Sexual and gender minority populations are disproportionately affected by the global syndemic of HIV and other sexually transmitted infections (STIs). We hypothesized that transgender women (TGW) and non‐binary individuals in Nigeria have more STIs than cis‐gender men who have sex with men (cis‐MSM), and that experiences of stigma and sexual practices differ between these three groups.From 2013 to 2020, TRUST/RV368 enrolled adults assigned male sex at birth who reported anal sex with men in Abuja and Lagos, Nigeria. Participants were tested for STIs and completed questionnaires about sexual behaviours and social stigma every 3 months. Participants were categorized as cis‐MSM, TGW or non‐binary/other based on self‐reported gender identity. Gender group comparisons were made of HIV, gonorrhoea and chlamydia prevalence and incidence; stigma indicators; and condom use during anal sex.p = 0.002). HIV incidence was 8.7 cases per 100 person‐years (PY) , 13.1 cases/100 PY (95% CI 6.5–23.4) and 17.6 cases/100 PY , respectively. Anorectal gonorrhoea incidence was lower in cis‐MSM than TGW (22.2 [95% CI 19.6–25.0] vs. 35.9 [95% CI 27.3–46.3]). TGW were more likely than cis‐MSM to report being affected by stigma, including assault (47.2% vs. 32.3%), fear of walking around (32.4% vs. 19.2%) and healthcare avoidance . TGW were more likely to report always using condoms than non‐binary/other individuals  during receptive anal sex.Among 2795 participants, there were 2260 (80.8%) cis‐MSM, 284 (10.2%) TGW and 251 (9.0%) non‐binary/other individuals with median age of 23 years (interquartile range 20–27). HIV prevalence among cis‐MSM, TGW and non‐binary/other participants was 40.8%, 51.5% and 47.6%, respectively (Sexual and gender minorities in Nigeria have heterogeneous sexual behaviours and experiences of social stigma that may influence the vulnerability to HIV and other STIs. There is a need for tailored interventions that acknowledge and are informed by gender. Further research is needed, particularly among understudied non‐binary individuals, to better understand disparities and inform tailored interventions to improve outcomes among these communities. PrioThere is an unmet need to elucidate risk factors and effective interventions to improve health outcomes among key populations, such as men who have sex with men (MSM), transgender individuals and gender non‐binary individuals . These iNigeria, Africa's most populous country, has institutional and cultural stigmatization of sexual and gender minority populations, including criminalization of same‐sex sexual practices . HIV preIn previous analyses from TRUST/RV368, significant differences were identified in HIV incidence and prevalence between cis‐MSM and TGW , 30, 32,22.1The TRUST/RV368 study was a prospective, observational cohort that enrolled sexual and gender minority participants at specialized community health centres in Abuja and Lagos, Nigeria, using respondent‐driven sampling (RDS) as previously described , 34, 35.2.2A validated, two‐step gender assessment was used for this study . All par2.3Sexual behaviours were assessed via a structured interview conducted by trained study staff. At enrolment, this interview included questions about sexual behaviours with men in the preceding 12 months. Participants who reported a specific sexual activity, such as insertive anal sex, were then asked how often condoms were used. Participants who answered “always” were categorized as having consistent condom use, while all other responses were considered less than consistent, recognizing that consistent condom use reduces HIV transmission , 39, 40.2.4Participants were tested for HIV at enrolment and every 3 months thereafter with fingerstick blood specimens utilizing a parallel algorithm of Determine®  and Uni‐gold®  , 29, 35.The local standard of care during the study relied on syndromic diagnosis of STIs, but study participation included universal screening for STIs every 3 months at up to three anatomic sites. Urine and anorectal swab specimens were used for real‐time PCR‐based testing for gonorrhoea and chlamydia every 3 months using the Aptima Combo® assay  , 35, 41.2.5At enrolment, participants were asked to report their lifetime experiences of stigma in the private, societal and healthcare settings using a tool validated in a population with similar geography, gender identification and sexual practices . Stigma 2.6Demographic characteristics at enrolment were summarized using means, medians with interquartile range and frequencies. Demographics were compared between gender groups using Pearson's Chi‐squared. HIV prevalence was calculated by dividing the number of cases at enrolment by the total number of participants tested and multiplying by 100 to yield a percentage. Prevalence of gonorrhoea and chlamydia was calculated at the time of first test, with separate calculations for each anatomic site . Pairwise comparisons of prevalence by gender group were made using the Tukey method.p values. The prevalence of stigma indicators and condom use at enrolment were compared between gender groups using the two‐sample test of proportions.Incidence rates were calculated by dividing the total number of cases by the population observation time at risk and multiplying by 100 to yield cases per 100 person‐years (PY). Participants with prevalent HIV contributed no observation time to incidence analyses. Participants with prevalent or repeated infections with gonorrhoea or chlamydia contributed observation time after documented clearance with a negative PCR. Incidence rate ratios with 95% confidence intervals were used to test for differences between gender groups. Among participants without HIV, Kaplan–Meier curves were generated to depict the time to incident infection, by gender, with global comparisons using log‐rank tests. Pairwise comparisons applied the Sidak correction to adjust p<0.05. Analyses were performed using Stata 15.0  and SAS 9.4 .For all analyses, statistical significance was defined as 2.7All participants provided written informed consent before enrolment. The study was approved by institutional review boards at the Walter Reed Army Institute of Research, Silver Spring, MD, USA; the University of Maryland, Baltimore, MD, USA; and the National Health Research Ethics Committee and Nigerian Ministry of Defense Health Research Ethics Committee, Abuja, Nigeria.33.1In total, 2795 participants were enrolled, including 2260 (80.8%) cis‐MSM, 284 10.2%) TGW and 251 (9.0%) non‐binary/other individuals. The non‐binary/other group included 215 participants who characterized their gender as “both male & female,” 18 “versatile,” 8 “don't know,” 5 “other” without specification, 2 “refused to answer,” and 3 with missing data. Gender groups differed by age, research site distribution and sexual orientation  and anorectal gonorrhoea . TGW also had a higher prevalence of oropharyngeal gonorrhoea than non‐binary/other individuals . There were no differences in urogenital gonorrhoea or site‐specific chlamydia prevalence between gender groups.Overall HIV prevalence was 42.5%, including 37.2% in Abuja and 58.2% in Lagos. Among cis‐MSM, TGW and non‐binary/other participants, HIV p = 0.025; Figure p = 0.010).Among cis‐MSM, TGW and non‐binary other participants, HIV incidence was 8.7/100 PY (95% CI 6.9–10.8), 13.1 cases/100 PY (95% CI 6.5–23.4) and 17.6 cases/100 PY (95% CI 9.8–29.0), respectively (p<0.001) and non‐binary/other participants . The incidence of anorectal chlamydia was higher among TGW than cis‐MSM participants . The incidence of urogenital chlamydia was higher among cis‐MSM than non‐binary/other participants .The incidence of anorectal gonorrhoea among TGW was 35.9 cases/100 PY (95% CI 27.3–46.3), which was significantly higher than among cis‐MSM , but there were no significant differences in time to first urogenital (log‐rank p = 0.419) or oropharyngeal (log‐rank p = 0.204) gonorrhoea infection. There were no significant differences by gender group in time to first chlamydia infection at the anorectal (log‐rank p = 0.21), urogenital (log‐rank p = 0.23) or oropharyngeal (log‐rank p = 0.98) sites.In time‐to‐event analyses, there were differences between the three gender groups in the time from enrolment to incident HIV . Cis‐MSM were more likely to report no participation in receptive anal sex in the last year (31.8%) than TGW (6.0%), or non‐binary/other people . Non‐binary/other individuals were also more likely to report no participation in receptive anal sex than TGW .Behavioural differences were noted between gender groups Figure . TGW werp = 0.045). Of participants who reported receptive anal sex, both cis‐MSM and TGW were more likely to report always using condoms than non‐binary/other individuals .Of participants who reported participating in insertive anal sex, TGW were more likely than non‐binary/other individuals to report always using condoms (48.5% vs. 36.4%, 4We found a high overall burden of HIV and other STIs among Nigerian sexual and gender minority populations. TGW had particular high prevalence and incidence of HIV, which is consistent with previous literature , 43, 44.There is a well‐recognized need for data regarding the syndemic of HIV and other STIs, particularly among sexual and gender minorities , 48. ConThe combinations of identities and orientations that were seen in our study demonstrate interesting relationships that are likely contributory to STI vulnerability. The majority of TGW in this study self‐identified as gay/homosexual, while the majority of cis‐MSM and non‐binary/other individuals self‐identified as bisexual. Although heterosexual was provided as a response option, only two participants identified with this sexual orientation. We did not ask about gender preferences for sexual partners, but recent data have suggested that birth males who are sexually attracted exclusively to men have heightened HIV risk, even when compared to other men and TGW who have sex with men but are sexually attracted to women .Despite reporting a higher frequency of consistent condom use than other gender groups, TGW had increased incidence of anorectal STIs. Although condom use was more consistent than in other groups, it may have been insufficient to reduce transmission driven by higher engagement in anal receptive sex. By extension, this may also suggest that condom‐related messaging alone is unlikely to be sufficient to curb HIV transmission, but must be accompanied by biologic interventions, such as PrEP with demonstrated safety and efficacy in sexual and gender minority populations , 57. FurThe high prevalence and incidence of gonorrhoea and chlamydia at the anorectal site merits further investigation of extragenital routes of transmission. Implementation of routine STI screening of extragenital sites for sexual and gender minority populations is recommended as extragenital STIs are less likely to be symptomatic, yet more common than urethral STIs in these populations . ProvidiStigma based on gender and sexual practices has garnered increased attention over the last decade, and efforts have intensified to quantify the impact of stigma on health outcomes , 63, 64.The Nigerian state criminalizes same‐sex sexual behaviour, prohibits gatherings related to same‐sex sexual relationships and imposes penalties for these activities that can include incarceration. Legally sanctioned stigma experienced by sexual and gender minorities negatively affects the health of these individuals , 72. NigThis study was conducted in urban clinics designed to engage marginalized, vulnerable and diverse sexual and gender minorities, so our findings may not be generalizable to other settings. Study eligibility and recruitment focused on individuals who were identified as male at birth and who participated in sex with men, which resulted in enrolment of some transgender and non‐binary individuals but may have excluded others who did not satisfy inclusion criteria. Gender identity may change over time but was only assessed at study enrolment; future studies should consider repeated longitudinal assessment of gender to explore relationships between gender fluidity and outcomes of interest. Analysis of non‐binary/other participants in aggregate may have obfuscated important differences between individuals with diverse gender and sexual identities, including participants who chose not to categorize their gender; future prospective research should be powered to characterize unique vulnerabilities of more granular gender groups. These analyses were exploratory in nature and each of the identified differences between gender groups merits further independent investigation and robust analysis. The multidimensional dataset available from TRUST/RV368 could be leveraged in future analyses using complex statistical approaches, such as machine learning, to further elucidate relationships between gender identity and STI risk, including the identification of underlying causal mechanisms. Marginalized sexual and gender minorities can be difficult to engage and retain, with previous analyses from our cohort showing 56% loss to follow‐up over the course of the TRUST/RV368 study, potentially biasing results of longitudinal evaluations . Sexual 5Sexual and gender minority populations, including cis‐MSM, TGW and non‐binary/other individuals in Nigeria, have heterogeneous sexual behaviours and diverse vulnerabilities to HIV and other STIs. Of these populations, sexual practices of the non‐binary/other individuals are the least understood and merit specific attention in future studies. The high incidence of HIV and other STIs among TGW and non‐binary/other participants in this study indicates that targeted prevention interventions should be prioritized toward these groups. Study designs that prioritize gender‐specific data gathering, and inclusion of diverse gender minorities, are necessary to inform tailored, rights‐affirming interventions to improve clinical outcomes among sexual and gender minority populations.There are no competing interests.EL analysed the data and authored the first draft of the manuscript. FH and ABM conducted additional data analyses. AK and SA oversaw the collection of clinical data and assisted with the interpretation of these analyses. HQ provided project management support and drafted sections of the manuscript. JAA, MLR, MEC, SDB and RGN designed the TRUST/RV368 study, provided general oversight of the study and assisted with the interpretation of these analyses. TAC coordinated TRUST/RV368 study activities, conceptualized these analyses and oversaw drafting of the manuscript. All authors reviewed this manuscript, provided feedback and approved the manuscript in its final form.The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army, the Department of Defense or the Department of Health and Human Services. The investigators have adhered to the policies for protection of human subjects as prescribed in AR‐70.


To investigate the safety and effectiveness of direct oral anticoagulants (DOAC) versus vitamin K antagonists (VKA) after recent stroke in patients with atrial fibrillation (AF) aged ≥85 years.Individual patient data analysis from seven prospective stroke cohorts. We compared DOAC versus VKA treatment among patients with AF and recent stroke (<3 months) aged ≥85 versus <85 years. Primary outcome was the composite of recurrent stroke, intracranial hemorrhage (ICH) and all‐cause death. We used simple, adjusted, and weighted Cox regression to account for confounders. We calculated the net benefit of DOAC versus VKA by balancing stroke reduction against the weighted ICH risk.≥85y = 0.65, 95%‐CI ) and < 85 years  in simple (pinteraction = 0.129), adjusted (pinteraction = 0.094) or weighted (pinteraction = 0.512) models. Analyses on recurrent stroke, ICH and death separately were consistent with the primary analysis, as were sensitivity analyses using age dichotomized at 90 years and as a continuous variable. DOAC had a similar net clinical benefit in patients aged ≥85 (+1.73 to +2.66) and < 85 years (+1.90 to +3.36 events/100 patient‐years for ICH‐weights 1.5 to 3.1).In total, 5,984 of 6,267 (95.5%) patients were eligible for analysis. Of those, 1,380 (23%) were aged ≥85 years and 3,688 (62%) received a DOAC. During 6,874 patient‐years follow‐up, the impact of anticoagulant type (DOAC versus VKA) on the hazard for the composite outcome did not differ between patients aged ≥85 (HRThe favorable profile of DOAC over VKA in patients with AF and recent stroke was maintained in the oldest old. ANN NEUROL 2022;91:78–88 In the absence of randomized data, we used prospectively collected, individual patient data pooled within an international collaboration of cohort studies on the use of OAC following ischemic stroke in patients with AF.We used prospectively collected, individual patient data pooled from an established international collaboration of investigator‐initiated cohort studies of patients with AF, recent ischemic stroke or transient ischemic attack (TIA) and OAC treatment, as described previously.In this study, we included consecutive patients with (1) an index recent  ischemic stroke or TIA Data were collected as described in prior researchFollow‐up data included length of follow‐up and absence or occurrence and timing of any of the following outcome events, which were defined in line with prior researchThe primary outcome was the time to occurrence of the composite of recurrent ischemic stroke, ICH and all‐cause death, in accordance with prior research.We stratified patients' characteristics by dichotomized age (≥85 vs. <85 years) and type of OAC (VKA vs. DOAC). We presented categorical data using frequencies and percentages and continuous data using the median and interquartile range (IQR) or mean and standard deviation (SD) as appropriate. We compared categorical variables using the χAs for the main analysis, we modelled time to primary outcome using Cox proportional hazards regression. For this, we analyzed time to first event after OAC initiation, without considering further events. To assess the effect of age on the performance of OACs, we included type of OAC (DOAC vs. VKA), dichotomized age (≥85 vs. <85 years) and an interaction term between these variables as fixed effects in the model. A significant interaction would indicate that the association between type of OAC and the composite outcome is modified by age and therefore differs in the oldest old compared to their younger counterparts. The model included the participating cohort study as a stratum.2DS2‐VASc scoreWe fitted the model three times according to the predefined analysis plan: (i) simple model including type of OAC and dichotomized age, with and without interaction term; (ii) adjusted model taking into account the known prognostic importance of sex, NIHSS at baseline and CHAWe performed the following secondary analyses:(1) We fitted the Cox models (i) – (iii) described above separately for the individual outcomes recurrent ischemic stroke, ICH and death. To account for competing risks, for these analyses we fitted Cox proportional cause‐specific hazards models treating competing outcomes as censored observations.(2) We analyzed the net clinical benefit (NCB) of DOAC over VKA in patients aged ≥85 and < 85 years. We calculated the NCB by subtracting the weighted rate of excess ICH attributable to DOAC from the rate of excess ischemic stroke prevented by DOAC according to the following formula, as in prior researchAs sensitivity analyses, we repeated the main analysis for the primary (composite) outcome using age as:(1) a categorical variable, dichotomized to ≥90 vs. <90 years. For this, we refitted all Cox models (i) – (iii) as described above.(2) a continuous variable, using cubic B‐splines to model the non‐linear association between age and log‐hazard for the composite outcome. For this, we fitted the weighted model (iii) described above twice, with and without the interaction OAC type by age, and compared the two models using a likelihood ratio test. We graphically present the predicted rate of the composite outcome by age stratified to OAC type.Statistical analyses were performed using R version 3.6.2 (2019‐12‐20) .We conducted this study in accordance with the STROBE Statement for observational studies.The NOACISP‐LONGTERM registry and the current analysis of pooled individual patient data were approved by the ethics committee in Basel, Switzerland . Patients provided written informed consent for participation in NOACISP‐LONGTERM. The requirement for additional local ethical approval and patient informed consent differed among participating studies and was acquired by the local investigators as necessary. CROMIS‐2 was approved by the National Research Ethics Committee, London Queen Square and patients with capacity gave informed written consent. When patients could not consent, written consent from a proxy was obtained as defined by relevant local legislation. The SAMURAI‐NVAF registry and the current collaboration were approved by the ethics committee in the National Cerebral and Cardiovascular Center (M23‐18‐3 and M29‐077).In total, 5,984 of 6,267 (95.5%) patients were eligible for analysis. Information on OAC type was complete. Seven patients were excluded for missing age and 125 patients for missing follow‐up data  and TIA in 391 (6.5%); 2,858 patients (47.8%) were female. The median age was 78 years ; 1,380 (23.1%) patients were aged ≥85 years and 4,604 (76.9%) were aged <85 years. OAC was initiated at a median (IQR) of 5 (2–11) days after the index event with DOAC in 3,688 patients (61.6%) and VKA in 2,296 patients (38.4%).2DS2‐VASc scores compared to younger patients. Diabetes mellitus was more common among younger patients, as was dyslipidemia and current smoking. Time to OAC initiation after index event was shorter in the oldest old.The baseline characteristics of the oldest old compared to their younger counterparts are displayed in Table p = 0.179), sex  and time to OAC initiation . Compared to patients with VKA treatment, DOAC‐treated patients had less often ischemic stroke as index event , reflected in their lower NIHSS scores . They had less often concomitant antiplatelets  and diabetes , but more commonly hypertension  and dyslipidemia . DOAC‐treated patients had higher CHA2DS2‐VASc scores . This was largely consistent in the subgroups of patients aged ≥85 vs. <85 years  outcome events, a primary outcome event rate of 14.5%/year. The follow‐up time, number and crude rate of events for the primary outcome stratified to age group and OAC type are given in Table interaction = 0.129). Consistent findings resulted from repeated analyses refined by adjustment for potential confounders  and weighting . Thus, the better performance of DOAC over VKA with regard to the composite outcome was not dependent on age and was maintained in the oldest old. The detailed results of all Cox models for the composite outcome are presented in Table In the simple Cox model, age < 85 years was associated with a significantly lower hazard for the primary outcome compared to age ≥ 85 years, as indicated by a HR of 0.46 . Likewise, DOAC‐treated patients had a lower hazard than VKA‐treated patients with a HR of 0.74 . There was no evidence for an interaction between age group and OAC type on their impact on the composite outcome . ). Likewiinteraction > 0.05). For the outcome death, there was evidence for a weak interaction only in the simple (pinteraction = 0.054) and adjusted models (pinteraction = 0.029), indicating that the lower hazard for death among patients treated with DOAC as compared to VKA‐treated patients was even more pronounced in the oldest old than in their younger counterparts . The detailed results of all Cox cause‐specific hazards models for the individual outcomes are presented in Table The point estimates for the NCB of DOAC over VKA were similar in patients aged ≥85 (+1.73 to +2.66) and < 85 years (+1.90 to +3.36 events per 100 patient‐years) and remained positive over the entire range of ICH weights used (1.5 to 3.1), with wide confidence intervals crossing zero in the smaller group of patients aged ≥85 years. Fig interaction = 0.283), adjusted (pinteraction = 0.514) and weighted (pinteraction = 0.433) models, consistent with the main analysis  vs. <90 years  showed no evidence for interaction between age and OAC type on the hazard for the composite outcome in the simple . The hazard for the composite outcome continuously increased with increasing age in a non‐linear fashion.Using age as a continuous variable, the favorable profile of DOAC over VKA regarding the composite outcome was maintained across the entire age spectrum in the weighted model Fig . There wThis study focused on the safety and effectiveness of DOAC versus VKA in the oldest‐old patients with AF and recent stroke or TIA in a real‐world setting. The key finding was that the benefits of DOAC over VKA were consistently maintained in the oldest old, even when potential confounders were accounted for.In our study, DOAC were associated with a lower hazard than VKA for the composite outcome of recurrent ischemic stroke, ICH and all‐cause death in patients with AF and recent ischemic stroke, independent of age. The favorable profile of DOAC was maintained in the oldest old, whether defined as aged 85 or 90 years or older. This observation is highly relevant for clinical practice as it contradicts the assumptions of many clinicians who are reluctant to use DOAC in this age group, particularly in multimorbid patients.Notably, there was no signal of a safety concern regarding ICH risk among the oldest‐old DOAC‐treated patients with recent ischemic stroke, which is a widespread concern.These results are clinically important because limited randomized data exist for such patients, as patients with recent stroke within 7 days,For patients with recent stroke, subgroup analyses in observational studies suggested the safety of DOAC versus VKA for the age groups of ≥75Our study has the following strengths: (1) we used individual patient data pooled within an established collaboration of prospective observational studies from Europe and Asia; (2) the high data completeness limits the risk of spurious findings; and (3) the consistency of results both in unadjusted and in adjusted and – most importantly – weighted analyses accounting for potential confounders, as well as in net benefit analyses and in sensitivity analyses focusing on patients ≥90 years or using age as a continuous measure, underlines the robustness of our key finding.We are aware of the following limitations: (1) as our data are observational rather than randomized, baseline imbalances in the allocation to the type of OAC that were unaccounted for might have introduced bias or confounding; (2) our study included exclusively OAC‐treated patients, so age‐matched stroke patients without OAC treatment were not available for comparison. Of note, the placebo‐controlled ELDERCARE‐AF trial suggested the benefit of anticoagulation even in very elderly patients with AF who were not appropriate candidates for standard anticoagulant treatmentIn conclusion, our study provides new and compelling evidence indicating that the benefits of DOAC over VKA in patients with AF and recent stroke are maintained among the oldest old.All authors contributed to study design, data acquisition and analysis, and critically revised the manuscript.MP: speaker honoraria from Sanofi‐Aventis, Boehringer‐Ingelheim, Bayer, BMS, Daiichi‐Sankyo, Pfizer . MK: speaker honoraria from Bayer, Nippon Boehringer‐Ingelheim, Daiichi‐Sankyo. MC: consulting fees from Boehringer‐Ingelheim, Pfizer/BMS; advisory board Daiichi‐Sankyo. GMDM: consultant/speaker honoraria from Bayer, travel honoraria from Pfizer. ST: travel grants from BMS/Pfizer. GYHL: Consultant and speaker for BMS/Pfizer, Boehringer‐Ingelheim and Daiichi‐Sankyo; no fees are received personally. LHB: consultancy or advisory board fees or speaker's honoraria from Bayer and BMS. PAL: research grants from Bayer, travel grants from Bayer, Pfizer, advisory board compensation from Bayer, Pfizer, Daiichi‐Sankyo, BMS. KT: lecture honoraria (modest) from Daiichi‐Sankyo, Bayer Yakuhin, Nippon Boehringer‐Ingelheim, BMS. DJW: personal fees from Bayer, Portola . STE: research support from Pfizer, Daiichi‐Sankyo; compensation from Stago (manufacturer of coagulation testing systems) for educational material; travel/speaker honoraria from Bayer, Boehringer‐Ingelheim, BMS, Daiichi‐Sankyo; advisory board Bayer, Boehringer‐Ingelheim, BMS. The remaining authors declare no relevant conflicts.Table S1 Participating cohort studiesTable S2. Cox proportional cause‐specific hazards models for time to recurrent ischemic stroke, intracranial hemorrhage and death, accounting for competing risksTable S3. Net clinical benefit of DOAC over VKATable S4. Cox models for time to composite outcome using age dichotomized at 90 yearsTable S5. Complete list of investigators of the cohort studies that contributed patients to this analysisClick here for additional data file.


P = .36; 9 month OS: 58% vs 63%, HR 1.08, 95% CI 0.48‐2.41, P = .86) and when combined with chemotherapy . No significant difference in response rate or ≥G3 immune‐related toxicities between Pem100 and Pem200 was observed. A cost minimisation analysis evaluating the degree of cost savings related to drug costs estimated a within study cost saving of SGD4,290,912 and cost saving per patient of SGD39,942 in the Pem100 group. A 100 mg of pembrolizumab appears to be effective with reduction in cost. A randomised trial should be done to investigate a lower dose of pembrolizumab.A dose of 200 mg 3‐weekly of pembrolizumab was approved by the Food and Drug Administration (FDA) as treatment for advanced non‐small cell lung cancer (NSCLC) without oncogenic drivers. This is despite evidence showing no difference in efficacy with 2 mg/kg. Our study aimed to assess the efficacy of a lower fixed dose of 100 mg, which is closer to 2 mg/kg weight‐based dose in an average‐sized Asian patient. All patients receiving pembrolizumab for advanced NSCLC from January 2016 to March 2020 in National University Hospital, Singapore, were included in this retrospective observational study. The effect of pembrolizumab 100 mg (Pem100) vs 200 mg (Pem200) upon survival outcomes, toxicity and cost were examined. One hundred fourteen patients received pembrolizumab. Sixty‐five (57%) and 49 (43%) received Pem100 and Pem200, respectively. There was no difference in progression‐free survival (PFS) and overall survival (OS) between Pem100 vs Pem200 as a single agent  compared with standard‐dose pembrolizumab. The results also confirm the clinical activity of pembrolizumab at a lower dose than 2 mg/kg every 3 weeks, which could provide considerable cost savings to patients and the health system. AEadverse eventDCRdisease control rateFDAFood and Drug AdministrationHRhazard ratioirAEsimmune‐related adverse eventsNSCLCnon‐small cell lung cancerOSoverall survivalPDprogressive diseasePD‐1programmed cell death protein 1Pem100pembrolizumab 100 mg 3‐weeklyPem200pembrolizumab 200 mg 3‐weeklyPFSprogression‐free survivalPRpartial responseRRresponse rateSDstable diseaseTPStumour proportion score1Worldwide, lung cancer is the most common cancer and leading cause of cancer death.Pembrolizumab is a fully humanised immunoglobulin G4 monoclonal antibody directed against the programmed cell death protein 1 (PD‐1) receptor, antagonising the interaction between itself and its ligand, resulting in anti‐tumour immune response.In the landmark KEYNOTE‐001, an ex vivo pharmacokinetics study of PD‐1 receptor saturation found complete peripheral target engagement at 1 mg/kg. In the initial dose escalation cohort, durable anti‐tumour activity across all patient cohorts from 1‐10 mg/kg once every 3 weeks was observed.Despite the pharmacokinetics profile showing PD‐1 receptor saturation at 1 mg/kg and clinical efficacy at 2 mg/kg, a flat dose of 200 mg 3‐weekly was used in Phase III trials leading to the FDA approval of 200 mg every 3 weeks.Goldstein et al performed an economic analysis comparing the FDA approved fixed dosing and personalised dosing at 2 mg/kg and estimated a cost saving of USD$0.8 billion annually to the United States healthcare system based on an average weight of 75 kg in an American adult patient.Given the lack of benefit demonstrated by pembrolizumab at doses above 2 mg/kg, the lower weight of Asian patients, economic benefits of a lower dose and packaging of pembrolizumab in 100 mg vials, a fixed dose of 100 mg pembrolizumab required evaluation in an Asian population.Using a retrospective observational design, our study aimed to evaluate the efficacy of low‐dose pembrolizumab (Pem100) compared with standard‐dose pembrolizumab (Pem200) in the treatment of NSCLC.22.1All patients receiving palliative intent pembrolizumab for advanced NSCLC with or without chemotherapy between January 2016 and March 2020 in an academic tertiary medical centre  were identified retrospectively from the pharmaceutical database. Baseline patient demographics, tumour and treatment characteristics were extracted from the electronic medical records.The dose of 100 mg was routinely delivered based on an approximate 2 mg/kg weight‐based dose, for patients who did not have an adequate financial reimbursement plan or based on physician's preference. Local protocols continue treatment until disease progression, unacceptable toxicities, death, patient's decision to stop treatment or after a total of 35 cycles of pembrolizumab although some patients who remained progression free after 35 cycles continued treatment.2.2Chest and/or abdominal CT scans were performed by clinicians every 8‐12 weeks as part of routine clinical care, to evaluate patient's response and assess for disease progression. The scans were evaluated by investigators retrospectively. In line with the KEYNOTE studies efficacy analysis was examined only in patients without an oncogenic driver mutation. A systemic response to pembrolizumab was measured by standard Response Evaluation Criteria in Solid Tumours (V.1.1).2.3The PD‐L1 IHC 22C3 pharmDx assay2.4P‐value of <.05 was considered statistically significant. All statistical tests were two‐sided and were performed using IBM SPSS Statistics Version 22.Differences in the baseline characteristics of patients receiving Pem100 and Pem200 were evaluated using the Fisher's exact test. Survival analyses were performed using the Kaplan‐Meier method and were compared using a log‐rank test. Multi‐variable Cox proportional hazard regression models were used to assess the relationship between baseline factors (including treatment) and survival. A Based on an acceptance of non‐inferior survival and toxicity outcomes, a limited economic evaluation was carried out using a cost‐minimisation approach.33.1One hundred fourteen patients received pembrolizumab for advanced NSCLC from January 2016 to March 2020 in National University Hospital (Singapore). Baseline demographics are shown in Table Tumour characteristics are summarised in Table 3.2P > .05). Patients in the Pem200 group were younger , with worse renal function (P < .001) compared to those in the Pem100 group  received pembrolizumab at a starting dose of 100 mg 3‐weekly (Pem100). The remaining 43% received pembrolizumab at a starting dose of 200 mg 3‐weekly (Pem200). The two cohorts were matched in terms of gender, smoking history, ECOG status and histological subtype  and received pembrolizumab as a monotherapy . Median dose received was 2.87 mg/kg vs 1.85 mg/kg (P < .001) and a <2 mg/kg dose was received by 4% vs 62% (P < .001) in the Pem200 and Pem100, respectively.Significantly more patients in Pem100 had a PD‐L1 (TPS) score of ≥50%  . PFS for Pem100 and Pem200 did not differ in all subgroups examined  for Pem100 vs Pem200, respectively   Figure .P = .71) for the Pem200 cohort. Median OS was not reached in either group. Nine‐month OS with chemotherapy was 85% vs 58%  for Pem100 and Pem200, respectively.Where Pembrolizumab was delivered with chemotherapy, the median PFS was not reached in the Pem100 cohort vs 11.9 months . This was however not statistically significant. Similarly, when pembrolizumab was combined with chemotherapy, there was also no difference in the RR and DCR   of 88 patients who received pembrolizumab in the first line setting. Forty‐six received pembrolizumab as a single agent and 42 received pembrolizumab combined with chemotherapy. RR and disease control rate (DCR) were numerically higher in patients who received single agent Pem100 compared to Pem200  in Singapore. The total number of cycles received by all the patients in our study is 1243, with 752 vs 491 cycles delivered in the Pem100 and Pem200 group, respectively. The median number of cycles was 7 .We estimated a total cost saving in the study population of SGD 4,290 912 based on the total number of cycles of pembrolizumab received by the Pem100 group. Assuming Pem100 was used instead of Pem200 across the entire study population, the cost minimisation analysis demonstrates a cost saving of SGD 7, 092, 558 and SGD 39942 per patient based on the median number of cycles received in our study population. A further sensitivity analysis of Pem100 in patients weighing ≤100 kg demonstrates a total cost savings of SGD 70065706.4To our knowledge, our study represents the largest cohort to date in which the efficacy of a lower fixed dose of 100 mg pembrolizumab given 3‐weekly is demonstrated.Many studies have demonstrated that dose selection of immune checkpoint inhibitors can be challenging with non‐linear relationships between dose and clinical outcomes. A pharmacokinetic analysis of doses of 200 mg and 2 mg/kg showed similar exposure distributions with no advantage to either dosing approach.In our study, the median patient weight was 59 kg. More than half of our patients received a fixed dose of 100 mg, with a median dose of 1.85 mg/kg  in the Pem100 group. This was close to a dose of 2 mg/kg and could explain why we did not see a difference in the efficacy in Pem100 vs Pem200. No efficacy difference between <2 and ≥ 2 mg/kg 3‐weekly. In fact, survival results numerically favoured <2 mg/kg.A fixed 100 mg dose of pembrolizumab appears to be cost efficient and logistically feasible, requiring a complete vial per patient. Goldstein et al demonstrated huge cost savings to the US healthcare system by using a personalised dosing of 2 mg/kg.Our study has its limitations. Despite the attractiveness of dosing pembrolizumab at a 100 mg fixed dose 3‐weekly or 200 mg 6‐weekly, fixed dosing must be interpreted with caution in a heavier patient. None of the patients in our study received a dose a <1 mg/kg, with a dose range of 1.24 to 1.99 mg/kg for patients who received <2 mg/kg pembrolizumab. Cross‐trial comparisons of our study against the other KEYNOTE studies are not valid given the different nature of the study design and heterogenicity of our study population. Moreover, the retrospective nature of the study, differing baseline characteristics and limited sample size does not allow for valid efficacy comparison among different dosing strategies. Also, there are some imbalances in our study groups with more patients in the Pem100 group having a PDL1 TPS score ≥50%. In a non‐randomised setting, this may result in selection bias, potentially accounting for the favourable outcome observed here for Pem100. However, most of these patients also received pembrolizumab as monotherapy and the subgroup analysis of PFS based on PDL1 expression in patients receiving pembrolizumab monotherapy did not differ. Thirdly, the relatively limited sample size limits the power of our study to demonstrate a statistically significant difference. Finally, given no difference was identified in the clinical outcomes of the two regimens a cost minimisation analysis was used to examine the cost saving provided by Pem100. This was not planned a priori and simply provides an indication of possible savings. The costs assessed are only those of the drug and do not include regimen related costs such as drug administration, premedication, clinic visits, subsequent therapy and AE management. Based on the study outcomes these costs are not anticipated to vary; however, further formal assessment of the cost‐utility of Pem100 should be considered alongside any future randomised study.Despite these limitations, our study is the first to suggest clinical efficacy of pembrolizumab at a fixed dose of 100 mg 3‐weekly. This lower dose could be efficacious and provide considerable cost savings to both patients and the health system more widely. Such savings could be redistributed to other health needs.5In our study, pembrolizumab had efficacy at a dose of 100 mg 3‐weekly. With the expanding role of immune checkpoint inhibitors in many tumour types, the principles and solutions discussed here will be highly relevant to oncologists, policymakers and patients alike. A randomised prospective trial is now required to further investigate the role and cost‐effectiveness of lower‐fixed dosing of pembrolizumab at 100 mg 3‐weekly or 200 mg 6‐weekly.A. Jeyasekharan has received consultancy fees from Turbine Ltd, Astra Zeneca, Janssen and MSD along with travel funding from Perkin Elmer and research funding from Janssen. R. Sundar is advisory board for Bristol Myers Squibb, Merck, Eisai, Bayer, Taiho, Novartis and MSD, and has received Honararia for talks from MSD, Eli Lily, BMS, Roche, Taiho and Astra Zeneca, travel funding from Roche, Astra Zeneca, Taiho and Eisai and research funding from Paxman Coolers, MSD. B. C. Goh has received research funding from Merck and Co, Bayer Healthcare, Taiho Pharmaceuticals, Squibb and Adagene, consultancy role in Adagene, Bayer Healthcare, Merck and co and Astra Zeneca, and has stock ownership in Gilead sciences, Moderna Inc. R. Soo has received honorarium from Astra Zeneca, Amgen, Bayer, BMS, Boehringer Ingelheim, Lilly, Merck, Novartis, Pfizer, Roche, Taiho, Takeda and Yuhan. W. P. Yong has received honorarium advisory board and speaker fee from Taihio, Ipsen, Novartis, Eisai, BMS, SD, Sanofi‐Aventis, Bayer, Astra Zeneca and royalty from miRXES.The study was approved by the National Health Group Domain Specific Review Board (NHG DSRB) (Reference number: 2017/012654) and was conducted in accordance to the Declaration of Helsinki provision. Informed consent was waived because of the retrospective design of the study.


No international standards include vitamin D levels at diagnosis or during treatment. It is included in the Children's Oncology Group long‐term follow‐up guidelines. However, bone health complications (like osteopenia and atraumatic fractures) can occur at diagnosis or during treatment as well.In this small case series, we illustrate the complexity of bone health complications among our broad paediatric oncology population. If the vitamin D level is low we supplement the patient with one standard oral dose . We do not adjust depending on diagnosis.Because of the potentially negative outcomes on short, medium and long term, we recommend checking vitamin D levels on diagnosis for all newly diagnosed patients. It is a simple, low cost test and one dose of oral supplementation can easily treat the deficiency. The aetiology of the reduced bone mineralization is multifactorial. It includes a direct effect of the disease itself, treatment effects , reduced sun exposure, decreased nutrition and immobility and hormonal therapy to suppress menstruation.Bone formation is affected by gender, the hormonal environment, nutritional status, vitamin D, weight‐bearing exercise and genetics. Strong links exist between reduced bone mineral density (BMD) and vitamin D levels.Vitamin D is converted to 1:25 cholecalciferol where it acts via the vitamin D receptor. The increased absorption of calcium and phosphate from the gut is the most important action for improving bone health and reducing osteopenia. All cancer therapies can decrease BMD through long‐term endocrine alterations and altered body composition. Endocrine, metabolic, and skeletal sequelae are among the most frequently described complications in long‐term survivors, affecting between 20% and 50% of the patients.22.1A 3.5‐year‐old Samoan girl diagnosed with B cell acute lymphatic leukaemia (ALL), started treatment per COG AALL0932 was found to have an insufficient 25‐OH vitamin D level 3 weeks after she was diagnosed. The 25‐OH vitamin D level was 37 nmol/L . Due to febrile neutropenia and a clinical suspicion of osteomyelitis, an X‐ray of tibia and fibula were made showing diffuse osteopenia. She received supplementation of vitamin D per local protocol, 300 000 IU in one oral dose. Despite this supplementation 4 months later the vitamin 25‐OH vitamin D level was 19 nmol/L, and supplementation of 300 000 IU was given per local protocol. The level was checked 2 months later and was found to be sufficient. The patient had entered the maintenance phase of her ALL treatment at that stage.2.2A 4‐year‐old New Zealand European girl, diagnosed with T cell ALL, received treatment per COG AALL0434, high‐risk arm, including cranial radiation at 12 Gy. The treatment was complicated by a PICU admission due to sepsis with cardiac arrest, renal failure and typhlitis with ileus and intravenous nutrition dependence due to a pseudomonas bacteraemia.Z‐score of −3.6 and more imaging revealed extensive central vertebral body collapse in her spine, particularly in the lower thoracic and lumbar spine, as well as osteopenic bones on X‐ray. She was treated for her osteopenia as per the local endocrine team with supplementation of vitamin D  and six monthly Zoledronate, 1 mg/kg/dose. The DEXA scan repeated 5 year later showed a Z‐score of +1.3  and she was clinically asymptomatic.During maintenance therapy she presented with back and leg pain and was found to have an insufficient 25‐OH vitamin D level (30 nmol/L). A DEXA scan revealed a 2.3A 8‐year‐old New Zealand European boy, diagnosed with medulloblastoma, received chemotherapy and radiotherapy per SJMB03 protocol. His treatment was complicated by severe acquired brain injury, resulting in immobility. At the end of his treatment, he had a fall from his bed and investigations revealed osteopenia and a transverse fracture of the distal femur. The 25‐OH vitamin D level was 52 nmol/L. He was started on calcium supplements per the local endocrine team. Follow‐up vitamin D level was 75 nmol/L 5 months later.2.4A 17‐year‐old New Zealand European boy, was diagnosed with stage 2a Hodgkin Lymphoma, treated per EURONET protocol, no radiation. He was diagnosed with avascular necrosis at the end of his treatment on MRI, 25‐OH vitamin D level was 50 nmol/L. He was also diagnosed with muscular weakness due to steroids; this improved over follow‐up.2.5A 16‐year‐old Samoan boy, treated for BCOR Sarcoma per AEWS0031 protocol. His treatment was complicated by multiple fractures due to trauma and osteopenia. His 25‐OH vitamin D level was <10 nmol/L and supplemented according to the local protocol with one single oral dose of 600 000 IU. Hereafter, he was lost to follow‐up.3The heterogeneity and complexity of low levels of vitamin D and bone health complications is illustrated here. Adequate vitamin D levels alone will not prevent all the bone health complications in this patient population; these complications are multifactorial. However, the size of adequate vitamin D levels during treatment or at the diagnosis has not been investigated and no published international guidelines or recommendations were found. Limited research is done to investigate the importance of bone health complications in this population during treatment, most published studies report about childhood cancer survivorship patients.Orgel et al. focussed specifically on bone changes during induction treatment in patients with ALL. No significant differences were found in assessment pre‐treatment compared to healthy individuals. After the induction phase, containing steroids, a significant decline was seen in the cancellous vertebral BMD, representing a median loss of 26.8%. The majority of the patients had insufficient or deficient levels of vitamin D (<20 ng/mL) at diagnosis (66%).Z‐score was reduced for the entire cohort when determined based on chronological age and bone age.The STeroid‐association Osteoporosis in the Pediatric Population (STOPP) research initiative is an extensive Canadian paediatric bone health research program.Currently, it is not standard to include vitamin D levels or BMD studies at diagnosis or during treatment for children diagnosed with a malignancy to assess their bone health. However, it is included in the Children's Oncology Group (COG) long‐term follow‐up guidelines. They recommend an initial evaluation of BMD at entry into long‐term follow‐up; acknowledging hereby that childhood cancer survivors are at risk for these bone health complications.In this brief report, we present a small heterogenetic case series. It illustrates the range of possible bone health complications in the broad spectrum of paediatric oncology during treatment. We recognize that not all bone health complications are solely related to low vitamin D levels, and have a multifactorial aetiology. However, we do feel that an adequate vitamin D level maintained during treatment can prevent complications.We acknowledge that an adequate vitamin D level will not result in the complete normalization of bone mineralization in our patient population.Long‐term outcome data are not yet available to support the benefit of an optimized vitamin D level during treatment. However, vitamin D deficiency is common and may result in negative bone health outcomes in the short, medium and long term for paediatric oncology patients. Given the potentially negative outcomes, the ease of treatment and its low cost, vitamin D deficiency should be identified and treated aggressively on presentation. We recommend prospective long and medium term studies to investigate the benefits for adequate vitamin D levels in paediatric oncology patients.All authors have no conflict of interest.Conceptualization, L.N., M.P.D.S., P.H.; Writing—Original Draft, L.N.; Writing—Review & Editing, L.N., M.P.D.S., P.H.; Supervision, P.H.; Data Curation, L.N.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. We received institutional approval for the retrospective study with no need for patients or parents consent.


We report on a young Ghanaian female who was diagnosed with thrombotic thrombocytopenic purpura (TTP) but had an ischemic stroke as the initial presentation. She was successfully treated with therapeutic plasma exchange. This case illustrates how TTP can masquerade as ischemic stroke and the application of PLASMIC score without ADAMTS‐13 assay in risk prediction. The initial working diagnosis was an acute ischemic stroke with left hemiparesis, Abnormal uterine bleeding from a uterine leiomyoma, hematological malignancy, and a bleeding diathesis. Samples were taken for blood film comment, blood film for malaria parasite, LDH, uric acid, clotting profile, HIV, ANA, and Hepatitis B and C. Additionally, an electrocardiogram, echocardiogram, abdominopelvic ultrasound, and chest X‐ray were requested. She was transfused with 1 unit of whole blood and neurological physiotherapy started. On days 2 and 3 of admission, her power improved to 4/5.The complete blood count revealed a low hemoglobin level of 4.3 g/dl (11.5–14.5), platelet count of 18 × 109/L, and platelet count of 22 × 109/L. Clotting profile results showed an INR of 1.16, PT – 9.8 s, APTT – 33.9 s, and PTT – 0.92 s. LDH was 2.6111 μ/L (135–214), Uric acid was 0.4 mmol/L (0.13–0.39), and both direct (19.86 μmol/L) and indirect (34.7 μmol/L) bilirubin were high with low globulin levels (25.7). HIV, Hepatitis B and C, ECG, chest X‐ray, blood film for malaria parasite, AST, ALT, AST, and ANA were all normal. A thyroid function test was normal. The working diagnosis was then modified to recurrent seizures with a differential of a recurrent stroke with hemorrhagic transformation, poststroke seizures, intracranial infections such as meningoencephalitis, electrolyte encephalopathy, and intracranial hemorrhage from idiopathic or acquired thrombocytopenia. A repeat head CT scan ‐cleaving protease,TTP results from either a congenital or acquired decrease/absence of the von Willebrand factor protease ADAMTS13. Low levels of ADAMTS13 result in microthrombi formation which leads to end‐organ ischemia or damage.Measuring ADAMTS‐13 activity levels may not guarantee initial diagnosis and therapeutic decisions, but it is important for the prognosis. An ADAMTS‐13 activity level of <5%–10% seems to have increased specificity for TTP, but it does not identify all patients at risk for relapsing. ADAMTS‐13 activity levels of >10% make the diagnosis of TTP unlikely in patients presenting with acute thrombocytopenia.Therapeutic plasma exchange is the main therapy for patients with TTP.4This case report shows how TTP can present as an ischaemic stroke which is an atypical presentation. It is interesting to note that our patient is the first Ghanaian to undergo plasmapheresis and the only case described in the literature and this makes our reported case a unique one. This may be due to late or missed diagnosis, the unavailability of screening tools, or inadequate funds to patronize plasma exchange. Rapid diagnosis and treatment are necessary for decreasing the risk of fatal outcomes in patients with TTP. This case illustrates the potential of TTP masquerading as ischemic stroke and it is necessary that clinicians are familiar with the clinical presentation and laboratory abnormalities of TTP, to make early diagnosis and initiate appropriate therapy. The diagnosis of stroke with TTP is atypical but a physician should suspect TTP in the setting of a stroke when the patient is a young female , those who come with abnormal uterine bleeding without any identifiable cause, intracranial bleeds, petechiae and purpuric patches on the skin, and two prior episodes of stroke events.Kwabena Oteng Agyapong: Conceptualization; formal analysis; investigation; methodology; project administration; supervision; validation; visualization; writing – original draft; writing – review and editing. Aba Folson: Conceptualization; investigation; methodology; resources; supervision; writing – original draft; writing – review and editing. Roland Wonkyi: Conceptualization; investigation; methodology; supervision; visualization; writing – review and editing. Kelvin Amenyedor: Conceptualization; investigation; methodology; validation; writing – original draft; writing – review, and editing. Jeffrey J. Boateng: Investigation; methodology; writing – review, and editing. Kate Fiador: Investigation; supervision; writing – review, and editing.There has been no significant financial support for this work.The authors declare that they have no conflict of interest.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy. A copy of the written consent is available for review by the Editor‐in‐Chief of this journal.


Taraxacum officinale (L.) were used in traditional medicine in various parts of the world for the treatment of health problems, and they possess significant biological activities. The present study aimed to estimate phytochemical and biological activities of T. officinale using different extraction solvents.  Different parts of in vitro methods, namely, DPPH assay, total reducing power (TRP) assay, and total antioxidant capacity (TAC). Toxicological analysis was done using the brine shrimp cytotoxic assay and radish seed phytotoxic assay.  Methanolic, acetone, and n-hexane extracts of selected species were prepared, and ten secondary metabolites were examined using standard protocols. The antioxidant activity was performed using three T. officinale methanolic extract showed the highest phenolic (178.27 ± 17.17 mg/GAE/g) and flavonoid (18.50 ± 1.64 mg QE/g) contents. Similarly, the methanolic extract also revealed the highest DPPH activity (32.80 ± 9.66 IC50), reducing potential (0.53 ± 0.02 mg/g), and TAC (19.42 ± 0.97 mg/g) as compared to the acetone and n-hexane extracts. The Pearson correlation analysis confirmed a strong positive correlation (r > 0.9) between total phenolic content (TPC), total flavonoid content (TFC), and all antioxidant assays. Furthermore, a heat map displayed the methanolic extract (red color) as a valuable source of phytochemicals and antioxidant agents. Moreover, the T. officinale methanolic extract also showed the highest (7.12 ppm) cytotoxic potential whereas both methanolic and acetone extracts were revealed as moderate phytotoxic agents when compared with the standard.  The T. officinale methanolic extract exhibited comparatively notable phytochemicals that are actively involved in antioxidant activities and possess toxicological properties. This upholds the folkloric use of T. officinale as a possible source to develop natural plant-based drugs. Further investigations to isolate bioactive compounds and elements and on their safety need to be conducted. The  Poonch is located in the northern mountainous region of AJK and is situated 1750–2500 m above the sea level between 33–36 north latitude and 73–75 east longitude [Azad Jammu and Kashmir (AJK) lies in the western part of the Himalayas, having an area of 13,269 kmongitude . The PooTaraxacum genus comprises wild medicinal plants with almost 2500 species reported worldwide [Taraxacum officinale var. erythrospermum; however, few are scientifically investigated nowadays. In the beginning, these species were used owing to the popular knowledge and experience from our ancestors. The first evidence of its therapeutic use is in Arabic medicine during the 10th century to cure diseases of the liver and the spleen. Later on, Fuchs in 1953 described its use to treat diarrhea, blister, gout, and spleen and liver diseases [th century, many researchers have diverted their attention from traditional knowledge toward the scientific explanations related to the mode of action of Taraxacum against diseases. Exploring the chemical composition and the action mechanism of this species against diseases using multiple in vitro and in vivo assays could establish its potential as a commercial herb [orldwide . Some ofdiseases . Since tial herb , 9.Uncontrolled production of reactive oxygen species (ROS) in the body causes oxidative stress which ultimately leads to diseases such as diabetes, aging, and myocardial infarctions . To counMoreover, phytochemicals also play a key role in plant adaptation under various growth conditions . Plant gTaraxacum spp. (family Asteraceae) is commonly known as dandelion and is considered as a weed in several crops [T. officinale is also used to treat malaria in Venezuela, for toothache in Kosova, and for hypertension in Ghana [4-induced hepatotoxicity potential of this plant in rats, and Khan et al. [T. officinale against cattle tick Rhipicephalus microplus infestations. Moreover, Kenny et al. [T. officinale against Staphylococcus aureus and Bacillus cereus.Despite the longstanding vegetation growing in the Poonch valley, only a handful of species have been investigated scientifically. al crops  while ital crops , 22. Danal crops . It is eal crops . In Nortal crops . In tradin Ghana . Glufrazin Ghana  revealedn et al.  determiny et al.  reportedT. officinale, while few studies have been performed to confirm its biological potential. Hence, the present study was designed to explore the chemical composition, antioxidant, cytotoxic, and phytotoxic potential of T. officinale (L.) collected from the Poonch valley, by using multiple extraction solvents, to verify the rationale behind the use of this plant as a cure for various diseases.Altogether, most of the previous studies have been focused on the ethnopharmacological potential of Fresh plant samples were collected from Rawalakot located in the Poonch district of Azad Kashmir. The plant sample was identified by Prof. Dr. Mir Ajab Khan (taxonomist), and a voucher number (QAU-AA-157) was assigned from the Herbarium of Pakistan, Quaid-i-Azam University (Islamabad). The plant material was washed, shade-dried, and powdered using an electric grinder. The plant material (20 gm) was extracted with 200 mL, each of methanol (polar), acetone (slightly polar), and n-hexane (nonpolar) solvents for 48 hours. Subsequently, filtration was carried out using a Whatman filter paper , and the whole process was repeated twice. Subsequently, the obtained filtrate was concentrated using a rotary evaporator  and crude extracts were stored at 4°C for experimental analysis.Qualitative tests were performed to detect the presence of various secondary metabolites using standard protocols, namely, alkaline detection assay for flavonoids, Mayer's test for alkaloids, Salkowski test for glycosides, gelatin test for tannins, foam test for saponins, ferric chloride test for phenols, Libermann's test for terpenoids and steroids, and sodium chloride (NaCl) was added to the extract to observe anthocyanins (bluish color) and coumarins (yellow color) , 29.μL) was added in the Folin–Ciocalteu reagent (90 μL) and then incubated for 5 minutes. Subsequently, 6% sodium carbonate (90 μL) was mixed and absorbance was measured at 630 nm. Gallic acid (standard) was used to obtain the calibration curve [μL) was dissolved in 1 M potassium acetate (10 μL), 10% aluminium chloride (Al2Cl3) (10 μL), and distilled water (160 μL), followed by incubation (30 minutes), and then, absorbance (405 nm) was noted.For TPC, each extract (20 on curve . The plaμL) was added to 0.004% of DPPH solution (190 μL), and the final volume was made up to 200 μL. The reaction mixture was placed in the dark for 30 minutes, and then, absorbance was measured at 517 nm wavelength. Ascorbic acid was used as a standard, and the scavenging activity was calculated by using the following formula:The procedure described by Tepe et al.  was follμL of each plant extract was added in potassium ferricyanide and phosphate buffer (500 μL each) and then incubated at 50°C for 20 minutes. Subsequently, 500 μL of trichloroacetic acid (TCA) was mixed followed by centrifugation  at 3000 rpm for 10 minutes. The supernatant was added in 0.1% ferric chloride (100 μL), and then, absorbance was measured at 630 nm. Ascorbic acid was used as a control, and results were expressed as μg AAE/mg of the extract [About 200  extract .μL) in 4 mM ammonium molybdate (0.25 g), 28 mM sodium phosphate (1.68 g), 1.63 mL of H2SO4, and 50 mL of distilled water, followed by incubation at 95°C for 90 minutes. Then, the samples were cooled at room temperature, and absorbance was measured at 630 nm.For the TAC assay, Prieto's method  was follμg/mL) of each extract were prepared in dimethyl sulfoxide (DMSO) to ascertain the cytotoxicity potential of the selected extracts. Artemia salina eggs  were released in a bipartitioned tray filled with artificial saline water  and placed for incubation at 32°C. The lamp was used as a light source for the hatching of shrimps. After 24 hours, 10 shrimps were added to each vial along with different concentrations of the plant extract (5 μL) and left for incubation for the next 24 hours. Subsequently, the number of alive shrimps was counted with the help of the naked eye, and (%) mortality and lethal concentration 50% (LC50) values were evaluated.For the cytotoxicity assessment, Sirajuddin et al.'s  method wFor the radish seed phytotoxicity assay, the Arzu and Camper method  was foll50 values were calculated using the GraphPad-Prism 5 software. Pearson correlation analysis was performed between phytochemicals and antioxidant assays using Microsoft Excel, whereas a heat map was prepared to observe variability among extracts in terms of antioxidant activities. Furthermore, LC50 values were determined to ascertain the toxicity potential of extracts by using Probit analysis software [All assays were performed in triplicate, and mean ± standard deviation was determined. Least significant difference (LSD) was measured using Statistix 8.1 by performing analysis of variance (ANOVA), and ICsoftware .Preliminary phytochemical tests revealed that secondary metabolites were present more abundantly in their methanolic extracts as compared to the acetone and n-hexane extracts. Saponins and tannins were not detected in n-hexane extracts, while glycosides were absent in acetone extracts. However, all other compounds were present strongly to moderately in most of the selected extracts .Phenolic and flavonoid contents were determined quantitatively, and results showed that methanolic extracts exhibited the highest phenolic (178.273 ± 17.17 mg GAE/g) and flavonoid (18.5 ± 1.64 mg QE/g) contents as compared to the other extracts. Overall, phytochemicals were observed in decreasing order of methanolic extract > acetone extract > n-hexane extract, as shown in T. officinale extracts was assessed using three methods, and among these, the DPPH activity was evaluated at three different concentrations to observe the percentage inhibition and IC50 values. Results revealed that all extracts exhibited a concentration-dependent percentage inhibition of free radicals in the DPPH assay. Among all samples, the highest activity was recorded in the methanolic extract (IC50 : 32.80 ± 9.66 μg/mL), followed by acetone (IC50 : 42.63 ± 5.55 μg/mL) and n-hexane (IC50 : 60.0 ± 8.37 μg/mL) extracts, respectively. The highest antioxidant potential of methanolic and acetone extracts was also found to be statistically significant when compared with that of the standard ascorbic acid  in all three antioxidant assays . However, the n-hexane extract revealed the lowest cytotoxic potential as it showed 14.02 ppm LC50 value , whereas in the case of seed germination, methanolic extract was found to be most potent showing 30% inhibition in the seed germination. In general, all extracts revealed a moderate phytotoxic potential when compared with the standard and presented significant statistical differences as shown in Figures T. officinale collected from the Poonch valley (AJK).For ages, nature has proved and served as a sumptuous repository of medicinal plants owing to the existing bioactive constituents that contribute toward the isolation of natural drugs . Till noT. officinale extracts were prepared using three solvents, namely, methanol (highly polar), acetone (slightly polar), and n-hexane (nonpolar) that were chosen by the difference in their polarity. Then, preliminary tests were conducted to ascertain the presence of ten secondary metabolites, and the results revealed that most of the tested compounds were abundantly present in the methanolic extract as compared to the acetone and n-hexane extracts of the selected plant. The presence of certain compounds in plant extracts can be attributed to the plant's physiological and biosynthetic reactions and the ecological conditions of the study area [T. officinale could be due to the low polarity of the solvent which impeded the extraction [Initially, udy area . Moreovetraction , 43. HenT. officinale extracts which is comparable with our study which showed TPC ranging from 45.675 mg GAE/g to 178.27 mg GAE/g in the selected extracts. However, Kenny et al. [T. officinale which is in disagreement with our study in which the whole plant was examined using different solvents.Among these bioactive compounds, phenolics and flavonoids are the dominant groups of phytochemicals that act as primary antioxidants or radical scavengers . The cony et al.  observedAntioxidant potential of the selected extracts was compared as antioxidant activity aids in scavenging free radicals in a specific reaction medium. DPPH assay can be viewed as a milestone for the determination of the antioxidant activity of test samples . SimilarT. officinale collected from Korea. Similarly, our results are also coherent with the previous findings of Hu and Kitts [T. officinale leaves and flowers collected from Canada and Poland. Moreover, Kenny et al. [T. officinale root extract prepared in ethyl acetate. Thus, it can be proposed that T. officinale is equally effective against oxidative stress-related diseases regardless of geographical location and seasonal variations. These antioxidant assays provide a basis and rationale for using these extracts as antioxidant ingredients in various food and medicinal products. The higher phenolic content in the polar extract is also responsible for the higher antioxidant capacity of the selected plant extracts [Our study correlates with the previous findings of Chon et al.  who docund Kitts  and Miłend Kitts  who repoy et al.  determinextracts . Howeverextracts .r > 0.9) correlation of phytochemicals with all antioxidant assays which is in parallel with the previous studies of Miłek et al. [r value above 0.8 between the antioxidant activities and TPC of T. officinale extracts. Therefore, it can be suggested that T. officinale collected from the Poonch valley also possesses a valuable reservoir of active compounds of pharmacological significance. In addition, differences between the three different kinds of T. officinale extracts were perceived by using a heat map which displayed high (red color) to low (green color) activities vividly  values are used to express their cytotoxic level, whereby low values represent high cytotoxicity. According to Meyer's criteria, plant extracts with LC50 > 1000 μg/mL are nontoxic, whereas plants with LC50 < 1000 μg/mL are toxic [50 > 1000 µg/mL, slightly toxic when LC50 500 to 999 μg/mL, moderately toxic when LC50 99 to 499 μg/mL, and highly toxic when LC50 0 to 100 μg/mL [T. officinale were highly toxic as they had LC50 values less than 100 μg/mL  extract of T. officinale exhibits less antioxidant potential and is moderately toxic. Thus, polar extracts of the selected species can effectively serve as a natural source to formulate antioxidant and toxicological agents; however, these results cannot be applied directly to humans.From this study, we came across a judgement that biological characteristics of plants are greatly influenced by the solvents used for extraction. As the solvent polarity reduces, the extraction process is hindered which eventually alters the chemical reactions inside the plants. The methanolic and acetone extract of T. officinale prepared in polar solvents have higher medicinal value than the extracts prepared in other nonpolar solvents; however, further empirical investigations using in vivo models are needed. Besides this, isolation of pure compounds and their characterization are required to analyze their mode of action against various diseases. All-inclusive, the selected plant exhibits multiple properties and thus could be utilized by humans and animals in their dietary items and in preparing pharmaceutical products.This research explored that the extracts of


TO THE EDITOR:Several predictive scores have been described for patients with myelofibrosis (MF): these include IPSS , DIPPS , MYSEC , MIPSS  and GIPSThe question is the following: can we identify a prognostic score, specifically designed for patients undergoing an allogeneic HSCT. A recent study has identified transplant and molecular characteristics predictive of transplant outcome, and is referred to as the MTSS or molecular and transplant scoring system. These include: age over 57, Karnovsky score < 90%, platelet counts < 150 × 10^9/L, leukocyte count > 25 × 10^9/L, an HLA mismatched donor, ASXL1 mutation, and non-CARL/MP genotype, to be independent predictors of outcome , The AutIn our own series of patients, we asked the question whether the same prognostic value could be achieved with less information as compared to the MTSS. We have previously reported that spleen size, , and transfusion burden (with a cut off of 20 red blood cell transfusions) were predictive of outcome after HSCT . A recenWe therefore analyzed 157 patients with myelofibrosis undergoing an allogeneic HSCT at a median interval of 925 days from diagnosis (116–8865). The clinical characteristics were as follows: 110 and 47 patients were aged < =/>60 years; 71 were prepared with a conditioning regimen including fludarabine and one alkylating agent   , and 86 n = 87) or high risk (n = 70). Finally 60 patients were grafted from HLA identical sibling and 97 from matched unrelated or mismatched related donors. We looked at three outcomes: disease free survival (DFS) the event being death or relapse; transplant related mortality (TRM) the event being death without relapse; and relapse, the event being relapse of myelofibrosis.The multivariate Cox analysis included TS, age >60 years, DIPSS score, conditioning regimen and donor type.Fifty two patients exhibited a maximum spleen size before HSCT of ≤ 22 cm and had received 0–20 red blood cell transfusion pre HSCT (TS = 0), whereas 105 patients had a large spleen (>22 cm) and/or a heavy transfusion burden (>20 transfusions) (TS = 1). Spleen size was recorded as maximum size, also if the patient was splenectomized pre-transplant. Patients were also stratified according to the dynamic international prognostic scoring system (DIPSS) as int1-int2 (p = 0.09) for patients aged < =60/> years, it was 60% vs 36% (p = 0.002) for patients prepared or not with TBF, and 59% vs 34% (p = 0.002) for patients with int1-int2 or high DIPSS. When looking at TS the 5 year DFS was 74% vs 36% (p = 0.0001) for patients with low or high TS .The 5 year DFS was 51% vs 42% (p = 0.02); the conditioning regimen had a strong impact on relapse  and therefore DFS ; TS had a significant impact on all three outcomes: DFS ; TRM ; Relapse . DIPSS predicted DFS, whereas donor type was the least predictive variable , the 5 years DFS was 79% for low TS vs 26% for a high TS (p = 0.007). Therefore, it seems possible to achieve excellent DFS also in older patients, given that they come to transplant without a heavy transfusion burden or a spleen that occupies the whole left abdomen. The problem remains for the older patients, over 60 years, with a high TS, in whom the TRM reaches 50%: one may have to reduce the intensity of the conditioning regimen in such patients.Causes of death in patients with a high TS as compared with patient with a low TS were as follows: infectious deaths were 13% vs 5%, multiorgan toxicity 11% vs 4%, graft failure 4% vs 2%, GvHD 3% vs 4%, other transplant related 5% vs 6%, and relapse 25% vs 8%. When looking at patients over 60 years of age, prepared with two alkylating agents (TBF) (As shown in the multivariate Cox analysis, TS predicts both transplant related death and relapse, possibly because spleen size and transfusion burden are surrogates for advanced disease and/or time from diagnosis, and at the same time, have implications for transplant events: a large spleen can delay hematologic recovery and increase infectious complications and a high transfusion burden generates sensitization to HLA antigens, and may increase the risk of graft failure.The drawback of the study is of course its retrospective nature, but on the other side the number of patients involved is relatively large.In conclusion we confirm that maximum spleen size and pre-HSCT transfusion burden are strong predictors of outcome for patients with myelofibrosis, and predict both transplant related toxicity as well as relapse of the original disease. Patients with a high TS may be eligible for programs of reduced toxicity conditioning regimens.


Primary Sjögren's syndrome (pSS) is a chronic inflammatory condition, which presents with symptoms of dryness, pain, fatigue and often symptoms of anxiety and depression. Health‐related quality of life (HRQoL) is significantly reduced in pSS and the direct and indirect health costs of pSS are substantial. This study aims to determine how symptom burden, disease activity and demographics associate with HRQoL longitudinally over a median of 24‐month follow‐up period in pSS.n = 377) were evaluated using a survival analysis strategy. Kaplan–Meier and Cox proportional hazards analysis were performed using baseline Newcastle Sjogren's Stratification Tool (NSST) subgroup, EULAR Sjogren's Syndrome Patient Reported Index (ESSPRI), EULAR Sjogren's Syndrome Disease Activity Index (ESSDAI), disease duration, age and sex as covariates including polypharmacy and comorbidity score, where data were available (n = 191).Longitudinal EuroQoL‐5 dimension (EQ‐5D)–3L data from the Newcastle pSS cohort (Of the 377 pSS participants analysed in this study, 16% experienced a decline in HRQoL to a health state comparable to or worse than death. NSST subgroup and ESSPRI score had a significant relationship with time to ‘EQ‐5D event’, whereas baseline ESSDAI, age, disease duration and sex did not.In pSS, symptom burden and to a great extent NSST subgroup, rather than systemic disease activity, has a significant relationship with HRQoL longitudinally. Improvements in symptom burden have the potential to produce significant impacts on long‐term HRQoL in pSS. Primary Sjögren's syndrome (pSS) is a chronic inflammatory condition that commonly presents with symptoms of dry eyes, dry mouth, pain and fatigue. Symptoms of anxiety and depression are also common. There is currently no effective treatment for pSS and the direct and indirect health costs of pSS are substantial.http://www.euroqol.org). Scores 0–1 represent death and full health, respectively, whereas scores below zero indicate health states 'worse than death'. EQ‐5D is a generic utility instrument and can be compared across diseases. EQ‐5D is also often used as a key metric in cost–utility analysis in therapeutic development.Health‐related quality of life (HRQoL) estimates are a key contributor to health economic evaluations. Cost–utility analyses for new treatments are assessed in quality‐adjusted life‐years (QALYs), which combine HRQoL and survival . EuroQoLIn pSS, HRQoL is significantly reduced . The mosResearch ethics approval was granted by the UK North‐West Research Ethics Committee.n = 507). All participants fulfil the American European Consensus Group classification criteria [http://www.sjogrensregistry.org) for a proportion of participants (n = 191). The following variables were available at baseline for all subjects: age, disease duration, sex, Ro/La status, ESSDAI, ESSPRI, HADS, EQ‐5D UK utility and Pro‐F.All longitudinal data were collected from a regional Sjogren's clinic based in Newcastle‐upon‐Tyne between August 2009 and December 2020 n = 07. All pcriteria , EULAR Scriteria , EQ‐5D UPolypharmacy score was calculated as the sum of medications at the time of clinic appointment. Details of comorbid conditions were collected prospectively, standardised and converted to clinically meaningful categories using Clinical Classifications Software Refined (CCSR) tools . A ChronLongitudinal health outcome data (EQ‐5D UK utility) were analysed. As the data were collected during routine clinic appointments, the time between visits and total number of visits differed between patients. The EQ‐5D UK utility scores were calculated as previously described  and evalPatients were involved in the planning and conduct of this research and have identified priority areas for pSS research that are most relevant to their experience of the disease, which include fatigue and general well‐being. Patient partner representatives are included in the project steering committee.The distribution of EQ‐5D scores in this pSS cohort is bimodal with a peak around 0 and another around 0.75; there is possibly a third peak of patients who report an EQ‐5D score of 1, perfect health. Of the 377 pSS participants analysed in this study, 69 (18%) experienced a low (<0.5) EQ‐5D utility score and 59 (16%) experienced a decline in quality of life to a health state comparable to or worse than death.p = <0.001) . In the PDF and HSB groups, the proportions of ‘EQ‐5D events’ are 15% (n = 15) and 41% (n = 41), respectively. The median EQ5D utility values at baseline and time of event or end of follow‐up are 0.85 and 0.80, respectively, for the LSB group and 0.74 and 0.69, respectively, for the DDF group; whereas the median EQ5D utility values at baseline and time of event or end of follow‐up are 0.52 and 0.19, respectively, for the HSB group and 0.69 and 0.68, respectively, for the PDF group. See Table NSST subgroup at baseline had a significant relationship with time to threshold EQ‐5D event p = <0.00 Fig. 1)p = <0.00p = 0.002). This effect was consistent after correction for polypharmacy score and CCI. CCI has a close relationship with polypharmacy score and is associated with a small but significant  risk of reduced EQ‐5D. Baseline ESSDAI, age at baseline visit, disease duration and sex were not significant risk factors for an ‘EQ‐5D event’ . NSST strata are calculated using five patient‐reported symptom scores. The impact of each score at baseline : (grant number: 022) and Medical Research Council, UK funding (grant number: G0800629). This study also received infrastructure support from the NIHR Newcastle Biomedical Research Centre and the NIHR Newcastle Clinical Research Facility.Jessica Tarn: conceptualization; formal analysis; writing – original draft; writing – review and editing. Dennis Lendrem: formal analysis; writing – review and editing. Peter McMeekin: formal analysis; writing – review and editing. Clare Lendrem: data analysis. Ben Hargreaves: data curation. Wan‐Fai Ng: funding acquisition; supervision; writing – review and editing. Wan‐Fai Ng: conceptualisation, funding acquisition, data interpretation, writing – review and editing.Figure S1. Histogram of EQ‐5D UK utility distribution showing 3 groupings of patients centered around EQ‐5D UK utility scores of 0, 0.6 and 1. A, First Visit; B, Last Visit; C, Baseline values are shown in teal with end of follow up values overlaid in red.Click here for additional data file.


Carcharias taurus of both sexes and a range of sizes caught in Delaware Bay . Mean hematocrit, total white blood cell counts, lymphocyte differentials, glucose, phosphorus, amylase, and aspartate aminotransferase levels were significantly higher in juveniles than in adults. Levels of estradiol, progesterone, testosterone, and differences in selenium and eicosapentaenoic acid (a polyunsaturated fatty acid) between males and females suggest that they are important parameters for improving Sand Tiger breeding success in managed care. Finally, blood metal levels for arsenic, cadmium, lead, and mercury suggest low levels of contaminant exposure for Sand Tigers during their summer residence in Delaware Bay. The results of this study provide baseline health parameters for wild Sand Tigers that will aid in effective maintenance of aquarium animals and contribute to a greater understanding of the biology of these sharks and efforts to accomplish sustainable management of their populations.Species‐specific hematological reference values are essential for diagnosis and treatment of disease and maintaining overall health of animals. This information is lacking for many species of elasmobranchs maintained in zoos and aquaria, thus reducing the effectiveness of care for these animals. Descriptive statistics and reference intervals were calculated for hematocrit and complete blood cell counts, biochemistry and protein electrophoresis parameters, trace minerals, vitamins, heavy metals, reproductive hormones, and fatty acids in the blood of 153 wild Sand Tigers  Species‐specific hematological parameters are important tools for evaluating and understanding health and nutrition in both wild and managed populations of animals. Diet, environmental features, activity level, social structure, stress, and parasite loads are all factors that may influence blood values in zoo and aquarium animal collections  provide a comprehensive suite of hematological, biochemical, and nutritional values  for wild Sand Tigers in Delaware Bay; (2) compare blood analytes between sexes and among demographic groups of wild individuals; (3) establish RIs for wild Sand Tigers for comparison with aquarium individuals; and (4) infer the overall health of individuals collected in Delaware Bay based on hematological parameters.N = 60) and 2012 (N = 93) in Delaware Bay  over 15 total days of effort using anchored longlines consisting of 366‐m, 0.64‐cm braided nylon, with 25 (16/0) circle hooks (barbs depressed) spaced 12 m apart. Hooks were baited with Atlantic Menhaden Brevoortia tyrannus or Bluefish Pomatomus saltatrix. Longlines were fished during daylight hours using soak times of approximately 2 h. Based on the time required for deployment and retrieval of the longline, however, some individuals could have been on hooks for periods exceeding 2 h. For each longline set, location (determined by GPS), surface water temperature, salinity, and dissolved oxygen were recorded.Sand Tigers were caught during August 2011 (Sharks were either immediately brought on deck (<150 cm TL) or secured adjacent to the boat with a tail rope (>150 cm TL). Animals were then rolled into dorsal or lateral recumbency, inducing a natural and temporary state of inactivity (tonic immobility), for workup. Individuals were measured (FL and TL), sexed, and tagged externally with conventional “spaghetti” identification tags; a subset of sharks received an internal acoustic transmitter for a separate study. Twelve milliliters of blood were collected  via caudal venipuncture using 18–21‐gauge needles of varying length  attached to a syringe via an extension set. For sharks that were processed onboard, the gills were irrigated with salt water via a hose inserted in the mouth and the skin was kept wet during handling. Total handling time for both onboard and in‐water processing was <15 min.g for 10 min), approximately 60–120 min after collection, and plasma samples were stored frozen at −80°C until analyzed.Whole blood was immediately transferred into a 1‐mL sodium citrate blood tube, a 10‐mL lithium heparin blood tube, and an i‐STAT cartridge  for blood gas analysis (see below). Sodium citrate whole blood was added to a cryovial containing a modified Natt–Herrick's stain  at a 1:100 dilution for total white blood cell (WBC) counts to be conducted at a later time. Natt–Herrick's stain was modified for elasmobranch osmolality  by mixing 25 μL of Natt–Herrick's stain with 0.79 g of urea and 0.34 g of sodium chloride . Blood smears were examined using light microscopy , and the differential count was read twice by identifying 100 individual WBCs and calculating the percentage of each cell type. White blood cells were classified as lymphocytes, neutrophils, basophils, monocytes, coarse eosinophilic granulocytes (CEGs), and fine eosinophilic granulocytes (FEGs) using criteria defined by Arnold . Segment2; mm Hg), partial pressure of oxygen , lactate (mmol/L), bicarbonate , total carbon dioxide (mmol/L), and percent oxygen saturation . Since mammalian conversion factors and constants are utilized by the i‐STAT to make temperature corrections to pH, pCO2, and pO2, water temperature was used as a proxy for body temperature so that values could be of diagnostic use in elasmobranchs  and a CG4+ disposable cartridge that measured pH, partial pressure of carbon dioxide (pCO+) + (glucose/18.0) + (BUN/2.8).Plasma biochemical profiles were performed at Michigan State University's (MSU) Diagnostic Center for Population and Animal Health (DCPAH) using an Olympus AU640e chemistry analyzer . This profile included blood urea nitrogen (BUN), creatinine, sodium, potassium, chloride, calcium, phosphorus, magnesium, iron, glucose, amylase  at the University of Miami's Avian and Wildlife Laboratory according to the manufacturer's instructions and as previously described . Similarly, for the quantification of progesterone and testosterone, Siemens Coat‐A‐Count reagents were used, which included calibrators with different levels of hormone, antibody‐coated polypropylene tubes, and 125I‐labeled progesterone or testosterone.Plasma hormone levels  were measured using solid‐phase Trace mineral and heavy metal analysis was based on the methods described by Wahlen et al. . Plasma Plasma vitamin A (retinol) and vitamin E (α‐tocopherol) levels were analyzed by MSU‐DCPAH using the methods described by Arnaud et al. . Fat‐sol3 [niacin], B5 [pantothenic acid], B12 [cyanocobalamin], and C [ascorbic acid]). Plasma vitamin B3 and B5 samples were prepared according to the methods described by Dawson et al. , in which nonspecific vitamin B12 binding R‐proteins were removed by affinity chromatography.Plasma samples were analyzed for water‐soluble vitamins  were explored using nonparametric Wilcoxon rank‐sum tests. Correlations between blood analytes, longline soak times, and shark size were explored using linear regression. For life history comparisons, females larger than 220 cm TL and males larger than 190 cm TL were considered mature  or by parametric/robust methods from native and Box–Cox transformed values (N > 40). The 90% confidence intervals around these limits were determined nonparametrically (N > 120) or through bootstrapping methods (N < 120) using Reference Value Advisor freeware  and ProUCL version 5 .Reference intervals encompass the central 95% of a healthy reference population. In nonnormally distributed (skewed) data, percentiles are used to establish RIs and the 2.5th and 97.5th percentiles are the upper and lower limits for the RIs  or TL  between sexes. Of the 153 animals captured, 103 were later categorized as juveniles and 53 were categorized as adults. Average ± SD surface water temperature, salinity, and dissolved oxygen were 26.2 ± 1.8°C, 25.9 ± 2.1‰, and 5.3 ± 1.6 mg/L, respectively.Blood samples were collected from 153 Sand Tigers ranging in size from 122 to 272 cm TL (101–233 cm FL) over the course of this study. Mean ± SD longline soak time was 2.9 ± 0.8 h, with a maximum soak time noted at 5.3 h for a single longline set. No differences were detected for any parameter based on year, and the data were pooled for subsequent analyses. The sex ratio of captured Sand Tigers was nearly equal , and there was no significant difference in FL . Juvenile sharks had significantly lower sO2 levels (47.7%) than adult animals , but a higher mean percentage of red blood cells was measured in juvenile animals (24%) compared with adult animals  and lymphocytes  and significantly lower nonsegmented FEGs  compared with adult sharks . Plasma phosphorus , glucose , amylase , ALP , and AST  levels were significantly higher in juvenile animals compared to adult sharks . Fraction 4 was significantly lower in juveniles  than in adults  compared to adults .Plasma protein fraction 1 (0.16 g/dL) and fraction 2 (0.13 g/dL) were low compared to other fractions. The largest globulin fraction was fraction 4 , with the exception of Se  and whole‐blood Se  were noted in males compared to females . Levels of Co, Mn, Zn, and As varied with life history stage , Mn , and Zn  levels were significantly higher in juvenile sharks, whereas As levels  were significantly higher in adult sharks. A large portion of the analyzed values for Cd (71%), Pb (67%), Hg (77%), and Tl (71%) were below the level of analyzer detection , and RIs were not determined.Trace mineral levels in Sand Tigers were similar between sexes for all minerals measured ; however, differences between life history stages were noted for vitamin E  compared to adults (1.03 μg/mL).No differences in vitamin levels were detected between male and female Sand Tigers , whereas significantly higher levels of both progesterone  and testosterone  were observed in males compared to females  than adult males (6.6 ng/mL). No other hormonal differences were observed between adult and juvenile sharks . Reference intervals were not determined for plasma hormone levels because those values would vary due to the seasonal reproductive cycle of the species.Plasma hormone levels varied based on maturity stage and sex. Significantly higher levels of estradiol were measured in females (both juveniles and adults) than in males  than females (8.0%). No differences in fatty acids were detected based on maturity .Twenty‐six individual fatty acids were detected in Sand Tiger plasma Table . Polyuns2). Sharks exposed to acute capture stress typically experience full physiological recovery within 12–24 h of release  were higher than counts previously measured in wild Sand Tigers , which can be influenced by physiological capture stress in sharks  than were reported for Sand Tigers captured by SCUBA divers  for assessment of ongoing inflammatory processes like infectious disease, parasitism, trauma, and/or poor nutritional plane . Notably, higher values for hematocrit, total WBC counts, lymphocytes, and glucose were measured in juvenile sharks compared with adults. Juvenile teleosts have been shown to exhibit higher counts of both total leukocytes and lymphocytes than adults, which may reflect an immune system that is not fully developed  than our sample size. The significance of variable plasma biochemistry values in our study is not clear, as variations in diet and age may influence phosphorus levels  between juvenile and adult Sand Tigers in our study. Otway  recordedvels NRC . AmylaseDifferences in blood analytes between sexes may offer insight into physiological processes related to reproduction. Sand Tigers have among the lowest reproductive rates known for sharks  and mature (mean = 777 pg/mL) female Sand Tigers in this study were similar to levels measured in serially sampled aquarium females that were identified as immature (<700 pg/mL) and mature  compared with females (230 ng/mL), although at lower circulating levels than measured in this study. The role of Se in fish reproduction is less well understood and limited to work with teleosts  and whole‐blood (746 ng/mL) levels of Se compared with females . Selenium is an essential trace mineral and an integral component of glutathione peroxidase (1.11.1.9), which, in conjunction with vitamin E, protects cells against oxidative damage and prevents nutritional muscular myopathy. As a result, Se compounds are capable of protecting tissues from the toxicity of heavy metals such as Cd and Hg , stearic acid (18:0), and oleic acid (18:1[n‐9]), which are preferentially oxidized in fish Tocher . DiffereSqualus acanthias  from the North Pacific  sampled off the Georgia coast  before migrating south (Teter et al. This study provides RIs for a comprehensive suite of blood analytes measured in wild male and female Sand Tigers representing a range of age‐classes and size‐classes. This information will improve the evaluation of nutritional status and overall health of Sand Tigers held in aquariums, especially with respect to the common problem of spinal deformity in this population of animals. Values obtained in our study provide a reference for comparison with other populations of wild Sand Tigers, some of which have experienced drastic declines or are extremely vulnerable to fishing pressure (Carlson et al.