a validation sample and a test sample (15% each). In our BPNN model for patients’ wishes for PO, the hidden layer neurons are in a range between four and 13. Theoretically, a higher coincidence rate (π) and a lower number of iterations lead to a higher BPNN model accuracy. A different number of hidden layer neurons results in different evaluation indexes ) and varying numbers of iterations. Thus, BP neural network training and repetitious data simulation processes were carried out under different numbers of hidden layer neurons. Matlab indicated that the highest BPNN model accuracy was achieved in a range between seven and nine neurons see . Another issue of the BPNN training is that different values of the weights coefficient (w) and the threshold value (b) lead to different learning process models. Since the initial value of (w) and (b) is a random number between −1 and 1, repetitious data simulation processes with different initial values of (w) and (b) and a number of hidden layer neurons from seven to nine were performed via Matlab. Comprehensively considering the evaluation indexes and the number of iterations, Network 3 with eight neurons in the hidden layer presents the optimal BPNN model for patients’ wishes for PO . It has The confusion matrices demonstrate a high coincidence rate (π) for each sample  in clinical settings that explored incidence rates of distress and the wish for PO in patients with cancer ,4,5,18. The large gap between reported distress and the wish for PO is of note. A possible explanation for this discrepancy might be a phenomenon characterized as a “normality paradox” [paradox” . Some paparadox” . Moreover, the cultural and medical context of PO involving different healthcare systems, treatment regimens and lifestyles in the U.S., Europe, Asia and across the globe should also be considered ,34. PercIn our study, we identified nine predictors that each independently influenced the wish for PO. Younger age (in line with a prior German study ) and femInterestingly, a new finding of our study is that patients’ requests for pastoral care and social services support predicted the wish for PO and additionally demonstrated the highest odds ratios (OR= 13.1 and 5.4). It is not surprising that distressed patients in need of psychosocial support request all the help they are offered. We also assume that patients might struggle to differentiate between the specific value of the different entities. On the other hand, PO, social services and pastoral care can be highly synergistic in reducing patients’ distress. For instance, social services reduced distress in patients with cancer significantly  . Cancer As a novelty, we established a prediction model for patients’ wishes for PO based on BPNN including all nine significant variables determined by logistic regression. In sum, it demonstrates a high level of discrimination, which is comparable to our regression analysis model. Although the established BPNN model is of clinical significance, it cannot reach its full potential due to the relatively low number of independent variables. However, the present study provides evidence that BPNN is able to accurately manage real-world data. Compared to conventional methods, BPNN has the advantage of better individual prediction accuracy and precise data fitting based on its adaptability and simulation of the human brain. Moreover, BPNN is better suited to handle a large sample with different participants’ characteristics due to its ability to identify complex non-linear relationships between variables ,40,41,42Strengths and LimitationsOur study has several strengths, such as the large study sample, including all types of cancer and treatment options and utilizing advanced statistical approaches. Still, there are some notable limitations. Our study utilized data from a single CCC in Switzerland, which limits generalizability. However, the quality of the data and of the obtained results is high, because the documentation of patient-related information in the present study followed strict institutional standards. An important limitation is the fact that some types of cancer, such as gynecological, urological and endocrinological malignant neoplasms, were underrepresented in the current study see . GüntherThe present study highlights a discrepancy between patients with a distress score above the cut-off value and those that wish for PO. With this study, we identified novel predictors for the wish for PO. Considering these factors could help increase PO utilization, thereby reducing distress and improving QoL in patients with cancer. Improved discrimination between the content of PO, social services and pastoral care as well as strengthening interprofessional collaborations might help to better meet patients’ individual needs. Future studies could also compare the percentage of patients who expressed the wish for PO with those who actually received it and investigate whether patients are involved in psychotherapy sessions. Patients might not be fully aware of all possible multifactorial causes for their wish for PO. BPNN is able to identify those complex relationships between variables that otherwise might have been overlooked. Moreover, the implementation of BPNN prediction algorithms in the hospital information system might help identify patients who need PO more easily.


The sterile group had higher levels of CSF interferon-gamma (IFN-γ), IFN-α, interleukin (IL)-6, IL-17, G-CSF, GM-CSF, and chemokine CXCL2 compared with non-sterile groups. Despite persons with sterile CSF cultures having higher CD4 counts, lower CSF opening pressures, and CSF cytokine profiles associated with better Cryptococcus control , mortality was similar to those with higher fungal burdens. This unexpected finding challenges the traditional paradigm that increasing CSF fungal burdens are associated with increased mortality but is consistent with a damage-response framework model.Cryptococcus is the leading cause of AIDS-related meningitis in sub-Saharan Africa. The clinical implications of a sterile cerebrospinal fluid (CSF) culture among individuals diagnosed with cryptococcal meningitis using CSF cryptococcal antigen (CrAg) are unclear. We prospectively enrolled 765 HIV-positive Ugandans with first-episode cryptococcal meningitis from November 2010 to May 2017. All persons were treated with amphotericin-based induction therapy. We grouped participants by tertile of baseline CSF quantitative Cryptococcus culture burden and compared clinical characteristics, CSF immune profiles, and 18-week mortality. We found 55 (7%) CSF CrAg-positive participants with sterile CSF cultures. Compared to the non-sterile groups, participants with sterile CSF cultures had higher CD4 counts, lower CSF opening pressures, and were more frequently receiving ART. By 18 weeks, 47% [26/55] died in the sterile culture group versus 35% [83/235] in the low culture tertile, 46% [107/234] in the middle tertile, and 56% [135/241] in the high tertile ( Cryptococcus neoformans is the leading cause of AIDS-related meningitis in sub-Saharan Africa ,2,3. CryDespite increasing access to ART throughout Africa in the last decade, many persons still present with advanced HIV disease . In geneDuring a large randomized trial , we founClinicalTrials.gov . All participants received antifungal induction therapy at meningitis diagnosis with amphotericin B deoxycholate (0.7–1.0 mg/kg/day) and fluconazole (800 mg/day) for up to 2 weeks, followed by fluconazole consolidation and maintenance therapy. All participants also received key supportive therapies including therapeutic lumbar pressures for elevated intracranial pressure, intravenous fluids, and electrolyte repletion. The COAT trial followed participants for 46 weeks, while the primary endpoint for the ASTRO-CM trial was 18-week survival. This analysis includes participants enrolled in any of the dose-finding pilot studies, the main interventional trials, or an open label compassionate care arm over that time period.We prospectively enrolled HIV-positive persons with cryptococcal meningitis into clinical cohorts from November 2010 to May 2017 in Kampala and Mbarara, Uganda. We screened hospitalized persons with clinical evidence of meningitis by lumber puncture for cryptococcal meningitis using the CrAg lateral flow assay (LFA) . Participants would then typically enter either of the two main trials occurring over that period. The Cryptococcal Optimal ART Timing (COAT) trial and the Adjunctive Sertraline for the Treatment of HIV-Associated Cryptococcal Meningitis (ASTRO-CM) trials have been previously described ,10,11. FQuantitative CSF cultures were obtained on all participants at baseline, day 3, 7, and 14, as described . CryptocTo assess whether persons with sterile CSF cultures had an immune response differing from those with fungal growth, we measured biomarkers from cryopreserved baseline CSF specimens. We analyzed 44 different cytokines using the Human XL Cytokine Magnetic Luminex assay  according to manufacturer’s protocols.p-value <0.05 and that were at least 2-fold different in the sterile group versus any of the fungal growth tertiles. Both 30-day mortality and 18-week mortality were compared by Chi-square testing of group proportions. We used a Kaplan–Meier time-to-event model with log-rank testing to visualize 18-week mortality by each group, and a Cox proportional hazards model to determine hazard ratios by tertile grouping. Survival analyses presented in the main manuscript are unadjusted. As a sensitivity analysis, we included two models adjusting for the impact of CD4 count and receiving ART. The first model adds baseline CD4 count as a co-variate. The second model adds both baseline CD4 count and baseline ART receipt as co-variates. Both models account for missing values using a multivariate imputation by chained equations (MICE) methodology. Analyses were completed using both SAS  and R .The primary analysis was mortality through 18 weeks, which accounts for 95% of 5-year mortality in cryptococcal meningitis . We cateAll participants provided written informed consent as part of the parent trials, including consent for the future testing of clinical samples for research purposes. IRB approval was obtained from the appropriate Ugandan and US institutions.2O (21% [10/55]) and lower overall mean opening pressure  participants had sterile CSF cultures. By CSF quantitative tertile, 235 participants were grouped in the low 1–14,700 CFU/mL category, 234 participants in the middle 14,701–206,000 category, and 241 participants in the high >206,000 CFU/mL category. Compared across the tertile groupings, those with sterile cultures had significantly higher CD4 counts  and were more likely to be receiving ART at time of diagnosis (71% [39/55]) . The ste0 mm H2O ,23,24). p < 0.01). Similarly through 18 weeks, 47% [26/55] died in the sterile culture group versus 35% [83/235] in low tertile, 46% [107/234] in the middle tertile, and 56% [135/241] in the high tertile groups (p < 0.001)  . When an< 0.001) . The hazPersons with sterile cultures were more likely to be receiving ART than persons in the other tertiles. Given the high mortality found in the sterile culture group, we analyzed the timing of ART initiation in this group. We found that persons with sterile cultures initiated ART a median of 2.3 months  prior to diagnosis, a shorter time frame as compared to the low tertile group at 4.1 months (0.8–19.7), although this difference was not statistically significant .A subset of 334 participants had baseline CSF cytokine measurements, including 24 persons in the sterile group. Of the 44 cytokines quantified from CSF in our participants, a total of 7 were found in statistically higher concentrations in the sterile group compared to those with any tertile of fungal growth . The cytOverall, our results demonstrate that persons with HIV-associated cryptococcal meningitis but without fungal growth in CSF have higher early 30-day mortality, comparable to those with the highest fungal burden. By 18 weeks, mortality falls between the middle and high tertile groups, but remains significantly higher than the low tertile group. The traditional paradigm considered increasing CSF fungal burden as having increased risk of mortality. However, with the introduction of highly sensitive, non-culture based diagnostics , cases o2O), compared to all other tertiles having at least 45% of persons with high opening pressures. Prior studies have demonstrated that high baseline opening pressures may be associated with increased mortality [We note some surprising features about this cohort. The first being that the sterile culture group paradoxically features several characteristics classically associated with lower mortality in cryptococcal meningitis. Only 21% 10/55] of the sterile culture group had substantially elevated CSF opening pressures  had increased cellular infiltrate and type 2 T helper responses in the CSF . AdditioPirofski and Casadevall proposed the damage-response framework model whereby clinical disease can occur by either uncontrolled pathogen dissemination or a detrimental host immune response, but with the common denominator being host damage  23,24].,24.23,24p = 0.13) [Cryptococcus may be driving the unique immune response. Nielsen and colleagues have demonstrated in mouse models that different clinically isolated human Cryptococcus strains yield different survival outcomes in the mice, and have identified numerous genes that may influence this differential pathogenicity [Alternatively, the unique pathogenesis of this sterile cohort may not be due to ART-associated unmasking IRIS, but rather some other immune related entity. In contrast to the Rhein et al. findings, the larger ACTA trial group observed only weak evidence of a trend toward increased mortality in those recently initiating ART, reporting a 2-week mortality of 23% for those initiating ART within 2 weeks prior to diagnosis compared to 15% initiating ART >2 weeks  . One posgenicity ,32. Unfogenicity .A limitation in studying the global cytokine profile of a severely immunocompromised person is the potential for other pathogens to influence the findings. All of our patients were admitted for presentations consistent with meningitis but may have had other opportunistic infections. In particular, the presence of IFN-α may suggest the presence of viral co-infections in higher proportions in the sterile culture group. Unfortunately, we do not consistently collect HIV viral loads in this cohort, which could also influence IFN-α. However, our large sample size of similarly immunocompromised persons should reflect a relatively even distribution of concomitant opportunistic infections. We cannot determine how cytokine profiles might have changed over time as our measurements are at baseline. We do not have paired blood cytokine measurements to determine if the CSF findings are a compartmentalized phenomenon versus a whole body response. It is possible that in rare situations cryptococcal yeast might not grow within 10 days of culture, and it is therefore possible persons in our cohort considered sterile might have had growth after 10 days. Lastly, we cannot generalize these findings to the subset of patients who have HIV-negative cryptococcal meningitis; however these findings may provide insight into the diversity of immune responses present in HIV-negative meningitis.The other major limitation of our study was our survival analysis. We recognize that there are limitations with either an unadjusted or partially adjusted survival analysis, but we felt it valuable to draw early attention to the surprising features of this group to facilitate future study. We performed a sensitivity analysis to determine if the hazard ratios were similar when adjusting for baseline CD4 count and ART receipt. In general, the hazard ratio for the sterile culture group is similar . We do tIn conclusion, we find that persons with sterile CSF cultures at cryptococcal meningitis diagnosis have high mortality, similar to those with higher fungal burdens. This finding is particularly unexpected given the higher CD4 cell count, lower CSF opening pressures, and IFN-γ predominant CSF pro-inflammatory cytokine profile of the group. Our observations challenge the traditional paradigm that increasing CSF fungal burdens are associated with increased mortality. However, sterile culture cryptococcal meningitis may rather represent a unique disease entity consistent with a parabolic damage-response framework model. Further research is needed from a prospective, controlled cohort.


Patients with coronavirus disease 2019 (COVID-19) frequently develop acute encephalopathy and encephalitis, but whether these complications are the result from viral-induced cytokine storm syndrome or anti-neural autoimmunity is still unclear. In this study, we aimed to evaluate the diagnostic and prognostic role of CSF and serum biomarkers of inflammation , and neuroaxonal damage (14-3-3 protein and neurofilament light [NfL]) in patients with acute COVID-19 and associated neurologic manifestations (neuro-COVID). We prospectively included 60 hospitalized neuro-COVID patients, 25 (42%) of them with encephalopathy and 14 (23%) with encephalitis, and followed them for 18 months. We found that, compared to healthy controls (HC), neuro-COVID patients presented elevated levels of IL-18, IL-6, and IL-8 in both serum and CSF. MCP1 was elevated only in CSF, while IL-10, IL-1RA, IP-10, MIG and NfL were increased only in serum. Patients with COVID-associated encephalitis or encephalopathy had distinct serum and CSF cytokine profiles compared with HC, but no differences were found when both clinical groups were compared to each other. Antibodies against neural antigens were negative in both groups. While the levels of neuroaxonal damage markers, 14-3-3 and NfL, and the proinflammatory cytokines IL-18, IL-1RA and IL-8 significantly associated with acute COVID-19 severity, only the levels of 14-3-3 and NfL in CSF significantly correlated with the degree of neurologic disability in the daily activities at 18 months follow-up. Thus, the inflammatory process promoted by SARS-CoV-2 infection might include blood-brain barrier disruption in patients with neurological involvement. In conclusion, the fact that the levels of pro-inflammatory cytokines do not predict the long-term functional outcome suggests that the prognosis is more related to neuronal damage than to the acute neuroinflammatory process. The frequency and spectrum of neurologic manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been described, but many questions remain unsolved regarding the underlying pathogenic mechanisms related to central nervous system (CNS) involvement . PatientIn this study, we aimed to characterize serum and CSF biomarkers of inflammation  and neuroaxonal damage (14-3-3 protein and neurofilament light-NfL) in patients with COVID-19 and acute neurologic conditions (neuro-COVID). We compared the profiles of these biomarkers with healthy controls, and among COVID-19 patients with different phenotypes, including those who developed encephalitis, encephalopathy or no CNS complications. We also evaluated the correlation of these biomarkers with the severity of the process at the time of acute COVID-19 and with patients’ long-term functional outcome. We hypothesized that distinct profiles of systemic and intrathecal proinflammatory cytokines and markers of neuroaxonal damage would help to differentiate phenotypes and assess severity in order to select a more appropriate therapeutic strategy, as well as to predict long-term neurologic disability.All patients with neurologic manifestations associated with COVID-19 whose serum and/or CSF were examined at the Hospital Clínic of Barcelona, Spain, between March 2020 and August 2020, were considered candidates for this study. All cases had definite COVID-19 as confirmed by positive SARS-CoV-2 RNA quantitative reverse transcription polymerase chain reaction (RT-PCR) test on nasopharyngeal swab. We included consecutive patients who provided informed consent for the use of clinical data and sample leftovers for research purposes. Serum and CSF were obtained during the acute stage of COVID-19 for diagnostic purposes as part of standard clinical care.Clinical information regarding sex, age, past medical history, premorbid functional status  , presencBrain magnetic resonance imaging (MRI), electroencephalogram (EEG) and routine CSF findings were also registered. In all cases, a wide microbiological screening with PCR array and cultures ruled out other infectious diseases in both serum and CSF. Functional outcome at 18 months of follow-up from neurologic symptom onset was assessed with the mRS based on a telephone interview with patients or caregivers conducted by the authors.For this study, encephalopathy was defined as diffuse brain dysfunction including decreased level of consciousness, cognitive impairment and/or behavioral alterations without signs of acute CNS inflammation, such as CSF pleocytosis and/or brain MRI changes. A diagnosis of encephalitis was given if the patient had decreased level of consciousness, cognitive impairment, behavioral alterations and/or focal abnormalities, along with CSF pleocytosis, inflammatory brain MRI changes and/or new epileptic activity.Forty-six serum samples from age-matched healthy subjects and 24 CSF samples from age-matched subjects with mild subjective cognitive complaints recruited and followed-up in our institution served as healthy controls (HC). They had objective cognitive performance within the normal range in all tests from a neuropsychological battery, no significant psychiatric symptoms or previous neurologic disease, and normal CSF core Alzheimer’s disease biomarkers, taking as reference the cut-off values described by Antonell et al. .®  custom MAP Human Cytokine/Chemokine/Growth panel and analyzed with LUMINEX® xMAP100. Both serum and CSF NfL concentrations were determined with Simple Plex™ Cartridge Kit containing NfL  on Ella™ instrument, according to the manufacturers’ instructions. 14-3-3 protein in CSF was measured by Circulex 14-3-3 Gamma Elisa Kit, MBL. IgG oligoclonal bands were detected by isoelectrofocusing and immunoblotting by Interlab CSF Isoelectrofocusing Kit. Intrathecal IgG synthesis was determined with the IgG/Albumin Ratio by nephelometry in serum and CSF. The following neuronal surface and synaptic antibodies were measured: AMPAR, amphiphysin, CASPR2, DNER, DPPX, GABAAR, GABABR, GluK2, GlyR, IgLON5, LGI1, mGluR1, mGluR2, mGluR3, mGluR5, neurexin 3α, and NMDAR. The following onconeural/intracellular and glial antibodies were determined: CV2, GAD, Hu, Ma1/Ma2, Ri, SOX1, Yo, AQP4, GFAP and MOG. The detection of neural antibodies  was performed by tissue immunohistochemistry, as previously described , granulocyte colony stimulating factor (G-CSF), interferon γ (IFN-γ), IFNα2, interleukin (IL)-1β, IL-10, IL-17a, IL-18, IL-1 receptor antagonist (IL-1RA), IL-6, IL-8 (CXCL8), IFN-γ–induced protein 10 (IP-10/CXCL10), macrophage chemoattractant protein 1 (MCP-1/CCL2), MCP-3, monokine induced by interferon (IFN)-gamma (MIG/CXCL9) and tumor necrosis factor-α (TNF-α) levels were quantified in both serum and CSF with a Milliplexescribed . Samplesescribed . CSF sampost-hoc analyses between groups were carried out with Bonferroni correction for multiple comparisons). Correlations between serum/CSF biomarkers and the clinical outcome (measured by mRS) at 18 months of follow-up were analyzed by Spearman’s correlation coefficient. Relative cytokine expression between groups was evaluated by principal component analysis and heatmaps. All analyses were addressed considering a two-sided type I error of 5% , using SPSS  and Prism .All data are described as median and IQR , or absolute frequency and percentage for quantitative and qualitative variables, respectively. Serum and CSF biomarkers’ levels were non-normally distributed as tested by the Shapiro-Wilk normality test. Comparisons between clinically defined groups for all biomarkers were performed using Mann-Whitney U test and Kruskal-Wallis test  with almost complete or totally complete recovery (mRS 0-1), 15 (31%) with mild-moderate neurological disability (mRS 2-3), 2 (4%) with severe functional dependence (mRS 4-5). Eight (16%) patients had died, all due to COVID-related complications; 11 (18%) cases were lost to follow-up.Seventy-five samples from 60 patients were collected and analyzed. Fifteen patients had paired serum/CSF samples, 30 only serum, and 15 only CSF. Eleven of 14 (79%) CSF samples of patients with COVID-19 associated encephalitis were tested for SARS-CoV-2 PCR and all were negative. Compared to HC, the COVID cohort had elevated IL-18, IL-6, and IL-8 levels in both serum and CSF, while IL-10, IL-1RA, IP-10, MIG and NfL were elevated only in serum, and MCP1 only in CSF Figure 1No differences were found in the serum and CSF levels of cytokines, NfL or 14-3-3 protein when comparing patients according to their neurologic diagnosis as classified in the 5 groups Table 2.Compared to HC, patients with encephalitis or encephalopathy presented elevated IL-6 and IL-8 in both serum and CSF, whereas IL-10, IL-1RA, IL-1b and MIG were elevated only in serum, and MCP1 only in CSF (data not shown). However, when specifically compared to HC, patients with encephalopathy had significantly increased levels of IP-10 in serum, and NfL levels in serum and CSF, and lower levels of IL-10 in CSF. In contrast, compared to HC, patients with encephalitis had elevated serum levels of IL-1b and CSF levels of G-CSF, IL-18 and MIG Figure 2The severity of the acute COVID-19 systemic disease according to the respiratory status was associated with CSF levels of 14-3-3  and NfL , and serum levels of IL-18 , IL-1RA , IL-8  and serum NfL . Finally, the long-term functional outcome, as measured by the mRS, significantly correlated with CSF levels of 14-3-3 protein  and CSF NfL , but not with serum NfL levels  Figure 3In this prospective cohort of 60 patients with COVID-19 and associated neurologic manifestations, we confirmed the presence of increased levels of several proinflammatory cytokines in both CSF and serum. This study also showed the association of neuroaxonal damage markers 14-3-3 and NfL, and proinflammatory cytokines IL-18, IL-1RA and IL-8 with COVID-19 severity in the acute phase, and interestingly, the significant association of CSF 14-3-3 and CSF NfL levels with the long-term functional outcome. In contrast, we did not detect the presence of antibodies against neural antigens in any of the patients who developed neurologic manifestations, including those with encephalitis and encephalopathy syndromes.Previous studies –15 analyRegarding neuronal damage in neuro-COVID-19 patients, we found increased levels of both the neuroaxonal marker NfL and the synaptic marker 14-3-3. Interestingly, unlike previous small cohort studies that focused primarily on the role of NfL as a biomarker of disease severity , 22, 23,Lastly, the fact that none of the patients harbored antibodies against neural antigens at the onset of neurologic involvement by acute COVID-19, including those patients who presented with inflammatory features of the CSF and/or brain MRI, would suggest that the infection by SARS-CoV-2 is unlikely to trigger an intrathecal B-cell-specific autoimmune response. This could be due to its low neurovirulence and the lack of viral-induced release of neural proteins during the acute CNS dysfunction, making cytokine storm the most likely cause of inflammatory CNS injury. However, we do not know whether a delayed neural autoimmune response may occur in some patients with persisting neurologic symptoms.There are a number of limitations to this study. Sample size, even though greater than previous studies, is still relatively small and limits the statistical analyses. The study did not include a control group of patients with COVID-19 without neurologic manifestations or a control group with a different viral infection. Another possible selection bias is the enrollment of patients with available serum/CSF samples, and that lumbar punctures for CSF examination were performed based on standard clinical indication . However, the study included a comprehensive serum and CSF testing at disease onset, and prolonged clinical follow-up. Further longitudinal studies with larger samples sizes are needed to confirm the correlations of CSF 14-3-3 and NfL levels in patients that progress to a long-standing neuro-COVID disease.In summary, this study provides further evidence of distinct systemic and intrathecal proinflammatory cytokine profiles in patients with acute neurologic manifestations associated with COVID-19, unrelated to neural autoimmunity. We also show that increased CSF levels of markers of neuronal damage during the acute phase of COVID-19 are associated with worse long-term clinical outcome of the patients, supporting their potential use as prognostic markers.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics’ Committee of Hospital Clínic de Barcelona. The patients/participants provided their written informed consent to participate in this study.Virginia Casado Ruiz , Marta Perez Moreno , Esther Catena Ruiz , Marta Palacín Larroy , Nicolau Guanyabens  Eloi Giné-Servén  Javier Villacieros-Álvarez , Natalia Hernando Quintana , Isabel Pellicer-Espinosa , Eloi Giné Servén , Nicolau Guanyabens , Elba Pascual Goñi , Delon La Puma .RS-V and MG designed and conceptualized the study, and obtained funding. RR-G, GM-S, EM-H and AS participated in the study design. MG, GM-S, RS-V, and RR-G interpreted the data and wrote the manuscript. MG, EM-H, DS, AC, AS, RS-V, and the Barcelona Neuro-COVID study group collected and reviewed the clinical data. RR-G, GM-S, MG, LN, UB, and MF laboratory studies and/or statistical analyses. MB (MSc statistics) reviewed the statistical analyses. All authors contributed to the article and approved the submitted version.This study was funded by the Fondo de Mecenazgo COVID-19 from Hospital Clínic de Barcelona, Barcelona, Spain. Dr. M. Guasp is a recipient of a Resident Award “Josep Font,” granted by Hospital Clínic de Barcelona, Research, Innovation and Education Departments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre‐transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I  assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation. The workshop, organized by the British Society for Histocompatibility and Immunogenetics (BSHI), was convened on 22 March 2019 in order to provide guidance on crossmatching for kidney transplantation from deceased donors in the United Kingdom. The brief was to resolve differences in crossmatch practices in relation to when a result becomes available (but not how crossmatching is performed). The overall aim was to minimize cold ischaemia time (CIT) and improve patient outcomes by agreeing ways to enable a crossmatch test to be performed as early as possible in the donation process.The United Kingdom has a single kidney offering policy that all transplant units follow . These two factors are of course related; highly sensitized patients have tended to wait longer than less sensitized patients. This means less concern with HLA mismatches, which is supported by evidence showing a reduced influence of HLA matching on transplant outcomes . Compatibility can be determined directly by testing the reaction in a crossmatch (XM) between recipient serum and donor cells, typically leucocytes (LXM). The LXM can be performed using cells from donor lymph nodes or spleen, or donor peripheral blood lymphocytes (PBLs). Lymph nodes and spleen are only available after organ retrieval, whilst PBLs can be taken from the potential organ donor prior to removal of the kidneys. Alternatively, HLA antibody compatibility can be assessed by prediction when a recipient's HLA‐specific antibodies have been sufficiently characterized and the donor's corresponding HLA alleles have been identified .All UK H&I laboratories regularly test waiting list patient samples from the transplant units they support  assays, as summarized in a review by South and Grimm . The incParallel to this, HLA typing methodologies have evolved with the introduction of rapid DNA‐based testing, such as PCR‐SSP, PCR‐SSOP and subsequently, real‐time PCR (or q‐PCR), which permit typing of HLA‐A, ‐B, ‐C, ‐DRB1/3/4/5, ‐DQ and –DP, enabling a detailed characterization of HLA for deceased donors in a rapid timeframe. More recently, methodologies that can provide DNA sequencing in the required time frame have been developed  XM with no day of transplant sample testing required. Others will perform a day of transplant Luminex antibody test to facilitate a VXM. In all situations, the effective use of a VXM is dependent on correct and adequate donor HLA typing together with up to date information regarding the potential recipient's HLA antibody screening and sensitization history ‐specific antibodies is currently problematic for two main reasons. If a target cell is used, then this must be known to express the relevant antigen or antigens. This approach has been tested for potential endothelial cell markers but the evidence that such a test is predictive of rejection or transplant outcome is mixed  alloantibodies, a specific incompatibility cannot be deduced as, currently, deceased donors are only typed for HLA, ABO and Rh. Pre‐transplant testing of these antibodies, whether allo or auto, is also required to reliably interpret a XM, and essential for a VXM, but this is not routinely undertaken in the United Kingdom as the level of evidence was insufficient at the time of development of the relevant guidelines  system has been used to rate the strength of evidence and the strength of recommendations .Grade B evidence means moderate‐quality evidence from randomized trials that suffer from serious flaws in conduct, consistency, indirectness, imprecise estimates, reporting bias or some combination of these limitations, or from other study designs with special strength.Grade C evidence means low‐quality evidence from observational evidence, or from controlled trials with several very serious limitations.Grade D evidence is based only on case studies or expert opinion.For each recommendation the quality of evidence has been graded as A (high), B (moderate), C (low) or D (very low):Level 1 (we recommend)Level 2 (we suggest)For each recommendation, the strength of recommendation has been indicated as one of:Not graded Level 1 recommendation is a strong recommendation to do (or not to do) something where the benefits clearly outweigh the risks (or vice versa) for most, if not all patients.A Level 2 recommendation is a weaker recommendation, where the risks and benefits are more closely balanced or are more uncertain.A 3Objective 1. Address the impact of the reporting time of a crossmatch on the overall time in the transplant pathway. The key issues identified are, first, where a prospective crossmatch is required; a transplant cannot proceed until said crossmatch is completed. Second, in the United Kingdom, the positive crossmatch rate is around 2.5% (based on data between 2010 and 2015) (ODT Statistics and reports) and the organs in these cases require re‐offering and potentially a further crossmatch. Third, there are logistical issues around access to theatres for transplantation with kidney transplantation not always being given sufficient priority for operating theatre availability. Therefore, the availability of a crossmatch result ahead of organ arrival is an aid to surgical planning.3.1To overcome this, we recommend that a crossmatch result (either VXM or LXM) for a particular recipient must be reported before the organ arrives at the transplant centre, both LXM and VXM procedures will allow this (Grade 1B).If a VXM is not possible then we recommend that where possible, pre‐retrieval donor blood samples must be used when a LXM is required (Grade 1B).Objective 2. Overcome concerns around quality assurance and accuracy of deceased donor HLA typing and virtual crossmatching strategies. It is now recognized that alloantibodies can be stimulated by all the classical, polymorphic HLA proteins   is generally required for crossmatch interpretation , or where the intended recipient has known donor HLA‐specific antibodies, a pre‐transplant LXM must be performed (Grade 1B).A retrospective LXM (e.g. using cells from the donor spleen or lymph node) may be undertaken when a pre‐transplant VXM has been used. However, if an audit of sufficient cases shows concordance between the initial VXMs and subsequent LXMs then the latter can be safely omitted (Grade 2B).Objective 3. Address issues relating to donor sample availability and quantity. The timeliness of reporting a prospective crossmatch will depend on early taking of donor blood samples arranged in advance between the laboratory, clinical and donation teams. There are many demands for donor blood samples and anecdotal evidence suggests that excessive blood taking may be harmful to the donor and compromise their donated organs.3.3We recommend that peripheral blood for crossmatch testing must only be requested and sent on demand. Therefore, when a VXM is to be used, crossmatch samples must not be taken from the donor pre‐transplant (Grade 1B).When PBLs for LXM are required, up to a maximum of 40 ml of peripheral blood in EDTA should be requested unless there are exceptional circumstances. In this scenario, the H&I laboratory can request a volume as required for locally validated procedures (Grade 1B).Pre‐retrieval crossmatch donor blood must be taken and sent as expeditiously as possible (Grade 1B).Spleen or lymph node samples must be taken at the time of organ retrieval in all cases  (Grade 1A).It should be noted that geographical considerations may need to be taken into account when considering feasibility of use of PBLs for LXM, namely time and cost taken to ship the samples, particularly where long distances are involved.Objective 4. Ensure fairness and equity for all potential transplant recipients as immunologically high‐risk cases tend to wait the longest for a transplant. The interpretation of a crossmatch in such cases tends to be complex, requiring more information. A comprehensive and up‐to‐date HLA serological history, according to national and other guidelines .The crossmatch test should be regarded as an enabler of safe transplantation, rather than primarily a tool to prevent transplantation, particularly for sensitized patients and local policy developed with this in mind . This can be achieved through the development of good working relationships between the clinical team and the laboratory, and the process documented in local policy.Non‐HLA donor‐specific antibodies are outside the scope of this guidance (Grade 2D).All authors participated in the working group, contributed to these guidelines, and have read and approved the final manuscript. SP and DB prepared the following text.


Fagopyrum esculentum) is crucial for crop yield. However, most published descriptions of early flower and inflorescence development in Polygonaceae are based on light microscopy and often documented by line drawings. In Fagopyrum and many other Polygonaceae, an important inflorescence module is the thyrse, in which the primary axis never terminates in a flower and lateral cymes (monochasia) produce successively developing flowers of several orders. Each flower of a cyme is enclosed together with the next-order flower by a bilobed sheathing bract-like structure of controversial morphological nature.Understanding the complex inflorescence architecture and developmental morphology of common buckwheat  and most cultivars of common buckwheat possess an indeterminate growth type with lateral thyrses produced successively on the primary inflorescence axis until cessation of growth. In contrast, determinate cultivars of F. esculentum develop a terminal thyrse after producing lateral thyrses. In contrast to F. esculentum, the occurrence of a terminal thyrse does not guarantee a determinate growth pattern in F. tataricum. The number of lateral thyrses produced before the terminal thyrse on the main axis of F. tataricum varies from zero to c. 19.Our data support interpretation of the sheathing bract as two congenitally fused phyllomes (prophylls), one of which subtends a next-order flower. In Fagopyrum and some other Polygonaceae require investigation using refined approaches to mathematical modelling of flower development. Our data on inflorescence morphology and development suggest contrasting evolutionary patterns in the two main cultivated species of buckwheat, F. esculentum and F. tataricum. The genus Fagopyrum offers an excellent opportunity for evo-devo studies related to inflorescence architecture.The nine stages of early flower development formally recognized here and our outline of basic terminology will facilitate more standardized and readily comparable descriptions in subsequent research on buckwheat biology. Non-trivial relative arrangements of tepals and bracteoles in  Fagopyrum include Common buckwheat (Fagopyrum esculentum Moench), the most economically important species of Polygonaceae, and F. tataricum (L.) Gaertn.  Meisn. and F. homotropicum Ohnishi, and has a centre of diversity in Sichuan, Yunnan and Tibet  and F. esculentum  share with F. cymosum and F. homotropicum the occurrence of articulate pedicels and shedding fruits  in Arabidopsis and CENTRORADIALIS in Antirrhinum, result in mutants with inflorescences that differ from those of the wild type in the presence of terminal flower. In Pisum sativum L. and some other legumes, mutations in homologues of TFL1 result in the development of a terminal raceme , whereas the wild type has only lateral racemes (Cajanus cajan (L.) Huth) is realized through gain of a terminal double raceme, whereas the wild type has only lateral double racemes  Gross, an example of the Urophyllum group.Fagopyrum flowers, Fagopyrum, Persicaria was investigated using SEM  are key players in the interaction between flowers and inflorescences, the two primary hierarchical levels of plant reproductive architecture . Surprissing SEM . A detaiPolygonum and related genera), the homologies of the bilobed sheathing organ that is immediately associated with the flowers is controversial. Traditionally, the bilobed sheath was interpreted as a fusion product of two bracteoles, of which one subtends a next-order flower  and the All-Russia Research Institute of Grain Legumes and Groat Crops . At least 15 individual plants of each accession were examined. The following cultivars of F. esculentum subsp. esculentum were studied: Batyr, Chatyr Tau, Dasha, Dozhdik, and Temp. In addition, an accession of the wild subspecies F. esculentum subsp. ancestrale was examined  as well as an accession derived from its cross with cv. Dasha (FAD_F2_2018). Three accessions of F. tataricum were used: K17 is a cultivated line , C9119 is a wild accession from Lixian district of Sichuan province, China (received from Kyoto University) and Zhd001 comes from a seed collection made in a ruderal location near a railway in Moscow Province, Russia . The accession of F. homotropicum examined here (С9139) comes from Yongsheng district of Yunnan province, China (received from Kyoto University). The accession of F. urophyllum (С9069) comes from southern China (received from Kyoto University).We examined plant material of tlb mutant of F. esculentum was conducted at the Royal Botanic Garden, Kew (UK). The material was dissected in 70% ethanol, dehydrated through absolute ethanol and critical-point dried using an Autosamdri-815B CPD . Material was mounted on SEM stubs, coated with platinum using an Emitech  K550 sputter coater and examined using a Hitachi cold-field emission SEM S-4700-II . SEM work on material other than the tlb mutant was conducted at the Laboratory of Electron Microscopy at the Biological Faculty of Moscow University. The material was dissected in 70% ethanol and transferred to 100% acetone using the following series: 96% ethanol (twice for 30 min), 96% ethanol: 100% acetone , 100% acetone (three times for 30 min). The material was critical-point dried using a Hitachi HCP-2 critical-point dryer , then coated with gold and palladium using a Eiko IB-3 ion-coater  and observed using a CamScan S-2  and a JSM-6380LA SEM .Whole plants and young shoot tips were fixed in 70% ethanol. SEM work on material of the F. tataricum (42 sheets) and F. cymosum (10 sheets) were examined in the Herbarium of Moscow University (MW) and the Herbarium of the Main Botanic Garden of the Russian Academy of Sciences, Moscow (MHA). In addition, available online resources of herbarium collections were examined. Inflorescence morphology of F. urophyllum was examined using the Chinese Virtual Herbarium (https://www.cvh.ac.cn/index.php). Fragments of herbarium material of F. tataricum were studied using high-resolution X-ray computed tomography (HRXCT). Dried herbarium material was rehydrated by soaking in boiling water and, transferred into an infiltration medium (1% phosphotungstic acid [PTA] in 70% ethanol) for a week to increase its contrast  a phyllome that bears in its axil a flower or an inflorescence module, or (2) a phyllome within an inflorescence that lacks a leaf lamina. Here, we use the term in the first sense, and always specify the nature of an axillary structure (i.e. flower-subtending bract or cyme-subtending bract).Term describing an inflorescence (or its module) with subtending phyllomes on the main axis represented by scale-like leaves.Fagopyrum, then the cyme is a monochasium (An inflorescence module with a primary axis that is terminated in a flower and branching occuring only in the axil(s) of the prophyll(s). If there is only one branch, which is the case of ochasium . By defiochasium , elementochasium  or cymosochasium , but theochasium .A module of the highest order that can be recognized in an inflorescence . The primary axis has a terminal flowering unit. Paracladia, which are lateral axes of often more than one order, also terminate in flowering units, but the occurrence of lateral flowering units is not mandatory and may in part depend on environmental conditions. The term ‘flowering unit’  is an EnPhyllome with a lamina.Inflorescence or its module in which the subtending the phyllomes on the main axis are represented by foliage leaves.Inflorescence or its module in which the lower subtending phyllomes on the main axis are represented by foliage leaves and the upper subtending phyllomes by scale-like leaves.Part of the plant body bearing flowers. The term ‘inflorescence’ is ambiguous because different authors use different characters to draw a boundary between an inflorescence and the vegetative part of a plant. Synflorescence is a precisely defined term describing the flower-bearing region of the plant body that develops during a season. In many annuals such as Common buckwheat, the entire above-ground part of the body is a synflorescence.Fagopyrum, these levels are flower, cyme, thyrse and flowering unit. The degree of similarity between modules varies. For example, Fagopyrum has both left- and right-handed flowers, whereas cymes may differ in the number of branching orders and are also left- and right-handed.A repeating part of the plant body. Modules can be of several hierarchical levels. In inflorescences of A lateral axis terminated by an inflorescence module that repeats the module that terminates the entire inflorescence. In the present paper, we do not use the term paracladium for lateral thyrses (see Discussion).General term for typical leaves and all their homologs .Two (in many eudicots) or one (in many monocots) phyllomes that initiate the pattern of phyllotaxis on a lateral shoot; they can be analogized with cotyledon(s), which initiate the pattern of phyllotaxis of the primary axis of the entire plant .An inflorescence or inflorescence unit in which the primary axis never terminates in a flower and the lateral structures are cymes with successively developing flowers of two or more orders. The buckwheat thyrse is sometimes called simply ‘inflorescence’ e.g., . The terFagopyrum and many other Polygonaceae, the thyrse represents an important inflorescence module. In a thyrse, the primary axis never terminates in a flower and there are lateral cymes with successively developing flowers of several orders. Each cyme is located in the axil of a cyme-subtending bract, which is also the subtending bract of the first flower  and its accession FAD_F2_2018.The following stages of early flower development can be recognized in Stage 1  possess inflorescences similar to that of F. cymosum, only the main inflorescence axis bears foliage leaves and does not produce a terminal thyrse. Lateral axes of all orders bear only scale-like leaves and are all terminated in thyrses.A few collections of F. tataricum from Southeast Asia are known as subsp. potanini, but apparently this name is not formally published. In addition, F. tataricum is widely distributed across temperate Eurasia as ruderal plant. We examined in detail living material of three accessions, K17 . These herbarium samples showed no thyrse terminating the flowering unit, but one cannot rule out its late appearance by analogy with our sample K17.This is an annual species that comprises both cultivated and wild accessions. Wild plants of In accession C9119, the main inflorescence axis is terminated by a thyrse at an earlier stage than that of K17 , the former comprising wild accessions from Southeast Asia and the latter cultivated accessions. In plants of subsp. ancestrale . The main axis apparently has foliage leaves only (frondose structure), though the uppermost leaves are retarded in development. Structures in the axils of these leaves are groups of two or three thyrses. Branching on these second-order axes takes place in the axils of scale-like leaves (bracteose structure). The main inflorescence axis lacks a terminal thyrse, at least at developmental stages available for investigation, but the second-order inflorescence axes always possess a terminal thyrse (F. urophyllum grown for the present study (The basic inflorescence module of F. urophyllum possess inflorescences with the axis terminated by a thyrse (https://www.cvh.ac.cn/). Rarely, the terminal thyrse is short and, in contrast with most other specimens, anthesis is delayed in terminal and uppermost lateral thyrses as compared to the long lower lateral thyrses (PE 01859339). The terminal and lateral thyrses form what can be interpreted as flowering unit. Some specimens show lateral units of this sort that can be interpreted as developing on paracladia . Since F. urophyllum is a perennial and often robust plant, the complete branching pattern not always can be inferred from the herbarium material. Observations in nature are necessary to obtain a complete picture.Many herbarium specimens of Fagopyrum (tribe Fagopyreae) and many other Polygonaceae , Fagopyrum cymosum, and suggested the occurrence of a single phyllome that forms the prophyllar sheath. Our In cymes of Groenlandia densa (L.) Fourr. (Potamogetonaceae), each lateral stamen arises as two separate primordia, whereas each median stamen initiates as a single primordium  position of the prophylls in the cymes.Sattler’s (1973) diagram shows the two lobes of the prophyllar sheath as symmetrically arranged in transversal positions. Therefore, the next-order flower is out of the median of the β-lobe in Sattler’s diagram. Our data on the earliest developmental stages reveal differences in the shape and position of the lobes. The β-lobe is shifted towards the abaxial side rather than being strictly transversal; it is also wider than the α-lobe  below the tepals, but no stalk below the prophyllar sheath. With the acquisition of tepal-like features, tlb plants tend to elongate the stem region below the prophyllar sheath and between the two prophylls. Sometimes a mosaic of features can be seen in mutant plants, including a situation when stem elongation takes place between two still fused prophylls, thus producing a distorted prophyllar sheath  adaxial one, followed by the two abaxial tepals  in buckwheat and other Polygonaceae merits special attention. Fagopyrum is located in almost exactly the same radius as the α-prophyll, a condition that differs from the two possibilities predicted by Bistorta officinalis and Coccoloba nitida Kunth (C. guianensis Meisn.) do not show a superposition of the α-prophyll and the first of the five tepals. In this respect, these diagrams differ from our diagram of F. esculentum and the diagram of Persicaria lapathifolia in Even though the initiation of the five outermost floral organs along a 2/5 spiral in Fagopyrum, though the prophylls of Persicaria were illustrated as symmetric by Fagopyrum and other Polygonaceae is the shape of the floral primordium and the young flower, which is pronouncedly elliptical rather than circular in outline. Some images even suggest a crescent-shaped outline of the young flower tightly packed between the cyme-subtending bract and the thyrse axis. Refined mathematical modelling of early flower development in Polygonaceae may shed new light on the problem, especially when considering aspects of pre-patterning as well as mechanical pressure and the peculiar shape of the floral apex , no evidence was found for similar superposition with the next-formed organs. For example, in F. esculentum. According to our data, the first evidence of initiation of these stamens can be seen at even earlier stages, before initiation of the fourth and fifth tepal  develops more paracladia than cultivars of F. esculentum subsp. esculentum in common garden experiments  as well as the closely related wild species F. homotropicum lack a terminal thyrse. These differences were already correctly described for F. esculentum by Our data show that the determinate growth pattern of cultivars Dozhdik, Temp and Dasha of F. tataricum and F. esculentum, with respect to the presence vs. absence of a terminal thyrse. Thus, our data suggest different evolutionary patterns in the two species. Selection towards fixation of the determinate growth pattern conditioned by the occurrence of a terminal thyrse took place in domesticated F. esculentum. In contrast, the cultivated accession of F. tataricum studied here (K17) is characterized by a delayed ontogenetic transition to terminal thyrse formation, compared with other accessions of this species. In theory, further heterochronic shifts in this direction may lead to permanent elimination of the terminal thyrse. It is tempting to suggest heritability of the striking differences in the timing of ontogenetic transition to terminal thyrse observed here among accessions of F. tataricum. Indeed, in a common garden experiment, at the stage when terminal thyrses were fully formed after 10-11 nodes of the flowering unit in C9119, no indication of their initiation was visible in K17. Furthermore, the ruderal accession Zhd001 initiated a terminal thyrse as early as starting from the third node above the cotyledons. Importantly, the occurrence of a terminal thyrse does not guarantee a determinate growth pattern in F. tataricum. The plant of F. tataricum C9119 illustrated in The present study highlights, apparently for the first time, important differences between wild accessions of Fagopyrum. Therefore, determinate inflorescences do not occur in this group. The presence versus absence of the terminal thyrse is related to the concept of axiality, which is a more general concept than that of determinate/indeterminate inflorescences  is a perennial species and the only perennial member of the Cymosum group. Its phylogenetic position does not preclude a plesiomorphic condition for (some) its morphological traits, including inflorescence morphology. If F. tataricum is derived from F. cymosum (F. cymosum could be plesiomorphic for F. tataricum. However, given the occurrence of infraspecific variation and complex evolutionary patterns in Fagopyrum that involve polyploidy, use of conventional approaches of character evolution are problematic here. It is more appropriate to develop studies in evolutionary developmental genetics of buckwheat in the context of microevolution, speciation, and natural and artificial selection.Inflorescences essentially similar to those of lineages . Fagopyr cymosum , the patFagopyrum is composed of two fused prophylls. The relative positions of the floral prophylls and tepals in buckwheat and some other Polygonaceae is uncommon among eudicots and at least at first glance cannot be easily interpreted by the Inhibitory Field theory. Our data can be used for mathematical modelling of early flower development that should incorporate parameters such as the non-spherical shape of the floral apex and mechanical forces. Our investigation provides clear and simple characters of inflorescence morphology that can be readily formalized and will provide advances in the taxonomy, evolutionary biology and breeding of buckwheat.We provide a formal classification of the early stages of flower development and define diagnostic characters that allow recognition of each stage. This classification will be important for further experimental studies of buckwheat. We confirm sequential tepal and stamen initiation with an overlap between these two processes in buckwheat, refine details of this phenomenon and illustrate it for the first time using SEM. Our study represents apparently the most complete SEM-based study of early flower development in Polygonaceae. The prophyllar sheath of Fagopyrum, and the same condition can be sometimes found in more than one species. At least in some instances, the variation is discrete; in the case of terminal thyrse development in Common buckwheat, the variation is determined by a single-gene mutation. Therefore, the conventional way to study character transformations against species trees is not appropriate in this case; it is further complicated by the occurrence of polyploidy and apparent reticulate evolution. Despite these limitations, the genus Fagopyrum offers an excellent opportunity for evo-devo studies related to inflorescence architecture. Our discovery of variation of inflorescence characters in self-pollinated species of F. tataricum offers a potential model system for the determination and molecular characterization of the genes responsible for this variation. Finally, we show that although the agriculturally important character, determinate growth pattern, is defined by the presence of a terminal thyrse in F. esculentum, in a wider context of the genus Fagopyrum, a terminal thyrse does not guarantee a more synchronized flowering pattern.Characters of inflorescence morphology can vary within species in The original contributions presented in the study are included in the article/tlb mutant of F. esculentum. CF performed X-ray microtomography. All the authors reviewed drafts of the paper and approved the final version.DS and ML designed the study. ML, AF and IF provided living plant material for the study. DS, RM, MR, AF, IF and ML made morphological observations and interpretations of inflorescence morphology. DS and RM conducted developmental studies in Moscow using scanning electron microscopy. DS, MR and PR studied and interpreted the reproductive development in the


Both chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) frequently occur and can lead to dose-limiting toxicity and even fatal chemotherapy side effects. The prophylactic use of recombinant human granulocyte colony-stimulating factor (rhG-CSF), including pegylated rhG-CSF (PEG-rhG-CSF), significantly reduces the risks of CIN and FN during chemotherapy in early-stage breast cancer (ESBC) patients. However, whether the prophylactic use of granulocyte colony-stimulating factor (G-CSF), especially PEG-rhG-CSF, can influence white blood cell (WBC) counts and absolute neutrophil counts (ANCs) after finishing the chemotherapy remains unknown. Therefore, exploring the development and recovery tendency of WBC counts and ANCs during and after chemotherapy is crucial.We aimed to investigate the variation tendency and recovery of WBC counts and ANCs during and after chemotherapy and evaluate the independent factors influencing leukopenia and neutropenia lasting longer after chemotherapy. We also aimed to provide individualized prophylactically leukocyte elevation therapy for breast cancer patients.This single-center retrospective cohort study evaluated 515 ESBC patients who received rhG-CSF or PEG-G-CSF for prophylaxis after adjuvant or neoadjuvant chemotherapy. Blood test reports were analyzed during chemotherapy, and on a 12-month follow-up period after finishing the chemotherapy. The WBC counts and ANCs were measured to assess their variation tendency characteristics and to identify independent factors that influenced the occurrence of leukopenia and neutropenia lasting longer than 12 months after chemotherapy.p < 0.001). During the 12-month follow-up after the end of the last chemotherapy, WBC counts and ANCs gradually recovered, but the group that used only PEG-rhG-CSF  or rhG-CSF  had better leukocyte elevation effects than the mixed treatment group (PEG-rhG-CSF mixed rhG-CSF). Besides, the short-acting group had a better neutrophil elevation effect than the longer-acting (pANC = 0.019) and mixed (pANC = 0.002) groups. Leukopenia was still present in 92 (17.9%) patients and neutropenia in 63 (12.2%) 12 months after the end of the last chemotherapy. The duration of leukopenia over 12 months was closely associated with the baseline WBC level (p < 0.001), G-CSF types (p = 0.027), and surgical method (p = 0.041). Moreover, the duration of neutropenia over 12 months was closely related to the baseline ANC (p < 0.001), G-CSF types (p = 0.043), and molecular typing (p = 0.025).Prophylaxis with rhG-CSF or PEG-rhG-CSF kept the mean values of WBC counts and ANCs within the normal range during chemotherapy, but a significant difference in WBC levels was detected before the end of the last chemotherapy compared to the prechemotherapy period (baseline) (The prophylactic application of G-CSF effectively stabilized the WBC counts and ANCs during chemotherapy in ESBC patients. Nevertheless, the recovery of WBC counts and ANCs after chemotherapy varied between different G-CSF treatment groups. The risk of leukopenia and neutropenia persisting for more than 12 months after chemotherapy was associated with G-CSF types, the baseline level of WBC count/ANCs, surgical method, and molecular typing. Breast cancer ranks first among female malignancies worldwide . NeoadjuLeukopenia and neutropenia are common chemotherapy side effects caused by the reduction of white blood cell (WBC) counts and absolute neutrophil counts (ANCs) during myelosuppressive chemotherapeutic treatments . MoreoveThe occurrence of CIN and FN during chemotherapy is closely related to chemotherapy regimen and intensity . HoweverFurthermore, the granulocyte colony-stimulating factor (G-CSF)—the principal cytokine responsible for the regulation of neutrophil production—boosts neutrophil counts by controlling the maturation, proliferation, and differentiation of hematopoietic stem cells and modulating bone marrow (BM) neutrophils into the circulation –12. PrevMany studies have indicated that age, baseline, cancer type, cancer stage, and molecular typing can influence the occurrence of CIN and FN during chemotherapy , 19. MeaA retrospective analysis was performed based on our prospectively collected 2015 patients with breast cancer who received adjuvant or neoadjuvant chemotherapy at the Second Affiliated Hospital of Zhejiang University School of medicine from November 1, 2017, to November 1, 2019. A total of 1,500 patients were excluded because of the following reasons: 1) were not at early-stage, namely, had any evidence of recurrence or distant metastasis; 2) were not dispensed and treated with PEG-rhG-CSF or rhG-CSF in our hospital; 3) had a follow-up time lower than 12 months after the last chemotherapy; 4) had incomplete baseline characteristics data; 5) were not regularly followed up as required; 6) suffered leukopenia or neutropenia before the treatment; 7) had a diagnosis of diseases that affect the WBC counts and ANCs, such as blood, immune, and infectious diseases; 8) underwent other systemic malignancies; and 9) changed their chemotherapy regimens during treatment Figure 1All enrolled patients received G-CSF for prophylaxis during each cycle of chemotherapy. The G-CSF types and application methods were 1) 100-μg subcutaneous injection of rhG-CSF  once a day for 5–7 days and 2) 3- or 6-mg subcutaneous injections of PEG-rhG-CSF  48 h after each cycle of chemotherapy.The demographic data of the 515 patients were prospectively collected, including age, body mass index (BMI), G-CSF types, baseline levels of WBC counts and ANCs before treatment, surgical method, pathological type, molecular typing, lymph node metastasis, chemotherapy regimen, and Herceptin use, and WBC counts and ANCs during chemotherapy and the 12-month follow-up period after the end of the last chemotherapy . Patients were divided into four groups according to different chemotherapy regimens and stratified according to G-CSF types. Blood routine results were collected and analyzed from the electronic medical record system (EMRS) database of the Second Affiliated Hospital of Zhejiang University School of Medicine.p < 0.05 was considered statistically different. The least significant difference (LSD) t-test was used for multiple comparisons. Fisher’s exact tests were used to compare the differences of categorical variables between groups. For continuous variables, independent sample two-tailed t-tests or Mann–Whitney U tests were used to compare the differences in median values between groups. Multi-pair sample non-parametric (Friedman) tests were used to describe the variation trend in WBC counts and ANCs during chemotherapy and the 12-month follow-up period after the end of the last chemotherapy. Repeated-measures ANOVA was used to compare inter- and intra-group differences between G-CSF treatment groups during the 12-month follow-up period after the end of the last chemotherapy. To analyze the independent factors influencing leukopenia and neutropenia 12 months after the end of the last chemotherapy, the variables that were potentially correlated with the development of leukopenia/neutropenia and had a univariate p < 0.1 were used in a multivariable logistic regression model.Statistical analyses were performed using SPSS 26.0. Two-tailed significance values were used, and a A total of 515 patients were enrolled in the present study. Their basic clinical characteristics are shown in The variation tendency of WBC counts and ANCs during the cycles of different regimens of adjuvant and neoadjuvant chemotherapies is presented in p < 0.001). The overall mean of ANC before the last cycle of chemotherapy did not significantly differ from the baseline . Therefore, after prophylactic administration of G-CSF, neutrophil levels can be maintained at normal or even baseline levels during chemotherapy in ESBC patients, ensuring adequate dose and scheduled cycle of the treatment.All patients prophylactically received G-CSF. Before the last cycle of chemotherapy, their overall mean WBC levels were significantly lower compared to baseline but remained stable within normal ranges .The means, SDs, and comparisons of WBC counts and ANCs levels between different time points are shown in According to the different G-CSF types used during chemotherapy, we divided the 515 ESBC patients into three different treatment groups: short-acting (used only rhG-CSF), long-acting (used only PEG-rhG-CSF), and mixed (used rhG-CSF or PEG-rhG-CSF in different cycles) groups.p = 0.002). No significant difference was detected in the pairwise comparison regarding the recovery of WBC levels between long-acting and short-acting groups. However, these groups significantly differed from the mixed treatment group. Furthermore, both long-acting (p = 0.012) and short-acting (p = 0.000467) groups presented excellent increases in WBC counts compared to the mixed treatment group . With the extension of follow-up time, the patient’s WBC level gradually recovered. The recovery of WBC counts in different G-CSF treatment groups is shown in Additionally, a significant intra-group effect was detected . The WBC levels at baseline or 3 months after the end of chemotherapy did not significantly differ among G-CSF treatment groups. From 6 months after the end of chemotherapy, the long-acting and short-acting groups had better effects in promoting the recovery of WBC counts than the mixed treatment group along the follow-up time.The repeated-measures ANOVA also revealed a significant interaction between time points and different G-CSF treatment groups . No significant differences were detected in the pairwise comparison for the recovery of ANC levels between the long-acting and mixed treatment groups. However, these two groups were significantly different from the short-acting group. Moreover, the increases in ANC in the short-acting group were better than in the long-acting (p = 0.019) and mixed (p = 0.002) groups . Along the follow-up time, the patient’s ANC level gradually recovered. The recovery of ANCs for different G-CSF treatment groups is shown in The repeated-measures ANOVA also indicated a significant intra-group effect; that is, within the same G-CSF treatment group, significant differences in ANCs were detected at different time points . In other words, there were significant differences in the ANC level measured at different time points and G-CSF treatment groups. The ANC level at baseline or 3 months after the end of chemotherapy was not significantly different among G-CSF treatment groups. From 6 months after the end of chemotherapy, along with the follow-up time, the short-acting group had the best effect in promoting ANC recovery, followed by the long-acting group. The mixed treatment group had the worst effect , including the baseline levels of WBC before treatment (p < 0.001), different G-CSF types , surgical method (p = 0.017), lymph node metastasis , and use of Herceptin (p = 0.077) . The baseline level of WBC counts was negatively correlated with the occurrence of leucopenia. The probability ratio of leukopenia was 0.468 between patients with a WBC counts one unit higher before treatment and patients one unit lower . Moreover, different G-CSF types also affected the occurrence of leukopenia (p = 0.027). Among them, the probability of leukopenia in the short-acting group was lower than that in the mixed treatment group. The probability ratio of leukopenia between the short-acting group and the mixed treatment group was 0.380 . The surgical method was also an independent influencing factor for leukopenia (p = 0.041). The probability of leukopenia in patients undergoing mastectomy and sentinel lymph node biopsy (SLNB) was lower than those undergoing mastectomy and axillary lymph node dissection (ALND). The probability ratio of leukopenia after chemotherapy in patients undergoing mastectomy and SLNB versus mastectomy and ALND was 0.470 .According to a multivariate logistic regression model, the independent factors influencing leukopenia in the 12th month after the end of the last chemotherapy are detailed in p < 0.001), different G-CSF types , and molecular typing (p = 0.022) were statistically significant factors (p < 0.1)  . Moreover, different G-CSF types influenced the occurrence of neutropenia (p = 0.043). Among them, the probability of neutropenia in the long-acting group was lower than that in the mixed one. The probability ratio of neutropenia between the long-acting group and the mixed treatment group was 0.461 . The molecular typing was also an independent factor influencing neutropenia (p = 0.025). Patients with hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (Her-2) positive) typing were more likely to have neutropenia than triple-negative patients. The probability ratio of neutropenia between HR-positive (Her-2 positive) and triple-negative mesocytopenia was 3.147 .The baseline level of ANC before treatment was a protective factor for neutropenia in the 12th month after the end of the last chemotherapy .In our hospital, the chemotherapy regimens used by ESBC patients generally contain epirubicin, platinum, or paclitaxel (high-risk factors for CIN). In the present study, ESBC patients received either prophylaxis with 3 or 6 mg of PEG-rhG-CSF or continuous 5–7 injections of rhG-CSF (100 μg) 48 h after the end of chemotherapy. The prophylactic use of G-CSF can effectively reduce the incidence of CIN and even FN to ensure the dose intensity and timeliness of chemotherapy . LambertMost of the current research has focused on the occurrence of CIN and FN during chemotherapy , 30, wheHere, all patients received G-CSF for prophylaxis during each cycle of chemotherapy. However, the employment of different G-CSF types affected the recovery of WBC counts and ANCs during the follow-up period after the end of the last chemotherapy. Through the line chart and repeated-measures ANOVA, we found that the WBC counts increased linearly after the end of the last chemotherapy in the long-acting and short-acting G-CSF groups, but the recovery effect was poor in the mixed G-CSF group . The timp < 0.0001). More seriously, some patients still had leukopenia or neutropenia after 12 months of the end of the last chemotherapy. In our current study, 92 (17.9%) and 63 (12.2%) patients still suffered from leukopenia and neutropenia, respectively, in the 12th month after finishing the last chemotherapy cycle. Therefore, it is necessary to assess the factors influencing the recovery of WBC counts and ANCs in ESBC patients after chemotherapy. In G-CSF guidelines, Smith et al. (Furthermore, we found that, compared with the last chemotherapy, along the follow-up time after the treatment, WBC counts and ANCs gradually recovered but did not reach baseline levels (h et al.  suggesteh et al.  reportedThe WBC count and ANC at baseline reflect the patients’ BM hematopoietic capacity. Thus, patients with lower baseline levels of these parameters are more likely to develop leukopenia and neutropenia following myelosuppressive chemotherapy , 37. ThiOur results indicated that the risk of long-term leukopenia after chemotherapy in the short-acting group and the risk of long-term neutropenia after chemotherapy in the long-acting group were lower as compared to the mixed treatment group. However, no significant difference was detected for the incidence of leukopenia and neutropenia 12 months after chemotherapy between the long-acting and the short-acting groups. PEG-rhG-CSF and rhG-CSF have the same mechanism of action: G-CSF binds to G-CSF receptors expressed on hematopoietic cells to stimulate the proliferation of hematopoietic progenitor cells and accelerate the transformation of immature metamyelocytes into mature neutrophils, thereby increasing functional neutrophils in circulating peripheral blood , 47. ComThe above repeated-measures ANOVA and multivariate regression analysis indicated that differences in the long-term effects of the short-acting and the long-acting groups on WBC counts and ANCs were small during the follow-up after the end of the last chemotherapy, but there is no denying that the treatment outcome of the mixed treatment group was the worst. The mixed treatment group had the worst long-term recovery of WBC and ANC levels and had higher incidences of leukopenia and neutropenia after the end of the last chemotherapy. Therefore, we propose the following probable mechanisms for the cause of poor treatment effects of the mixed treatment group. For one thing, the main reasons for changing G-CSF treatment regimens in the mixed treatment group patients during the chemotherapy cycle were poor patient compliance and limited economic conditions. Patients with poor compliance may not strictly follow the doctor’s advice after being replaced with short-acting rhG-CSF, and patients may voluntarily reduce or delay the injection due to the increased number of rhG-CSF injections. Standard administration of G-CSF can promote the recovery of the hematopoietic function of BM and the protection of chemotherapy . HoweverOverall, the regularly prophylactic use of PEG-rhG-CSF or rhG-CSF can effectively stabilize WBC counts and ANCs during chemotherapy and ensure treatment’s RDI and tolerance. The alternating use of different G-CSF types during the cycle affected the recovery of WBC counts and ANCs after the end of the last chemotherapy. Long-term leukopenia and neutropenia after chemotherapy was associated with WBC count and ANC at baseline, G-CSF types, surgical method, and molecular typing. The application and management of G-CSF in ESBC patients after chemotherapy still need further research.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author.This study was reviewed and approved by the Institutional Review Board of the Second Affiliated Hospital of Zhejiang University School of Medicine. The ethics committee denoted that since this study was a retrospective cohort study, which did not involve human experiments, the informed consent of individual patients was not required.WT and YD conceptualized the research project. YW drafted the manuscript. YW, YZ, and YY participated in the data collection. WT, YW, and YZ reviewed and modified the manuscript. YD supervised the research and led the discussion. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.