There is no clear data on the optimal duration of treatment with anti-interleukin-1 drugs in colchicine-resistant familial Mediterranean fever patients, as well as on the dose interval. This study aimed to assess patients whose canakinumab dose interval was adjusted according to a specific protocol, with the objective of evaluating the effectiveness of implementing this protocol for the patient care.The files of 45 patients whose canakinumab treatment interval was opened with a standard protocol previously determined by the Delphi method were retrospectively reviewed.Canakinumab treatment was initiated once a month for all patients. In the sixth month of canakinumab treatment, a dose interval extension was introduced; however, 7 patients (15.5%) experienced an attack, and consequently, no further interval extension was administered to them. For 29 patients, the dose interval was successfully extended to once every three months, as they remained attack-free for a year after the first interval extension. Nine patients continued receiving the drug every 2 months, as they had not yet completed one year since the first extension. The study found no significant correlation between experiencing an attack during the dose interval extension protocol and the number, duration of attacks, or autoinflammatory diseases activity index score.Extending treatment intervals with canakinumab in colchicine-resistant familial Mediterranean fever shows promise for favorable outcomes.The online version contains supplementary material available at 10.1186/s12969-023-00925-5. MEFV gene, which is responsible for encoding the pyrin protein, is often mutated in patients with FMF. MEFV mutations are known to affect pyrin-mediated regulation of caspase 1 activity in inflammasomes, which can lead to excess production of interleukin-1 (IL-1), a pro-inflammatory cytokine [Familial Mediterranean fever (FMF) is the most common hereditary autoinflammatory syndrome and primarily affects individuals from the Mediterranean basin. The condition manifests as recurrent inflammatory episodes typically marked by fever and serositis . The MEFcytokine .MEFV variants on the RhoA-induced pyrin inflammasome and NLRP3 inflammasomes are still unclear [Colchicine is the recommended treatment and has proven effective in preventing inflammatory attacks as well as the development of amyloidosis, the most important complication associated with this condition –5. Howev unclear . Neverth unclear –12. Howe unclear . The proThe objective of this study was to assess patients whose canakinumab dose interval was adjusted based on the aforementioned protocol.The study included patients diagnosed with FMF before the age of 18 and receiving canakinumab treatment from seven different centers. All patients met at least one of the Tel-Hashomer or Eurofever/Paediatric Rheumatology International Trials Organisation (PRINTO) 2019 diagnostic criteria for FMF , 15.In order to be included, patients are required to comply with the protocol for extending the dose interval described in the Delphi study. In this Delphi study, recommendations were developed for the diagnosis, follow-up, and colchicine treatment of FMF patients, as well as for determining the dose intervals of anti-IL-1 treatment. According to the Delphi study, a consensus was reached on the dose intervals for anti-IL1 treatment in FMF patients resistant to colchicine: (1) For patients without any attacks and no laboratory evidence of subclinical inflammation within the last 6 months following the initiation of biologics, treatment intervals can be extended to twice the original dose intervals. (2) After the first extension of the treatment intervals, for patients without any attacks and no laboratory evidence of subclinical inflammation within the last 1 year, intervals can be further extended to three times the original dose intervals. In order to evaluate a standard protocol, the first inclusion requirement regarding the canakinumab dose regimen for this study is to initiate canakinumab treatment on a monthly basis and then, after six months, extend the dose interval to two months. The second requirement was that the dosing interval was extended to every three months for patients who were one year after the first dose interval extension and had no subclinical signs of inflammation or a new FMF attack. Patients who did not comply with the mentioned schedule, meaning they did not experience an attack or show any subclinical signs of inflammation, but whose dose range was not extended according to the schedule were excluded from the study. According to protocol, if a patient had at least one attack or showed signs of subclinical inflammation while on the current dosing schedule, the dosing range was not reextended and the previous dosing range was reverted. The diagram of the canakinumab dose interval extension protocol examined in the study is shown in Fig. FMF attacks were defined as high fever with clinical findings of serositis/arthritis with high C-reactive protein (CRP) level. Subclinical inflammation was defined as elevation of acute phase reactants  between attacks , 13, 16.t-test and Mann-Whitney U test were used to compare numerical variables. A value of p < 0.05 was considered statistically significant.Data was collected using Microsoft Excel  and SPSS 17.0  for the analysis. Descriptive statistics including mean, mode, median, minimum and maximum were conducted according to the distribution of the variables. Categorical variables were compared with the Pearson chi-square test or Fisher’s exact test where appropriate. The Student The study was carried out complied with the Declaration of Helsinki. Approval was obtained for the study protocol from the Ethics Committee of Istanbul University, Istanbul Faculty of Medicine (approved: 21/05/2020/19).A total of 45 patients were included in the study. The patient population consisted of 55.6% females and 44.4% males. The median age of the patients was 15.2 (range 3.5–21) years and the median age of diagnosis was 3.66 (range 0.9–17.5) years. The median follow-up period for patients was 90 (24–223) months, and the median follow-up period after initiating canakinumab was 25 (9–56) months. All patients were using colchicine in addition to canakinumab throughout the protocol. The reason for starting biologic therapy was colchicine resistance in all patients. The percentage of patients who received other anti-IL-1 treatments (anakinra) before starting canakinumab was determined to be 51.1% with a median duration of 3 (1–23) months. In all patients, canakinumab treatment was initiated as once a month with a dose of 2–4 mg/kg (maximum 150 mg).MEFV gene. None of the patients had amyloidosis or nephrotic proteinuria. Demographic and baseline clinical and laboratory characteristics of the patients who received canakinumab treatment are detailed in Table All patients had at least one exon 10 mutation in the After six months of receiving canakinumab treatment, 45 patients who showed no evidence of subclinical inflammation and had not experienced any attacks had their dose intervals extended to every two months. Out of the 45 patients, 7 15.5%) experienced an attack while receiving canakinumab every two months. Consequently, the dose interval was reverted back to once a month and could not be extended again. Out of the remaining 38 patients, 9 were still taking the drug every 2 months because they had not yet completed one year since switching from the previous dosing schedule. The dose interval for 29 patients was extended to once every three months as they had not experienced any attacks for a year, and there were no signs of subclinical inflammation observed during this period. Out of the 29 patients who received canakinumab every three months, one patient experienced an attack on this dosing schedule, therefore the patient’s treatment was switched back to once every two months. The remaining 28 patients (96.6%) continued on the every-three-months schedule, with a median follow-up time of 8 months (range 3–38 months). None of these patients have had any further attacks, and there are no indications of subclinical inflammation. When patients who had prior received anakinra treatment and those who were biologically naive were assessed separately, out of the 23 patients who had used anakinra before transitioning to canakinumab, 16 completed the protocol period, and 11 of them had their dose intervals extended. In the evaluation of 22 biologically naive patients, 18 out of 20 who completed the protocol period were eligible for second dose interval extension. No significant difference was observed between the two groups in terms of achieving a second dose interval extension (Table 5.5% expeThere was no correlation between failure to extend the dose interval to three months due to an attack and age, age at diagnosis, number of attacks, duration of attacks, AIDAI score, laboratory values, prior use of other anti-IL-1 agents before canakinumab, duration of use of colchicine or other anti IL-1 agents, and clinical findings during an attack Table .In this study, patients with familial Mediterranean fever who were treated with canakinumab and whose dose intervals were adjusted according to a standard protocol were evaluated. The study focused only on patients who followed the protocol, demonstrating that it is possible to successfully integrate this protocol with canakinumab therapy. Our study represents the largest number of patient evaluation of canakinumab dose interval extension using a standardized protocol. It is worth noting that the protocol used in this study was developed using the Delphi method, a consensus-building approach that involved pediatric rheumatologists from multiple centers, and this protocol has been implemented in their clinical practice , 18.The use of anti-interleukin-1 therapies in patients with colchicine-resistant FMF is a relatively new concept that has gained significant attention in recent years. Currently, guidelines recommend the use of anti-IL-1 therapy for patients who do not respond adequately to colchicine , 17. RanIn the randomized controlled CLUSTER trial evaluating the efficacy and safety of canakinumab, patients who achieved remission at week 16 had their dose interval extended to once every 8 weeks. The study found that an extended dosing interval of canakinumab every 8 weeks was effective in maintaining disease control in 46% of patients with colchicine-resistant FMF . In the Our study demonstrated the efficacy of the canakinumab dose interval extension protocol in a large series of pediatric patients. The dose interval was effectively extended to once every 3 months in 28 patients. The fact that success of dose interval extension was not associated with disease severity, attack frequency before treatment, acute phase marker levels, or disease duration indicates that this dose interval extension protocol can be considered in patients regardless of these factors. In fact, our study showed that even patients who had 24 attacks in 6 months prior to biologic treatment were able to successfully extend the interval to 1 in 3 months (Supplementary file While our study offers valuable insights into the efficacy of the dose interval extension protocol, there are some limitations to our findings. Specifically, our study did not provide information on the discontinuation of the drug after the dosing interval was increased, and we lacked data on the follow-up period after the drug was stopped. Additionally, due to the retrospective design of our study, not all patients completed the full treatment schedule, as some had not yet reached the second dose interval extension point. Therefore, we are planning to provide long-term results of our protocol to further investigate the efficacy of the protocol. Another limitation of our study is the small number of patients who were unable to undergo a second dose interval extension, which may have limited our ability to thoroughly investigate the factors that influence the success of a second extension. For a more comprehensive understanding of the long-term effects of the protocol, it is essential to conduct larger and global multicenter clinical studies with extended follow-up periods.This study suggests the feasibility of the protocol designed to extend the dosing interval of canakinumab. It demonstrated that patients who tolerated the first dose interval extension also exhibit good tolerance to subsequent dose interval extensions. Nonetheless, further studies incorporating additional data are warranted to ascertain the optimal dosing intervals and cessation strategies for this patient population.Below is the link to the electronic supplementary material.Supplementary Material 1: The status of all patients in the extended dose interval protocol.


Background and aimChronic patients with thalassemia major were mainly recognized as more prone to poor mental health during this global pandemic. This study aims to evaluate causal relationships leading to poor outcomes and how they manage to tackle this.MethodsIn-depth face-to-face semi-structured interviews were conducted with 21 thalassemia patients selected through probability consecutive sampling from Pakistan Thalassemia Welfare Society Centers. The following criteria served as the basis for the interview: (1) solitude at home, (2) interruption of transfusion services, (3) alteration of appetite and interests, (4) lack of control and uncertainty, (5) extensive media coverage, (6) deterioration of pre-existing health issues, difficulty in sleeping, (7) practicing gratitude, (8) participation in activities and hobbies, (9) connectivity with others, and (10) ability to recognize social support. All interviews were recorded, transcribed, and analyzed with reflexive thematic analysis.ResultsThe commonly described mental health conditions were depression and concern about the overall health status. The following seven elements associated with the pandemic contributed to the deterioration of mental health: (1) isolation at home, (2) disruption in transfusion services, (3) change in appetite and interests, (4) lack of control and uncertainty, (5) intensive media reporting, (6) worsening of pre-existing health problems, and (7) difficulty in sleeping. The following four coping strategies were identified for maintaining mental issues: (1) practicing gratitude, (2) involvement in activities and hobbies, (3) connectivity with others, and (4) discerning social support.ConclusionsThalassemia major patients had been negatively affected during the pandemic. Only a small number of people modified their management techniques to maintain steady well-being. Thalassemia is a chronic and inherited disease in which there is a reduced tendency of the body to produce adequate amounts of hemoglobin. This disease has a high occurrence in South East Asia, South Asia, Middle East, and Mediterranean countries . WHO staIn December 2019, in the city of Wuhan, China, severe acute respiratory syndrome (SARS) erupted, which is known as the coronavirus disease 19 (COVID-19). This pandemic affected the entire world and badly affected the daily life of every individual . To contThere is a great association between mental health disorders and thalassemia patients, and COVID-19 pandemic has added fuel to the fire. To be healthy and to cope with the burden of disease, mental health is very crucial. COVID-19 has affected both the physical and mental health of people. Quarantine and lockdown caused fear among masses and affected the thalassemia patients as well. These patients are more prone to be affected with mental health disorders and are subject to special care and attention. The purpose of this research is to deeply analyze the condition of thalassemia patients in the COVID-19 era and its association with mental stability and well-being of the patients, along with the strategies adopted by the patients to cope with the ongoing situation. This area is still needed to be explored as little work is done in this domain. With the help of this research, we can find out the problems of thalassemia patients and identify the strategies to overcome those problems related to mental health and well-being of patients .A study conducted by Alsaad in 2020 showed that there is an upraised ratio of psychiatric disorders, specifically depression and anxiety, in thalassemia patients. Among all, depression is the most common psychological mental disorder . DepressA qualitative descriptive study was conducted using a convenient sample of 21 volunteer subjects  with thalassemia major, with a mean age of 14.1 years (SD = 6.5 years). The initial data of the patients were taken from Pakistan Thalassemia Welfare Society Centers. In-depth face-to-face semi-structured interviews were conducted to illustrate the impact of living with thalassemia major during the era of COVID-19 crisis.Ethical approval of the study was taken from the School of Public Health, Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan (Reference No. ERC-63/AST-21). The questionnaire designed was simple, clear, and convenient enough to assess psychiatric health and well-being of thalassemia victims and to identify the coping strategies to overcome the effects. Table The designed questionnaire was developed for this study using existing theories on health and social networks , behavioA total of 21 people with thalassemia major took part in the study, the majority of individuals were males n=13, 61.9%), and the mean age of volunteers was 14.1 years (SD = 6.5 years). Summary of themes and subthemes present in Table , 61.9%, Themes relating to the deterioration of mental healthTheme 1: Isolation at HomeOut of 21 participants, 18 (96%) described isolation at home as one of the most common deteriorating factors of mental health. Most of the participants stated that staying at home for long hours is disrupting their routine as well as their interpersonal relationships in their lives. This theme includes acceptance of patients of the situation, the number of days they were isolated at home, and the impact COVID-19 and isolation had on them. All the patients described the pandemic as a stressful situation as it had the potential to take a toll on their physical as well as mental health.“I thought the situation would get better with time but it didn't. lack of normal human interaction as usual like meeting my friends made me feel lonely and tired throughout the day, I am still trying to adapt to the pandemic and accept the situation.” (Patient 2)“I have been isolated for about two weeks now. I worry a lot about the well-being of those who live far away from me. Not being able to see my family who has been affected by COVID-19 had a lot of impact on me.” (Patient 7) “Being an introvert I don't feel the effects of isolation very early on but at a times I was really worried if I would ever get any help if I fell sick.” (Patient 13)Theme 2: Disruption in Transfusion ServicesInsufficient storage of the blood,unavailability of blood donation services, and unavailability of public transportation resulted in disruption in transfusion services, leading to deterioration of mental health in around 73% of the patients.“I used to go for a blood transfusion once every two weeks, but due to lack of proper public transportation, someone else had to drive me to the hospital. So, I missed the transfusion sessions.” (Patient 9)“We needed permission letters to travel to the hospital during complete lockdown; this lack of extra paperwork had led to few problems during travelling to the hospital and I worried a lot about my health.” (Patient 18)Theme 3: Change in Appetite and InterestsDisruption in lifestyle and a decrease in quality of life have contributed to the deterioration of mental health in 58% of the patients. “I used to have at least 3 meals a day before pandemic, but due to lack of physical activity and minimal interaction with people, my appetite has gone down and I also started losing weight” (Patient 15)“My appetite has gone down drastically. I eat around one to two meals a day and sometimes even in the middle of the night, which has caused me a lot of health problems.” (Patient 16)“I used to go for a walk every evening before the pandemic but now I stay at home and try to watch television or listen to some music.” (Patient 20)“I have lost interest in my hobbies like reading books. I don't get to visit my grandchildren and children like before, which has led to loss of interest in my daily interests.” (Patient 17)Theme 4: Lack of Control and AnxietyAround 89% of the participants reported that the pandemic has led to disruption in various areas of life causing them a feeling of lack of control over situations, lifestyle, and deterioration of mental health and well-being.“Waking up in the morning was very hard during the pandemic; the number of deaths due to COVID-19 had added to my anxiety. I haven't felt anxious at this frequency before.” (Patient 12)“It was months since I saw my close family and friends. This made me lonely and sad a lot of times during the pandemic.” (Patient 18)Theme 5: Intensive Media ReportingThis contributed to the highest (96%) level of impact on patients’ mental health. Patients reported having experienced feelings of anxiety and depression due to intense and extensive media coverage during the pandemic.“I was a nurse by profession, and the misinformation that was spread through media made me worry a lot about mental health effects of COVID-19 on other people who aren't a part of healthcare.” (Patient 16) “The continuous reporting on television and seeing the lack of facilities in hospitals for patients with COVID-19 and the high rate of deaths had made me anxious a lot of times.” (Patient 20)Overall health: All the factors mentioned above indirectly contributed to the disruption of the regular lifestyle of the patient, thereby having an overall impact on the patient's health which was reported by 83% of the patients.“I don't feel great about my health due to disruptions in my daily lifestyle. I would rate my health as 7/10. Due to the pandemic, I don't exercise anymore, which has led to negative effects on my health.” (Patient 8)“I don't feel healthy and active like before. It has been difficult to carry out my daily activities. I would rate my health 4/10.” (Patient 11)Theme 6: Difficulty in SleepingAbout (69%) of the patients reported disturbances in the sleep cycle, as well as difficulty in falling asleep.“I sleep for about five hours now. I used to sleep for about eight hours before the pandemic. I have great difficulty in falling asleep on most of the days.” (Patient 15)“I wake up multiple times during the night. I have a broken sleep cycle now, which is also leading me to have low energy levels during the day.” (Patient 12)Themes relating to coping strategiesThe themes relating to coping strategies to maintain mental health were as follows: (1) practicing gratitude, (2) involvement in hobbies and activities, (3) staying connected with others, and (4) discerning social support. Around 87% of them were engaged in practicing hobbies and other activities to cope with the lasting effect of a pandemic.Theme 1: Practicing GratitudeAround 69% of participants reported practicing gratitude as a method of dealing with COVID-19.“By starting a gratitude journal and filling it daily made me feel less anxious about the pandemic and sleep well at night.” (Patient 3)“I expressed gratitude through daily prayers; this kept my mental health stable during these times.” (Patient 4)Theme 2: Involvement in Hobbies and Other ActivityMore than 87% of the participants used this as a coping mechanism.“By starting daily yoga, I used to keep my body and mental health fresh and it helped me feel less anxious.” (Patient 5)“I used to try new recipes everyday; this helped keep me busy and my mind occupied.” (Patient 2)Theme 3: Stay Connected With OthersAround 55% of people used staying connected with others as a coping mechanism.“I used to video call my parents every day and talk to them about what I did throughout the day and other work-related stuff.” (Patient 17)“I and my friends had a group chat on Instagram where we would share funny posts daily and talk to each other.” (Patient 12)Theme 4: Discerning Social SupportAround 48% looked for social support as a means of coping mechanism.“During COVID-19, I joined many groups on Facebook where people shared their COVID-19 experiences and it felt good to know about others going through similar experiences and that you were not alone.” (Patient 15)“I found support mainly from my family who would always give me words of encouragement whenever I felt down.” (Patient 9)This is the first time, to our knowledge, that any study was conducted in Pakistan on thalassemia major patients to assess their stability physically and mentally. This study highlighted a range of themes that were specific to the mental health and well-being along with coping strategies of people living with thalassemia major .Themes included anxiety and depression created by intensive media coverage of COVID-19. According to a study, the anxiety in the general population was noted to be 6.33-35.1%, with patients focusing on family members getting infected with COVID-19, while in patients with thalassemia, the levels of anxiety were noted to be higher . The impAccording to our study, the factors that exacerbated the factors related to the deterioration of mental health are home isolation, disruption in transfusion services, change in lifestyle, quality of life, feelings of anxiety, and overall health and sleep patterns. Hossain in 2020 in Bangladesh found that there is a disruption in blood supply because recruiting volunteers for blood donations is a big challenge in this pandemic. Moreover, samples of blood become unserviceable owing to insufficient storage, underutilization, and the short shelf-life of blood. Our results are compliant with his research in that there are fewer blood donations, thus causing difficulties for thalassemia patients and disruption in transfusion services .Our findings are also similar to the study conducted by Maheri et al. in 2016, who explored the association of health-promoting healthy lifestyle among adults with beta-thalassemia major and found that both are not at an acceptable level among thalassemia patients, thus lifestyle change, uncertainty, and lack of control  are not up to the mark, thus increasing deterioration of mental health and well-being in patients .In 2019, Gunez conducted a case-control study regarding disturbed sleep patterns in children and adolescents with beta-thalassemia major and concluded that sleep problems are more prevalent in children and adolescents and can lead to behavioral disorders, sleep disturbance, and anxiety . The resAccording to the research by Hashemi et al. in 2015, a randomized control trial including 87 adolescents with thalassemia major concluded that teaching coping strategies to patients and self-care programs are advantageous as they can manage their anxiety, stress, and depression in a more effective manner. Problem-focused and emotion-focused strategies were intervened so that patients with thalassemia can adopt different ways to cope with the stress [In new research from Ahmadi in 2020, a qualitative study among 18 patients, in-depth interviews were conducted with thalassemia major patients. They explored coping strategies in terms of spiritual coping, psychosocial coping, and knowledge acquisition. Spiritual coping includes prayers, hope, and acceptance of providence. This shows that practicing gratitude is also compliant with this. Psychosocial coping was another aspect in which patients tried to cope by engaging themselves in things they love to do such as sports, listening to music, thinking positively, and comparing themselves with those who had more severe problems. They also found that support from family and society played an important part in coping with stress. This also supports the fact that engaging in hobbies, discerning social support, and being connected with others help in reducing stress and anxiety among thalassemia patients .Limitations of the studyIn this qualitative study, the cause-and-effect relationship cannot be established. The results cannot be generalized to all thalassemia patients as the focus group consisted of only 21 patients. Convenient sampling was done due to COVID-19, being restricted to the Pakistan Thalassemia Welfare Society Centers. A cross-national study including various institutes would yield more reliable results. There is a probability of researcher bias in these types of qualitative studies. The difficulty in identifying patterns and trends is another drawback of qualitative studies. It is always recommended to do follow-up interviews and a full-scope investigation employing statistical measures and large samples. Previous studies conducted on the neurological impact of COVID-19 on patients with mental illness can also shed light on the population of our study, and future studies can be conducted focusing on patients with thalassemia . EffectsData suggest that, in accordance with previous beliefs, living with thalassemia major during the COVID-19 pandemic has had detrimental effects on the health of individuals, both physically and psychologically. Depression was a common symptom, and engaging in various daily activities was the most common coping strategy employed by many patients. This research highlights the correlation between physical and mental health by age and emphasizes the importance of stress management and help-seeking. Additionally, thalassemia major patients have extended periods of isolation as a precautionary measure. This is essential in order to ensure that they are sufficiently prepared. We recommend regular screening of all thalassemia major patients. Such screening can detect areas where intervention is required in order to preserve the stability of patients during this global pandemic.


Near-infrared spectroscopy (NIRS)-based regional cerebral oxygen saturation (rSO2) can non-invasively examine cerebral oxygen content and has the potential for high spatial resolution. Past studies examining the relationship between PbtO2 and NIRS-based parameters have had conflicting results with varying degrees of correlation. Understanding this relationship will help guide multimodal monitoring practices and impact patient care. The aim of this study is to examine the relationship between PbtO2 and rSO2 in a cohort of TBI patients by leveraging contemporary statistical methods. A multi-institutional retrospective cohort study of prospectively collected data was performed. Moderate-to-severe adult TBI patients were included with concurrent rSO2 and PbtO2 monitoring during their stay in the intensive care unit (ICU). The high-resolution data were analyzed utilizing time series techniques to examine signal stationarity as well as the cross-correlation relationship between the change in PbtO2 and the change in rSO2 signals. Finally, modeling of the change in PbtO2 by the change in rSO2 was attempted utilizing linear methods that account for the autocorrelative nature of the data signals. A total of 20 subjects were included in the study. Cross-correlative analysis found that changes in PbtO2 were most significantly correlated with changes in rSO2 one minute earlier. Through mixed-effects and time series modeling of parameters, changes in rSO2 were found to often have a statistically significant linear relationship with changes in PbtO2 that occurred a minute later. However, changes in rSO2 were inadequate to predict changes in PbtO2. In this study, changes in PbtO2 were found to correlate most with changes in rSO2 approximately one minute earlier. While changes in rSO2 were found to contain information about future changes in PbtO2, they were not found to adequately model them. This strengthens the body of literature indicating that NIRS-based rSO2 is not an adequate substitute for PbtO2 in the management of TBI.Brain tissue oxygen tension (PbtO Traumatic brain injury (TBI) is the preeminent form of neurotrauma globally and is a leading cause of death and disability worldwide ,2. Conte2), with recent guidelines incorporating its use into treatment algorithms [2 monitoring to improve outcomes following TBI [2 measures diffusible oxygen content in the extracellular space through a Clarke-type electrode. As a result, it is speculated that it has a slower response to physiologic variations than other monitoring modalities. This also necessitates its invasive placement into viable brain tissue and results in its ability to only sample a small volume of brain tissue [The most widely adopted of these a”xili’ry modalities is brain tissue oxygen tension  as a cerebral monitoring modality, in the setting of TBI, has grown for the past three decades . The non studies ,17,18,19 studies ,21,22.2 and rSO2 data, the likes of which have not previously been reported in the literature. The primary objective of this study is to utilize contemporary time series analysis techniques to better characterize the relationship between PbtO2 and NIRS-based regional cerebral oxygen saturation (rSO2). The secondary objective of this study is to determine if PbtO2 can be adequately modeled by rSO2 in the setting of moderate-to-severe TBI.One possible etiology of this discrepancy is a failure to leverage time series analysis techniques that allow for the utilization of high-resolution data streams while accounting for the autocorrelative and hierarchical nature of this type of physiologic data. Without accounting for hierarchical and autocorrelation structures in the data, the assumptions of the regression methods utilized in these studies, mainly the independence of samples, is not entirely valid. It can be hypothesized that this may have resulted in an erroneously strong correlation between these parameters. Presented here is a study utilizing the Canadian High-Resolution Traumatic Brain Injury (CAHR-TBI) Research Collaborative database, which is a multi-institutional database of moderate-to-severe TBI patients. A highly unique feature of this dataset is its concurrently measured high-resolution PbtOUsing data from the Canadian High Resolution TBI (CAHR-TBI) Research Collaborative, a retrospective multicenter cohort study utilizing a prospectively collected database of critically ill TBI patients was performed, similar to recently published work from this group. Retrospective analysis was determined to be appropriate based on availability of data. Additionally, given the exploratory and observational nature of the study, an entirely prospective study would not be particularly beneficial at this time. Patients included in this study were admitted to one of the following university-affiliated hospitals: Vancouver General Hospital (University of British Columbia), Foothills Medical Centre , and Health Sciences Centre Winnipeg (University of Manitoba). Institutions collaborating in this research have committed to prospective collection of high-resolution physiologic data in TBI patients. Research ethics approval at the University of Manitoba has been obtained for this database (H2017:181 and H2017:188). Additionally, ethics approval was obtained for retrospective access to the database, as well as for anonymous data transfer between centers .2 was managed based on local practice norms which varied from aggressive management to purely observational. PbtO2 monitors were placed into viable brain tissues based on CT scans or under direct inspection in the operating room. NIRS-based rSO2 was not actively used to guide management at any of the institutions. While granular patient-specific data are not available, similar vasopressor, sedative, and hyperosmolar/hypertonic agents were utilized at all participating institutions. Patient data were entered into the database from 2011 to 2022.The CAHR-TBI database is composed of moderate-to-severe TBI patients  treated at an adult intensive care unit (ICU). All patients in this database had invasive ICP and ABP monitoring and were cared for using management strategies based on Brain Trauma Foundation (BTF) guidelines [2 monitoring and NIRS-based rSO2 monitoring. Those without PbtO2 or NIRS monitoring were excluded. Age, biological sex, admission Glasgow Coma Score (GCS), admission pupil exam, and follow-up Glasgow Outcome Score (GOS) were extracted. Sample size calculations were not possible and therefore not performed due to the exploratory nature of this study.Included in this study were all patients in the CAHR-TBI database that had concurrent invasive PbtO2, as well as left and right rSO2. ABP was measured utilizing radial arterial lines. ICP was monitored using intra-parenchymal strain gauge probes  placed in the frontal lobe or using external ventricular drains . PbtO2 was measured using intra-parenchymal brain tissue oxygenation probes  placed in viable frontal lobe tissue. rSO2 was measured using NIRS regional cerebral oximetry of both the left and right frontal lobes (Covidien INVOS 5100C or 7100) when possible.High-resolution physiologic data-streams included ICP, ABP, and PbtO2 and rSO2) using analogue-to-digital signal converters  when required. This digitized data were linked and stored in time series using Intensive Care Monitoring (ICM+) software . For the purposes of this study ICP and ABP were included for the sake of cohort characterization and were not utilized in subsequent data analysis.Data streams were recorded in digital high-frequency time series  files for data analysis. The minute-by-minute data were utilized throughout the analysis as it is generally considered the standard for cerebral multimodal monitoring signal analytics as it provides a good balance between data size/computational time and temporal resolution ,24. The astsa, blandr, forecast, lmtest, nlme, tidyverse, tseries, and zoo. The Intel oneAPI Math Kernel Library  was utilized for the Basic Linear Algebra Subprograms (BLAS) and the Linear Algebra Package (LAPACK) to improve computational performance. Data streams were further filtered to exclude PbtO2 values less than 0 mmHg and greater than 60 mmHg, as these values were felt likely to be erroneous based on clinical expertise. rSO2 values less than 25% were excluded, as this is the lower limit of output for the INVOS devices used. This, along with the previously mentioned manual artifact clearing, resulted in discontinuities in the data streams. As subsequent time series analysis required continuous data streams, discontinuities were filled through basic linear interpolation through the approx function. For all models, alpha was set to 0.05 without correction for multiple comparisons. Additionally, no sample size or power calculations were performed. This was due to the exploratory nature of this study, which leveraged available data sets.The data analysis was performed using R statistical software  with the following packages: 2 and rSO2 was characterized through time series analysis and inferential statistical modeling. Linear regression assumes independent sampling. In the setting of frequent resampling from individual subjects, as is the case in all high-frequency cerebral physiologic monitoring, this assumption is invalid in two regards. Resampling from the same subject in a cohort leads to a hierarchical structure, as samples from the same subject are likely to be more similar than those taken between subjects. This is because intrasubject sampling has random unaccounted-for effects held constant that are not constant in intersubject sampling. Beyond this, when samples are taken with a high frequency, there is a tendency for samples to be correlated with previously taken samples from the same subject. This is known as autocorrelation. The simplest means by which to reinstitute the validity of these assumptions of linear regression is to average the data over large epochs of time  for each subject and then regress over these averages. In the dynamic setting of the critically ill TBI patient, this can result in a significant loss of information.In this study, the relationship between PbtO2. The details of this analysis are described further in the subsequent sections. However, an in-depth review of the theoretical background of these methodologies is beyond the scope of this paper. We refer the interested reader to previous works on this subject [Fortunately, there are statistical methods to account for these deviations from the assumption of independent sampling. In this study, two such methodologies were utilized. First, hierarchical linear modeling, also known as linear mixed-effects modeling, can help account for the random effects experienced by each subject. Additionally, time series-based autoregressive integrative moving average (ARIMA) modeling can account for the autocorrelative structure of the modeled data stream, in this case PbtO subject ,27,28,29 subject .2, were stationary. This is necessary, as there is an assumption of signal stationarity in the utilized time series modeling techniques. If signals are not stationary, they must be transformed to be made stationary prior to any modeling. Testing of signal stationarity was accomplished through examination of the autocorrelative function (ACF) plots for each patient’s PbtO2 data. For all ACF plots, significance levels were set to a correlation level of +/−(2/N1/2), where N is the number of samples. Generally, for each patient, there was no rapid drop off to zero in lag significance, indicative of non-stationarity of the series. This was confirmed with the Augmented Dicky–Fuller (ADF) and Kwiatkowski–Phillips–Schmidt–Shin (KPSS) testing for stationarity.Prior to performing time series modeling and analysis, it was necessary to determine if the response data streams of interest, PbtO2 (ΔPbtO2) and the change in rSO2 (ΔrSO2). While this is not the same as PbtO2 and rSO2, it was felt that the data obtained from examining how ΔPbtO2 relates to ΔrSO2 would provide insight into the relationship between these physiologic parameters.To make the series stationary, the first difference was taken for each data series. Following first differencing, the stationarity of the data streams was confirmed through inspection of the ACF plots which now showed rapid decay in the significance of subsequent lags. Additionally, ADF and KPSS testing both confirmed stationarity of the first differenced signals. Physiologically, the data streams can now be thought of as the change in PbtO2 samples the extracellular fluid of brain parenchyma and NIRS-based rSO2 reflects changes in the brain microvasculature, it is conceivable that changes in these parameters would be asynchronous [2 and ΔrSO2, utilizing the minute-by-minute data over the entire cohort. The cross-correlation function (CCF) of the minute-by-minute data shows the largest correlative magnitude at a lag of 1 (ΔrSO2Lag1). This can be interpreted as ΔrSO2 being most strongly correlated with ΔPbtO2 a minute later. The CCF plot of the 10-seconds-by-10-seconds data reinforces this conclusion, as the most significant lag is seen at lag 6, corresponding to a one-minute delay between ΔrSO2 and ΔPbtO2. Since ΔrSO2Lag1 was found to contain the most information about ΔPbtO2, it was used in subsequent linear modeling.Given that PbtOchronous ,32. To i2 and ΔPbtO2, impulse–response function (IRF) plots were created based on a multivariate vector autoregressive (VAR) model. These plots examine the modeled response of ΔrSO2 and ΔPbtO2 to a sudden impulse of ΔABP. The high-frequency 10-seconds-by-10-seconds data streams of interest  were utilized as the cerebral vasoactive response was being examined in this analysis and acts on a frequency scale of approximately 0.1 Hz [To further provide insights into the relationship between ΔrSOy 0.1 Hz ,33. GiveVAR function in R. Finally, using the irf function in R, this VAR model was utilized to model and plot the response in ΔPbtO2 as well as ΔrSO2 of an orthogonal impulse in ΔABP over the subsequent 10 lags.To determine the appropriate autoregressive order of the VAR model, the Akaike Information Criterion (AIC) was determined for vector autoregressive models of order 1 to 15. There was diminishing marginal improvement in AIC as model order increased past 6. As such, following the principle of parsimony, a VAR model of order 6 was constructed utilizing the lme function, was performed with ΔPbtO2 as the dependent variable and ΔrSO2Lag1 as the independent variable. Next, to evaluate whether there was any autocorrelation of the model’s residuals, ACF and partial autocorrelative function (PACF) plots were made of these residuals. As with the ACF plots, for all PACF plots, significance levels were set to a correlation level of +/−(2/N1/2), where N is the number of samples. There was an autocorrelative structure to the residuals, indicating that there was unaccounted for autocorrelation in the response variable, ΔPbtO2.Given the hierarchical nature of this dataset, a linear mixed-effects model with random slope and intercept, utilizing the 2 were constructed, as has been done in previous cerebral physiology studies [2 for each individual subject and make inferences about the relationship between PbtO2 and rSO2 based on the general findings of these models.Population-level linear mixed-effects models that incorporated the autocorrelative structure of ΔPbtO studies . However2 as the dependent variable and ΔrSO2Lag1 as the independent variable. In order to account for the autocorrelative nature of ΔPbtO2, the ARIMA structure of the residuals needed to be determined. This was done through the auto.arima function for the residuals of the linear model in each subject. Next, the autoregressive and moving average orders were utilized in the arima and sarima functions, with ΔPbtO2 as the response variable and ΔrSO2Lag1 as the external regressor, to produce a linear model that accounted for the autocorrelative structure of ΔPbtO2. For each subject, the significance the ΔrSO2Lag1 coefficient was determined. Additionally, for each model, ACF and PACF plots of the residuals were examined to determine if there was any remaining autocorrelative structure.For each subject, an initial simple linear regression was performed with ΔPbtO2 and actual values of ΔPbtO2 for each subject, a Pearson correlation coefficient was obtained. Next, a Bland–Altman plot was produced to evaluate agreement between the predicted values of ΔPbtO2 and actual values of ΔPbtO2.To examine the correlation between the predicted values of ΔPbtO2 and rSO2 measurements without interpolation. The full demographic data of the cohort can be found in A total of 20 subjects were included in the study, with a total of 114,136 min of time with concurrent PbtO2 data, can be found in 2 data also uniformly indicated non-stationarity. In most patients, ADF testing indicated no presence of a unit root. As such, a transformation to the data was required before the assumption of stationarity could be fulfilled and time series models of the data constructed. This pattern of ACF plots and KPSS and ADF testing results is consistent with a difference stationary series, and so the first-order difference of the data was taken to transform the data.The ACF plots, as well as the ADF and KPSS test results for each patient’s PbtO2 data, can be found in 2 data for each patient. This indicated that the first-order-differenced data fulfilled the assumptions of stationarity. As a result, subsequent analysis was carried out using both ΔPbtO2 and ΔrSO2 data.The ACF plots, as well as the ADF and KPSS test results for each patient’s ΔPbtO2 and ΔrSO2, over the entire cohort, indicated the ΔPbtO2 was most strongly correlated with ΔrSO2 one minute earlier. In other words, ΔPbtO2 and ΔrSO2Lag1 shared the strongest cross-correlation. In the 10-seconds-by-10-seconds data, a similar pattern was seen with ΔPbtO2 correlating with ΔrSO2 six lags earlier, equivalent to one minute earlier. The CCF plots of the minute-by-minute data and 10-seconds-by-10-seconds data can be seen in 2 and ΔrSO2Lag1 were utilized for modeling.Cross-correlation analysis between ΔPbtO2, and ΔrSO2 with an order of 6 was utilized to construct the IRF plots. The IRF plots of the modeled response in ΔrSO2 and ΔPbtO2 to a sudden impulse in ΔABP can be seen in 2 indicates an almost instantaneous response to an impulse in ΔABP followed by a sharp decrease and eventual return to equilibrium by approximately lag 6. This is in contrast to ΔPbtO2, where an impulse in ΔABP results in a delayed and prolonged response that only peaks at approximately lag 6 or 7.The AIC for the multivariate VAR models from order 1 to 15 can be seen in 2Lag1 to be a significant positive linear regressor of ΔPbtO2 . However, the ACF and PACF plots can be seen in 2. This indicates that the autocorrelative structure of ΔPbtO2 needs to be accounted for, prior to valid inferences being made about the relationship between ΔPbtO2 and ΔrSO2Lag1.The linear mixed-effects model (population level model) with random slope and intercept did find ΔrSO2 at the population level were unsuccessful. This was due to computational complexity with failure to converge. Creating independent inferential linear models for each patient was more successful. The details of these models can be found in 2 was found to be a significant regressor in 16 of the 20 patients. However, in three of these patients, the coefficient was negative, which is not consistent with the expected relationship between rSO2 and PbtO2. Examination of the ACF and PACF plots, found in Attempts to create a linear mixed-effects model that adequately incorporated the autocorrelative structure of ΔPbtO2 are also summarized in 2 for each subject can be found in The results of the correlation analysis between actual and predicted values of ΔPbtO2 from ΔrSO2Lag1.An example of a Bland–Altman plot for a single model can be seen in 2 and change in NIRS-based rSO2 was performed in this multi-institutional cohort of 20 moderate-to-severe TBI patients. There are three key insights brought about by this study. First, changes in PbtO2 are correlated with changes in rSO2 that occur one minute earlier. Second, changes in rSO2, in a linear way, contain information about changes in PbtO2 that occur one minute later. Finally, while changes in rSO2 have this delayed linear relationship with changes in PbtO2, changes in rSO2 are not adequate for predicting changes in PbtO2.A statistically rigorous exploration of the relationship between the change in PbtO2 are often best correlated with changes in rSO2 one minute earlier. This finding is both consistent with the theoretical mechanisms of each modality and previous findings in the literature. In a study of 42 TBI patients, Budohoski and colleagues noted that NIRS reacted earlier to changes in ABP and ICP as compared to PbtO2 [2 is both delayed and prolonged. In the case of change in rSO2, it is probable that the initial step rise, subsequent overcorrection, and eventual return to equilibrium may be reflective of cerebrovascular reactivity. The delayed and prolonged nature of the change in PbtO2 may explain why continuous cerebrovascular reactivity metrics based on PbtO2 have been found to be so dissimilar to those based on more responsive surrogates of cerebral blood flow or volume [2 is a measure of the extracellular content of oxygen in brain tissue as it only measures dissolved oxygen in the interstitial fluid of the brain. NIRS-based rSO2 measures microvascular oxygen saturation over a volume of brain as it utilizes deoxyhemoglobin (DeOxHgB) and oxyhemoglobin (OxHgB) as chromophores to scatter and the NIR light [2, oxygen would need to disassociate from the OxHgB and diffuse into this extracellular space. A similar delay in decreases in cerebral oxygen content might also be explained by this mechanism. This mechanism is consistent with the findings of changes in PbtO2 being correlated with changes in rSO2 that occurred one minute earlier. This is a significant finding that may help guide further research into the flow of oxygen through the cerebral microenvironment.In this study, changes in PbtOto PbtO2 . This war volume . As for IR light ,32. Oxyg2 may, in a linear way, contain information about a change in PbtO2 approximately one minute later. The statistical significance of this relationship held true even when the autocorrelative structure of PbtO2 was accounted for. While this was not found in every patient, there are several reasons why this may be the case, the most obvious of which is that NIRS-based rSO2 is prone to interference from extravascular blood collections, such as those seen in subgaleal, epidural, and subdural hematomas, as well as intraparenchymal hematomas. In the setting of TBI, these forms of interference are common.This study found that changes in rSO2 are inadequate on their own to predict upcoming changes in PbtO2. Despite using a methodology that may tend towards overfitting of the models, measures of change in rSO2 were unable to reasonably predict changes in PbtO2. This was primarily evident when examining the degree of agreement between actual values of change in PbtO2 and predicted values. This adds to the body of evidence that indicates rSO2 is not an adequate alternative to PbtO2 [2 and PbtO2 are in some ways measures of cerebral oxygenation, they interrogate entirely different compartments. There are likely several factors that influence changes in PbtO2 that were not utilized in the models in this study. Hemoglobin concentrations (HgB), cerebral metabolic rate of oxygen (CMRO2), partial pressure of oxygen in the arterial blood (PaO2), and microvascular cerebral blood flow velocity (CBFV) are all factors that may modulate how changes in rSO2 relate to changes in PbtO2. It is understandable why, despite containing information about upcoming changes in PbtO2, changes in rSO2 could not adequately predict changes in PbtO2 in this cohort.Finally, this study indicates that changes in NIRS-based rSOto PbtO2 ,21,22. O2 and PbtO2 provide related but not equivalent information about brain physiology, and as such, rSO2, as a raw parameter, is not a viable non-invasive alternative to PbtO2 for the monitoring of TBI patients. Further work is needed to better elucidate the potential role of rSO2 in the multimodal monitoring of critically ill TBI patients. The related nature of rSO2 and PbtO2 may be leveraged in the future to improve care, such as in the prehospital setting where invasive monitoring is not possible. Secondly, given the identifiably delayed reaction of PbtO2 to changes in ABP as compared to rSO2, as demonstrated in the CCF analysis and IRF plots, it is likely that rSO2 is better suited for continuous indices of cerebrovascular reactivity. These indices require response parameters that react quickly to changes in ABP. There is a significant degree of interest evolving in the area of continuous cerebrovascular reactivity indices in the monitoring and management of moderate-to-severe TBI.The findings of this study have significant clinical implications. The first is that rSO2 and PbtO2 monitoring is relatively uncommon, with this study being the first such analysis of high-resolution concurrent recordings in the literature. This cohort represents less than 7% of the full CAHR-TBI database indicating the rarity of simultaneous recordings of these parameters. This likely reflects both the relatively recent global adoption of PbtO2 as a means of cerebral monitoring in TBI and the paucity of evidence for the use of NIRS in monitoring moderate-to-severe TBI.There are limitations to this study that need consideration when interpreting its findings. The first is the relatively small size of the cohort in this study, with only 20 patients included. This necessitates the validation of these findings in a larger cohort before the findings can be fully incorporated into patient care as generalizability might be limited. However, concurrent NIRS-based rSO2 measurements, such as extravascular blood collections, was not available. Additionally, the side of PbtO2 monitor placement needed to be assumed, as this was also not available for all patients.A second limitation, brought on by the type of analysis performed, was the need to interpolate data. This, obviously, injects some inherent uncertainty into the study findings. Another limitation of this study is that information about factors that may interfere with rSO2 and changes in PbtO2. This was done due to the lack of evidence suggesting a more appropriate alternative structure to this relationship. It is possible that a mathematically more complex model may prove more suited to describing this relationship. Complex supervised machine learning algorithms might be an obvious method to map this relationship better. While more complex mathematical models might provide an accurate prediction of PbtO2 values based on rSO2 levels, they are likely to increase computational complexity. This may limit utility at the bedside if computations are not possible in real time due to this increased complexity.Finally, the models utilized in this analysis assumed a linear relationship between changes in rSO2, PaO2, HgB concentration, and microvascular CBFV, would also be concurrently measured to fully elucidate the relationship between these two modalities. With a larger cohort and better characterized physiology, the complexity of this relationship may be better captured.The findings of this work lay the groundwork for additional research. First, these findings need to be validated in a larger multi-institutional cohort, where information about sources of NIRS interference is also available. Ideally, additional parameters, including CMRO2 has not gained traction as a stand-alone parameter in the management of TBI, there is increased interest in leveraging NIRS as a means of non-invasively interrogating cerebrovascular reactivity in TBI [2 and PbtO2 monitoring may become available in the future. In such complex multimodal monitoring datasets, supervised and unsupervised classification and regression machine learning algorithms may provide additional insights into how these parameters interact with one another. While complex computational models may not be deployable at the bedside, they may act as inferential models to drive our understanding further. This may ultimately lead to new treatment paradigms in the management of moderate and severe TBI in the acute phase, including specific molecular targets.While NIRS-based rSOy in TBI ,38,39,402 and PbtO2 monitoring, changes in PbtO2 were found to correlate most significantly with changes in rSO2 approximately one minute earlier. Through mixed-effects and time series modeling, changes in rSO2 were found to often have a statistically significant linear relationship with changes in PbtO2 that occurred one minute later. This was the case even when the hierarchical and autocorrelative structure of the data was considered. However, changes in rSO2 were inadequate on their own to predict changes in PbtO2 based on the poor agreement between modeled and actual changes in PbtO2. Given the uniqueness of this dataset, only a small number of subjects were available for analysis, limiting the confidence of these findings. In the future, a larger cohort, with additional parameters that influence cerebral oxygenation, is required to validate and better explain these findings.In this multi-institutional exploratory analysis of a cohort of 20 TBI patients with concurrent rSO


Day-case tonsillectomy is becoming popular with varying patients' satisfaction. Whether day-case or overnight tonsillectomy is safe and preferred by patients has been debatable among otorhinolaryngologists. To date, majority of otorhinolaryngologists in Tanzania are practicing overnight tonsillectomy unlike what is being practiced in other parts of the world. There is scarce literature regarding whether day-case or overnight surgical option is preferred by patients.To assess the time preferred by patients to undergo surgery by ascertaining the proportion of patients who would prefer day-case to overnight tonsillectomy or adenotonsillectomy.We conducted a hospital based cross sectional study at Ekenywa Specialised Hospital from January to December 2021. The study recruited 200 patients who underwent elective tonsillectomy or adenotonsillectomy. A structured questionnaire adopted from previously published studies and thereafter modified accordingly to fit the current study was used to collect relevant data. Data were analysed using Statistical Package for Social Sciences (SPSS) version 21st day post surgery while 65 (32.5%) preferred discharge on the 2nd postoperative day or later. Similarly, five (2.5%) would have wished day-case tonsillectomy/adenotonsillectomy while 195(97.5%) preferred an overnight surgery. Patients with postoperative pyrexia, older patients and those discharged on the 2nd day post surgery or later were more likely to prefer a longer postoperative hospital stay.The study had more male participants 104(52%) than females, 96 (48%) with a male to female ratio of 1.1:1. Majority of the study participants were aged below 5 years. Male participants aged below 5 years accounted for 50% while females aged <5 years accounted for 65.6%. Of the 200 (100%) patients who returned the questionnaires, 135(67.5%) preferred discharge on the 1Day-case surgery seems feasible due to less risk of postoperative complications such as haemorrhage and fever. Majority of the participants in this study preferred overnight tonsillectomy/adenotonsillectomy. Although patient selection in day-case surgery is based on strict and relatively uniform criteria, patient satisfaction with same-day discharge tend to vary. In a study conducted in Portugal, overall satisfaction following day surgery was at least 95% at discharge and at 30 days. However, complete satisfaction was present only in 75% at discharge and it reduced to 62% at 30 days.3 Controversies on whether to adopt day-case or overnight tonsillectomy/adenotonsillectomy have necessitated studies to determine acceptability and safety of the two. A study conducted in Jordan involving 419 paediatric patients, 103 inpatients and 316 day-case group reported that only one patient (0.32%) among the day-case patients had secondary post-tonsillectomy bleeding. Similarly, there was only one case of this reported for the inpatient group (1%), and with none reactionary hemorrhage reported in either groups. The study found no significant association between the inpatient and day-case surgery groups in terms of post-tonsillectomy bleeding.4 Another study conducted in England to assess the safety of day-case paediatric tonsillectomy found the rates of adverse postoperative outcomes to be similar for those who discharged >70% children the day-case post tonsillectomy compared with those who discharged <50% children the day-case post tonsillectomy and therefore no consistent evidence that day-case tonsillectomy is associated with poor outcomes.5 A study conducted in France to audit changes in practice following the opening up of a day-surgery unit with dedicated operating room found that the opening of the day-surgery unit led to changes in practice, with most tonsillectomies being performed on an outpatient basis, without increased complications, and notably immediate complications.6 Similarly, day-case tonsillectomy in children showed a 30-day complications rate comparable to those reported in the literature (8.3% postoperative hemorrhage), with a higher rate in adults (35.3%).6 A systematic review of outpatient tonsillectomy in children aiming at determining the level of evidence regarding the safety of outpatient paediatric tonsillectomy having pooled 17 articles found that the current level of evidence supports the practice of outpatient tonsillectomy in properly selected children.7Day-case tonsillectomy/adenotonsillectomy is becoming increasingly popular worldwide. Published studies elsewhere are reporting day-case tonsillectomy/adenotonsillectomy to be safe in both adults and children.2A study that was conducted in Finland to determine patients' preferences for length of hospital stay post adenotonsillectomy found 39.8% of the study participants to have preferred discharge on the first day, while 59.7% preferred discharge on the second postoperative day or later, and only one patient (0.4%) wished for day-case tonsillectomy.8 Whether the day-case tonsillectomy and adenotonsillectomy being reported by otorhinolaryngologists is preferred by patients or not, has not been assessed. Our study aims to address this existing knowledge gap.There is scarce literature on the same topic in Africa. A study conducted in Tanzania on the survey of tonsillectomy care patterns reported 34.6% of the otorhinolaryngologists to have never performed day-case tonsillectomy while 65.4% sometimes performed day-case tonsillectomy.To address this gap, this study evaluated patients undergoing elective tonsillectomy and adenotonsillectomy over 12 months and their opinion as to whether they would prefer day case or overnight tonsillectomy/adenotonsillectomy. This is the first study in our country to assess the time preferred by patients to undergo the mentioned surgery with emphasis on what proportion would accept day-case or overnight tonsillectomy/adenotonsillectomy.The study was conducted at Ekenywa Specialised Hospital, Department of Otorhinolaryngology from January to December 2021. The study recruited 200 patients that were undergoing elective tonsillectomy or adenotonsillectomy. The department of otorhinolaryngology attends to about 200 outpatients on daily basis and performs an average of 6 elective surgeries per day.A hospital based cross sectional study was conducted to determine the time preferred by patients to undergo tonsillectomy/adenotonsillectomy by ascertaining the proportion of patients who prefer a day-case or an overnight option for the scheduled surgery.Healthy patients who underwent elective bilateral tonsillectomy with or without adenoidectomy upon obtaining their consent to participate were included in the study.Patients who underwent elective bilateral tonsillectomy with or without adenoidectomy and were unwilling to participate were excluded. Also patients who were mentally unfit to consent to participate were also excluded.Data was collected using convenient sampling technique. Study participants were recruited on convenient basis until the desired sample size was achieved. Moreover, the sample size for this study was calculated using the formula below;Where;n = sample sizez= standard normal deviate=1.96 for 95% confidence level4p = proportion, P=75.4%, proportion of day-case tonsillectomy/adenotonsillectomy.E= margin of error which is 5%2 ×75.4(100 75.4)/52 = 285Therefore, N=1.96The minimum estimated sample size was 285 study participants; however, only 200 participants were recruited.9 The first version of the questionnaire was prepared in English and the final draft was translated to Swahili since the study participants attending the health facility were more conversant with Kiswahili. The questionnaire comprised of 2 main parts with sub-sections: (i) Age and sex distribution of the study participants (ii) Clinical profile of the study participants and this included; (a) Indications for tonsillectomy/adenotonsillectomy (b) Discharge summary of the study participants particularly the specific day of discharge post surgery (whether first day or second day post surgery) (c) their preference on when they should be discharged post surgery (d) Postoperative outcomes such as postoperative pyrexia, postoperative haemorrhage. The questionnaires comprised of closed ended questions and were self-administered. The procedure included self-introduction by the principal researcher, introduction of the topic and purpose of the study. Participants were assured of their free participation and withdrawal will from the study at any time if they wish to do so. Tool validity was assessed by reviewing the literature as well as pilot testing the instrument prior to the study by involving 10% of the actual sample size from the specialised hospital and these were excluded from the actual study.A structured questionnaire adopted from previously published studies and thereafter modified accordingly to fit the current study's goal and objectives was used to collect data.Patients were mandated to report at the hospital one (1) day before surgery and on admission each patient received details of the anticipated procedure including the course of recovery and possible postoperative complications and for those under the age of 18 years, same information was conveyed to them and to their respective parents/caretakers. Tonsillectomy was performed under general anaesthesia with a bipolar electro dissection technique.st day post surgery and provided them with verbal information on the course of recovery. Prolonged hospital stay was considered if the patient had a medical reason such as post-tonsillectomy haemorrhage or pyrexia (body temperature higher than 37.5 degree Celsius). Each patient received a prescription for non-steroidal anti-inflammatory analgesic and an oral antibiotic post tonsillectomy/adenotonsillectomy and for those who underwent adenotonsillectomy, were prescribed with a nasal decongestant . All patients and, in the case of those under the age of 18 years had their parents/caretakers providing a written informed consent for the planned surgery.An otorhinolaryngologist at the ear, nose and throat (ENT) department discharged all patients at the earliest 1The collected data were cleaned and analysed using SPSS version 21 software package. Descriptive statistics were performed to present frequency distribution for demographic characteristics and clinical profile of the study participants.Chi-square test was performed to establish the relationship between the selected independent and dependent variables. All the independent variables with p-value <.05 were regarded to be statistically significant. Statistical limits were set at a 95% confidence interval and a 5% significance level.th December 2020 with number Ref: ESH/2020/12. Participants were assured of their privacy and confidentiality. Anonymity was maintained by the use of code number on the questionnaire instead of the participant's name and the participant had an absolute freedom and right to withdraw from the study at any time without any compromise of the medical care they were receiving at the hospital.A written informed consent was obtained from all participants. For participants below 18 years, written informed consent was obtained from their respective parents/guardians. The aim of the study was explained to patients, parents and caretakers. Ethical approval from the hospital ethics committee was granted on 10In this study, there were more males, 104(52%) than female, 96 (48%) participants. Male to female ratio was recorded at 1.1:1. Majority of the study participants were aged below 5 years, where males aged below 5 years accounted for 50% while females aged below 5 years accounted for 65.6% of the respective gender. On the other hand, of all the 200 surgical patients, 188 (94%) patients underwent adenotonsillectomy but 12(6%) underwent tonsillectomy. The indication was obstructive tonsillitis in 6 patients (50%), recurrent acute tonsillitis in 2 (16.7%), and chronic tonsillitis in 4 (33.3%). Obstructive adenotonsillitis was the commonest indication in younger age groups and obstructive tonsillitis in those aged 11+ years. Majority of patients across all age groups in this study were discharged on the first day postoperatively. 1 patient (0.5%) had primary post-tonsillectomy hemorrhage and 2 patients (1%) had primary post-adenotonsillectomy hemorrhage and all the 3 patients were aged <5years. Similarly, 3 patients had secondary post adenotonsillectomy hemorrhage (1.5%). No hemorrhage post adenoidectomy was encountered in this study. Postoperative fever was encountered in 7(3.5%) patients and all were aged <5 years. st day after surgery. Similarly, those patients who had 2 nights hospital stay post surgery and those with postoperative fever preferred discharge to happen longer (2nd day or thereafter post surgery) .  preferred discharge to happen 1e=.001). Tonsillectomy being one of the commonest surgery in otorhinolaryngology has been performed invariably in different parts of the world with other surgeons preferring patients to be admitted a day before surgery (overnight) and other being admitted on the same day when surgery has to be performed. Whether patients prefer day-case tonsillectomy/adenotonsillectomy or an overnight surgical option remains to be unknown in Tanzania and this study aimed at addressing such gap. Regarding patients' views on whether they would prefer overnight or day-case tonsillectomy, majority (97.5%) reported preference of overnight surgery and thus day-case surgery to patients was found to be undesirable.2 On the other hand, since our patients had no systemic illnesses (ASA class 1), majority of them would have been suitable for day-case surgery but because in the private health facility where the study was conducted day-case surgery was not an option to be considered but rather all patients have to be admitted a day before adenotonsillectomy/tonsillectomy and this might have probably influenced patients' attitudes on discharge preference. Patients' acceptability of day-case adenotonsillectomy is higher in a study that was conducted in a day-surgery unit in the United States 10, than involving in-patient operations in a study that was conducted in the United Kingdom. 11This finding appears to be similar to what was established in a study that was conducted in Finland. st day after surgery. Such finding appear to correlate with what was found in studies done elsewhere. 12 In our study, those patients who had 2 nights hospital stay post surgery and those with postoperative fever preferred discharge to happen longer (2nd day or thereafter post surgery) Regarding patients' discharge preference after elective surgery, majority, (67.5%) preferred discharge to happen 12Pertaining patients' and suitability for day-case adenotonsillectomy, our study found children aged <5 years unsuitable for day-case surgery since both children who suffered from postoperative hemorrhage were aged <5years and similarly all the seven children who developed postoperative fever were aged <5 years. Such findings appears to be dissimilar to those found in a study which was conducted in Finland where children younger than 10 years seemed to be the best group for day-case tonsillectomy because they were the least likely to suffer from postoperative fever or primary postoperative hemorrhage. Regarding whether day-case adenotonsillectomy/tonsillectomy should be adopted or not from our findings where majority of the study participants found overnight surgery to be the preferred choice should be taken with caution since these are findings from a single health facility and therefore multi-centered studies should be conducted on the same topic and this must be coupled with adequate preparations if at all day-case surgery has to be adopted in our country. Preparations should include preparedness of health workers in accepting the new treatment policy and also make meticulous preoperative preparations to patients' who are to undergo day-case adenotonsillectomy/tonsillectomy. Pilot health facilities to perform day-case surgery may be designed countrywide to check feasibility of such surgical strategyOvernight tonsillectomy/adenotonsillectomy has been the preferred surgery in this study and on the other hand day case tonsillectomy/adenotonsillectomy from our findings seems feasible due to less risk of postoperative complications such as postoperative hemorrhage and fever. Large multicentric studies should be designed to validate these findings and ascertain on the possibility of implementing day-case surgery in our settings.The study was conducted in a single health facility and considered a limited sample size. Thus, the results from the study cannot be generalised. Larger multicentric studies incorporating relevant socio-demographic information like residence, level of education and profession should be designed so as to assess their influence on patients' discharge and type of surgery opted for.


This cross-sectional study evaluated the growth and distribution of physicians in the Conrad 30 Waiver program during the past 2 decades. Noncitizen IMGs can legally train in US medical residency programs through the H-1B and J-1 visa pathways, with the vast majority entering through J-1.2 The Immigration and Nationality Act requires that those trainees return to their country of origin for 2 years when they complete their training. During that time, they may apply for a traditional immigrant visa to return to the US. However, the Conrad program waives the requirement by allowing those trainees to convert their J-1 to H-1B nonimmigrant status in exchange for a 3-year full-time commitment to practice medicine in underserved areas.3Physician shortage is a persistent challenge facing the US.4 Since 2003, Congress has increased the waiver cap to 30 per state per year. However, there is little information on the growth and distribution of Conrad physicians during the past 2 decades. This study aimed to fill this evidence gap.The Conrad program, authorized by the 1994 Immigration and Nationality Technical Corrections Act, is both the predominate pipeline for noncitizen IMGs with J-1 visas to enter the domestic workforce and the largest supplier of physicians to shortage areas.STROBE) reporting guideline. We assessed trends in the number of Conrad physicians and their distribution by specialty and geography. All tests were 2-sided, used an α level of .05, and were conducted in Stata statistical software, version 17.0 (StataCorp). The data were analyzed between May 30, 2022, and November 2, 2022.This retrospective cross-sectional analysis of all US states and the District of Columbia used data from 2001 to 2020 from the Rural Recruitment and Retention Network. These data were publicly available and determined as not human participant research by the corresponding author’s institutional review board. Informed consent was waived because the data were deidentified. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology  supported by the program fell from 82% (n = 471) to 28% (n = 402) in the study period . The num5 However, considering the importance of specialty care physicians on health outcomes and continued shortages of those physicians, states should be encouraged to use the Conrad program to recruit those physicians to meet their needs.6 Declining Conrad physicians in rural areas suggests that the program may not have fulfilled its original goal of improving physician supply in HPSAs, of which the majority (65%) are in rural counties.This retrospective cross-sectional study revealed 2 trends in the Conrad program during the past 2 decades: the declining proportions of recruited PCPs and rural physicians. The decreased proportion of Conrad PCPs is a substantial finding given the severe shortage of primary care clinicians in the nation, with more than 86 million (26%) Americans living in primary care Health Professional Shortage Areas (HPSAs) in 2021.Although the present study assessed national trends of the Conrad program, we did not analyze state-level drivers of variations in program implementation. Further exploration of the Conrad program is needed, such as discussions with state officials about their definition of program success, their use of 10 additional FLEX slots, and their efforts to balance between PCPs and other specialists and between rural and urban areas. Identifying and sharing best practices across states can make the Conrad program more effective for improving physician supply in underserved areas.


We developed a functional ex vivo anthracycline-based sensitivity test. Surgical resection material of primary breast cancer (BC) was used to determine criteria for the ex vivo sensitivity assay based on morphology, proliferation and apoptosis. Subsequently, a proof-of-concept study was performed correlating results of this assay on primary BC biopsies with in vivo response after treatment with anthracycline-containing neoadjuvant chemotherapy (NAC). Cut off values for the ex vivo anthracycline-based sensitivity test were established based on analysis of 21 primary breast tumor samples obtained after surgery. In the proof-of-concept study based on a new set of tumor biopsies, 41 patients were included. Eight biopsies did not contain tumor cells and three patients could not be biopsied for various reasons. In the remaining 30 biopsies, the success rate of the ex vivo test was 77% (23/30); six out of seven failed tests were due to excessive apoptosis, our pre-specified test criteria. Of the 23 patients with a successful ex vivo test result, three patients did not undergo NAC after the biopsy. Here we report the ex vivo anthracycline-based sensitivity assay is feasible on biopsy material and shows 75% concordance between ex vivo outcomes and in vivo MRI response. Unfortunately, the percentage of unsuccessful tests is rather high. This study provides the foundation for further development of ex vivo sensitivity assays. Approximately 30–40% of patients whose disease would recur after surgery can now be cured by pre- or post-operative chemotherapy. Unfortunately, this also means that 60–70% of these patients will develop recurrent disease in spite of the chemotherapy. Despite the encouraging developments of targeted therapies, chemotherapy is expected to remain the cornerstone of BC treatment for decades to come. It is therefore essential to develop biomarkers which assess the sensitivity of breast tumors to chemotherapy, preferably before starting the treatment.The prognosis of primary breast cancer (BC) has improved over the past decades, largely due to better locoregional and especially systemic treatment options5. However, the efficacy of anthracycline has also been demonstrated in patients with tumors without TOP2A abnormalities. Desmedt et al. focused on developing the so-called A-Score, a combination of three gene expression profiles with a strong negative predictive value, for selecting anthracycline-resistant tumors6. Although its predictive value was confirmed using three other retrospective studies, the results of these studies were less clear. Moreover, prospective validation is lacking and the test itself is still only performed on frozen tumor material.The search for predictive biomarkers for response to chemotherapy has predominantly focused on alterations in its main target. The most extensively studied example for BC is anthracycline sensitivity of tumors and the genomic alterations in those tumors to the main anthracycline target, topoisomerase 2-alpha (TOP2A). For example, patients with tumors harboring TOP2A aberrations benefitted more from anthracycline-based therapy than patients with tumors without TOP2A aberrations in an individual patient-level pooled analysis from five adjuvant phase III trials comparing anthracycline-based therapies with combination therapy of cyclophosphamide, methotrexate and fluorouracil9, keeping cells in their natural (micro)environment. This allows testing of tissue responses to various types of treatment9. With this technique we previously showed that outcome parameters could be developed for the response of these human BC slices to anthracycline-based chemotherapy, platinum salts and taxanes10. A correlation of these ex vivo sensitivity assays with the in vivo response, however, was still lacking.An alternative approach to predict sensitivity is direct analysis of tumor response to chemotherapy. Recently, we developed a technology to maintain viability, tissue integrity and proliferation of BC tissue slices ex vivo for up to 1 weekIn the current study we further optimized the ex vivo sensitivity assay for anthracycline-based chemotherapy and performed a clinical proof-of-concept study for the correlation with the in vivo response. The BREAST study correlates this ex vivo sensitivity assay on tumor biopsies with the in vivo tumor response by breast MRI after treating these patients with anthracycline-containing NAC. The main goal was to determine whether this sensitivity assay was suitable for further validation or whether structural adjustments were needed before further validation.We first developed a scoring system for ex vivo anthracycline-based sensitivity testing. We determined morphology, proliferation and apoptosis after incubation for 3 days with various concentrations of anthracycline-containing chemotherapy for 21 primary BC samples. In the primary setting, a total of two samples did not lead to a successful test. The three concentrations depicted in Fig. To establish the criteria for predicting ex vivo response, we took into account the limited availability of tumor material in biopsies. Therefore, two concentrations were chosen for ex vivo anthracycline treatment. Some tumors did not respond to the 5× SFC, while other tumors already responded to 1× SFC. This resulted in criteria to select anthracycline-resistant tumors, shown in Table After defining these starting parameters on 21 primary resected tumors, we took the next step toward clinical validation by comparing the ex vivo sensitivity to in vivo tumor responses. A total of 41 new BC patients signed informed consent for the BREAST study. Three patients ultimately did not undergo a biopsy. The lack of tumor cells in eight biopsies left 30 patients with an adequate tumor biopsy Fig. .Fig. 2OvThe success rate of the ex vivo sensitivity assay was 77% (23 out of 30 samples). Infection during culturing in one of the samples prevented an interpretable ex vivo test. The other six failed tests were due to the (pre-specified) test criterion that the fraction of apoptotic cells should not exceed 40% in the untreated slice. We did not find any significant differences in tumor characteristics between successful and unsuccessful test results Table . No resiOf the 23 patients with a successful ex vivo test result, three patients ultimately did not undergo NAC before resection. The in vivo tumor responses based on MRI response after four courses of anthracycline-containing chemotherapy showed sensitivity ( > 50% decrease in tumor size) to anthracyclines in four patients (20%) and intermediate sensitivity ( < 50% decrease and <20% increase in tumor size) to anthracyclines in 16 patients (80%). No tumor showed anthracycline resistance.In our proof-of-concept study, the ex vivo sensitivity assay and the in vivo tumor response was concordant in 75%  and discordant in 25% of the cases  in response to anthracycline was explored. This classification might have the advantage that it can take into account the potentially large variation in TUNEL positivity of the untreated slice between biopsies of individual tumors, which might result in a better classification of tumors with relatively high apoptotic levels in untreated samples. We evaluated a threshold of 20% increase of TUNEL positive DAPI pixels relative to the untreated slice. This resulted in two tumors 3 and 18) going from an intermediate sensitive to a sensitive ex vivo test result . Further improvement of the rate of concordance by adapting thresholds for outcome parameters could not be achieved. The number of unsuccessful ex vivo test results was unfortunately rather high, mainly due to high levels of cell death in the tumor samples. This study is a starting point for further development of predictive ex vivo chemotherapy sensitivity tests and identifies several aspects that require further improvements.In our proof-of-concept study, as many as 41 patients were needed to select 20 suitable patients. Six patients (6/41 = 15%) dropped out due to logistical reasons . Unexpectedly, eight out of 38 (21%) biopsies did not contain tumor cells and therefore these patients could not be included in our analysis. This is a high percentage and might be explained by the fact that the biopsies for this study were taken after previous biopsies to establish the primary diagnosis and mostly at exactly the same location. The previous biopsies may have caused local changes in the ultrasound image , increasing the likelihood of sampling errors.Of the 30 biopsies which could undergo the ex vivo sensitivity test, seven biopsies (23%) were found to yield an unsuccessful test result. Six of the seven test results (86%) were unsuccessful due to the predefined test criteria, and thus only one biopsy was unsuccessful due to the failure of the test itself. The predefined criteria for scoring the ex vivo assay were a sufficient number of proliferating tumor cells and less than 40% apoptotic cells in the biopsy material. This was not a problem for the surgical tissue sections (21 of 23 tumors), but proved to be much more challenging in the relatively small breast tumor biopsies. Increasing the apoptosis limit to <50% in the pre-treatment biopsy sample did not improve the outcome. We therefore propose to start from at least two biopsies in follow-up studies, one in the center and one at the periphery of the tumor.Five out of 20 tumors showed discordant results, either overestimating or underestimating tumor sensitivity. This meets our predefined requirement of at least 55% concordance and is therefore sufficiently hopeful to warrant further investigation of the ex vivo assay.12; this would influence the concentration of the chemotherapeutic compounds in vivo, whereas the ex vivo concentration is only dependent on diffusion into the tumor slice. Insufficient vascularization in vivo would therefore mainly result in overestimation of the tumor sensitivity ex vivo. Including vascular markers in the analysis could provide more insight into this. Third, systemic differences, caused by differences of various organs, influence the in vivo response. For example, detoxification or metabolic activation in the liver or secretion via urine or faeces is not mimicked in the ex vivo assay and could lead to both under- and overestimation of the ex vivo sensitivity test result. Fourth, the ex vivo tissue slice system has not been optimized for immune components, such as tumor-infiltrating leukocytes (TILs), which are an important factor in determining chemotherapy sensitivity13. Although TILs can be present in tumor slices, changes occur during treatment in the patient14, which is not mimicked faithfully in our ex vivo system. It is interesting that concordance is high (75%) in spite of all these possible factors that could cause differences between ex vivo and in vivo responses, which probably means that intrinsic tumor cell sensitivity is the overruling parameter determining therapy response in the majority of patients.We considered several issues that may negatively influence the degree of concordance between ex vivo and in vivo outcome, which can be partially avoided. First, tumor heterogeneity may hamper correct prediction and could be responsible for both under- and overestimation of the ex vivo sensitivity test results. Previously mentioned two biopsies at different positions could better probe this heterogeneity. Second, differences in drug accessibility of the tumor can be caused by the vasculature15, it is not likely that this would change the conclusions significantly. Second, pathological response could not be used in this study because the anthracycline-based therapy was directly followed by taxane treatment, before surgical removal of the tumor. A study performed on MRI size prediction, in comparison to pathological measurement, stated a wrong size prediction in 28% of the cases. Of these 28%, tumor size was overestimated in 93%. This ensures us that response to FAC therapy would more likely be underestimated instead of overestimated16.Two limitations should be mentioned. First, the ex vivo test was set up with a FAC regimen, because this was the standard of care when the test was developed. It is possible that this is significantly different from an AC sensitivity assay. However, as the effect of 5-Fluorouracil (F) in addition to AC is very limited according to the literatureAn interesting observation is that simplification of the ex vivo test seems possible. Based on our results shown in Fig. 10. Since both taxanes and anthracyclines are often used in the (neo)adjuvant setting in BC treatment, it might be of interest to use both ex vivo sensitivity assays simultaneously on different slices of the same biopsy to explore the possibility to optimize treatment by selecting the most optimal treatment for each patient. However, the limited availability of tumor material is the limiting factor. In the future, culture conditions could be optimized using a Cancer-on-Chip approach, that we recently developed17. This would require a minimal amount of tumor material; we estimate that two biopsies should be enough to test several conditions. Cancer-on-Chip could also be combined with direct microscopic imaging and/or analysis of outflow media to maximize the amount of information extracted from a single experiment. The assay conditions determined in this study provide a starting point for such a multiplexed chemotherapy sensitivity test.Recently, we have taken the first steps in developing ex vivo sensitivity assays for taxanes and platinum derivates, two chemotherapeutic agents which are frequently used in BC treatmentIn conclusion, in this study we showed that the ex vivo anthracycline sensitivity assay can be performed on small biopsies and that the concordance with in vivo outcome is high. Further development should focus on not only expanding the number of patients to confirm the presented data but also on further developing other ex vivo chemotherapeutic sensitivity tests. Using multiple different drugs in one assay would have the ultimate goal of predicting which chemotherapeutic agents have the highest potential for efficacy at the individual patient level.8. After surgical removal of the primary BC, viable tumors were sent to the pathology department. Samples from breast tumors, where sufficient tumor material was present, were donated for our study, approved by the Erasmus MC Medical Ethical Committee (MEC-2011-098). Samples were collected in customized breast tissue medium7, sliced into 300 µm slices using the Leica VT 1200 s Vibratome, and cultured as described8. Briefly, slices were cultured on an orbital shaker (60 rpm) in a 5% CO2 and 37°C atmosphere. Slices were treated with 5-fluorouracil (F), doxorubicin (A) and 4-hydroperoxy cyclophosphamide (C) at various concentrations for 3 days. The concentrations differed, but the ratio (46:1:22) remained the same between the three chemotherapeutics and was based on the clinically used ratio18. Concentration 1× is 23 µM F, 0.5 µM A and 11 µM C and was called the standard FAC concentration (SFC). This combination of chemotherapeutic agents was the standard treatment in daily practice at the time of test development. 5-Ethynyl-2′-deoxyuridine  was added 2 h before formalin fixation as a marker for proliferation. After paraffin embedding, 4 µm sections were used for staining. The outcome of the ex vivo assay was determined by evaluation of three parameters; histology (HE-staining and keratin staining), proliferation (EdU staining combined with the keratin staining to identify tumor cells) and apoptosis (TUNEL staining)8. The first 14 primary BC samples have been described in detail elsewhere8. With the results from both the previously published samples and the newly added samples, criteria have been established with which the proof-of-concept study was performed.A set of previously published tumors was combined with new samples7. The ex vivo response per tumor was obtained by performing the ex vivo anthracycline-based chemotherapy sensitivity assay developed in part I on at least three fresh 300 µm tumor slices; untreated and at least two different FAC concentrations. Before the sensitivity assay could be performed, the untreated tumor slices had to contain (1) tumor cells, proven by HE and keratin staining, (2) EdU positive cells in the tumor area and (3) <40% TUNEL positive DAPI pixels. Three subgroups were defined based on morphology, proliferation and apoptosis; sensitive, intermediate sensitive and resistant. The in vivo response was evaluated from contrast enhanced breast MRI before starting NAC and after four courses of anthracycline-containing treatment. In vivo response was defined as complete response, partial response, stable disease and progressive disease using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. on MRI19. The ex vivo response was considered concordant when one of the following three results was obtained: a sensitive response was coupled to an in vivo response of >50% decrease in tumor size  of the breast was collected in customized breast tissue mediumResults of the ex vivo anthracycline sensitivity test can be known between 7 and 14 days.20. With an α and β of both 0.10, the required number of patients with a successful ex vivo test result to be included was 20. Of these 20 patients, 55% needed to be concordant to draw a final conclusion. One interim analysis was planned after the results from the first nine evaluable patients were available. If ≤2 patients had a concordant result or >5 patients had tumors classified resistant by the ex vivo anthracycline-based sensitivity assay showing a good in vivo response, the study would have been terminated prematurely. Only patients with a representative biopsy and a useful test result were included in the final analysis. A biopsy was considered representative if a pathologist confirmed the diagnosis of breast malignancy.For this pilot, we aimed to find a concordance between the ex vivo and in vivo response of at least 55% (P1), which was considered sufficiently promising to warrant further research. If concordance was <25% (P0) the ex vivo assay in its current format has no advantage over patient selection according to present standards. For the sample size calculation, the optimal Simon 2-stage design was appliedP values of <0.05 were considered significant.Statistical analyses of ex vivo test outcomes were all two-sided and performed using IBM SPSS v25. Significance was calculated using a Fisher exact test for categorical data. Further information on research design is available in the Supplementary figuresReporting Summary