Chronic myeloid leukemia (CML) accounts for 2-3% of childhood leukemias. About 5% of cases present in a blastic phase of CML which clinically and morphologically mimics more common acute leukemias of childhood. We report a case of a 3-year-old male who presented with gradual onset swelling of the abdomen and extremities along with generalized weakness. Examination revealed massive splenomegaly, pallor, and pedal edema. Initial workup showed anemia, thrombocytopenia, and leukocytosis  with a blast percentage of 35%. Blasts were positive for CD13, CD33, CD117, CD34 and HLA-DR, and stained negative for Myeloperoxidase and Periodic Acid Schiff. Fluorescence in situ hybridization was positive for b3a2/e14a2 junction BCR-ABL1 transcript and negative for RUNX1-RUNX1T1/t, clinching the diagnosis of CML in myeloid blast crisis. The patient expired within 17 days of diagnosis and initiation of therapy. The blastic phase is defined as the presence of at least 20% blasts (>30% according to European LeukemiaNet [ELN] recommendations) in the blood or bone marrow or the presence of blasts in extramedullary sites.4 Hence such cases require a thorough evaluation. Here we report a rare case of a 3-year-old male presenting in the blastic phase, posing clinical and diagnostic challenges.CML in the blastic phase (CML-BP) behaves clinically like acute leukemia and resembles morphologically acute leukemia in children.A 3-year-old male child presented with a history of gradual swelling of the abdomen for the past eight months and swelling of the hands and feet with a generalized weakness for the past two months. There was no history of fever, rash, bleeding, or jaundice. The patient’s appetite, bowel and bladder habits were normal. There was no history of similar episodes or recurrent infections in the past. According to his growth charts, the child’s growth rate had been within the normal percentile range. Physical examination revealed the presence of pallor, pedal edema, and massive splenomegaly  but no significant lymphadenopathy or palpable hepatomegaly. The remainder of the physical examination was within normal limits.Initial laboratory investigations showed anemia , thrombocytopenia , and leukocytosis . A shift to the left was noted on differential leukocyte count with a blast count of 35% and basophilia (4%). The blast cells were round to oval, having coarse nuclear chromatin and prominent nucleoli, with a few cells containing indented nuclei . Blast cBone marrow aspirate showed suppressed erythropoiesis, subtle megaloblastic reaction, and mild dyspoiesis. Blasts accounted for 24% of the cellularity, with a myeloid: erythroid ratio of 14:1 . MegakarFlow cytometry of the bone marrow aspirate was performed using BD FACScalibur, and blasts were gated by CD45/Side Scatter gating strategy for immunophenotyping (IPT). On IPT, the blasts were positive for CD13, CD33, CD117, CD34 and HLA-DR but were negative for Tdt, CD3, CD4, CD7, CD19, and CD20  and 2C. Fluorescence in situ hybridization (FISH) with dual fusion probe on peripheral blood (PB) sample for BCR/ABL gene rearrangement was performed considering the probability of acute leukemia vis-a-vis CML-BP. Abnormal clones with BCR-ABL1 (Breakpoint cluster region - Abelson gene) fusion were noted in 30.5% of cells . FISH waCorrelating the clinical presentation of massive splenomegaly, the patient’s hematological parameters, IPT, and the presence of p210 BCR-ABL1 transcripts, a final diagnosis of CML in myeloid blast crisis was made.2 and planned to be re-evaluated at the end of three months, but he died due to febrile neutropenia after 17 days of initiation of therapy.The patient was started on Imatinib mesylate 400 mg/m4 CML blast transformation is lymphoid in 30% of cases and myeloid in 70% of cases.4 There are only a few isolated case reports of pediatric CML either presenting or transforming into myeloid blast crisis. An extensive search in PubMed and Google scholar with the keywords ‘pediatric’, 'chronic myeloid leukemia’ and ‘myeloid blast crisis’ revealed 4 cases in English language, which had all the laboratory and clinical details.6 The details of these 4 cases are summarized in CML-BP is the last stage of CML's progression, and it acts like acute leukemia, with rapid progression and may morphologically resemble acute leukemia in children.In the present case, the myeloid nature of the blasts was confirmed by flow cytometry. The blasts were positive for myeloid markers CD13, CD33, and CD117. All the lymphoid markers, including B-lineage and T-lineage markers, were negative.2 This is partly because BCR-ABL1 suppresses c-Jun, a monopoiesis-promoting transcription factor in both CML neutrophils and blasts.1 As a result, in CML, the BCR-ABL1 fusion gene is expected to be detected in mature neutrophils also unlike the blasts of de novo AML.1 Furthermore, mature eosinophils and basophils also possess the BCR-ABL1 fusion gene, which causes them to proliferate in CML.1 BCR-ABL1 in CML usually encodes p210 fusion transcript (b3a2/e14a2 junction).7 It is also seen in 2-4% of pediatric acute lymphoblastic leukemia cases and is usually p190.7 In the present case, BCR-ABL1 gene rearrangement was demonstrated by FISH and p210 was demonstrated by RT-PCR. However, BCR-ABL1 was demonstrated in only 30.5% of the PB cells. This could be attributed to the sample tested and the technique used. Although the common practice is to use PB instead of bone marrow aspirate to detect BCR-ABL1 in CML, it has been questioned due to relevant differences at specific cut-offs that might influence the sensitivity of molecular testing in CML. FISH is a faster method for BCR-ABL1 detection, but it suffers from human observer bias.8The biology of pediatric CML to BP progression is still unknown; however, it is thought to be comparable to that of adults. Because of its inherent tyrosine kinase activity, the Philadelphia (Ph1) chromosome and the resultant BCR-ABL1 fusion gene induce granulocytic as well as blast proliferation in leukemic stem cells.9 “AML with BCR-ABL1” has been included as a provisional entity in the updated World Health Organization (WHO) 2016 classification of myeloid malignancies.10 Since no precise criteria have been developed, it can be challenging to distinguish between “de novo AML with BCR-ABL1” and “myeloid CML-BP” in many circumstances.9 In the present case, splenomegaly and basophilia favored a diagnosis of “CML-BP ” over ‘AML with BCR-ABL1’. Moreover, deletions of IKZF1 and CDKN2A have been reported in AML with BCR-ABL1 and seem to be absent in the myeloid blast crisis of CML.9 These could help establish the diagnosis in complex cases.About 0.5-3% of all AML cases also have the BCR-ABL1 fusion gene.9 CML-BP is treated by tyrosine kinase inhibitor followed by an early allogeneic stem cell transplant, whereas AML with BCR-ABL1 is treated with intense induction chemotherapy.9 The outcome with current tyrosine kinase inhibitors is often poor, and survival after presentation with CML-BP is generally less than 1 year. The present patient died after 17 days of initiating therapy due to febrile neutropenia.It is of paramount importance to differentiate CML-BP from AML with BCR-ABL1 as these have different treatment modalities.CML-BP is a rare presentation in the pediatric demographic mimicking more common diseases in that age group, including AML. Physicians should keep a high index of suspicion as treatment, as well as prognosis for CML, is distinct from these other diseases.


In this study, we aimed to implement and evaluate a Web-based partnership support program to enhance the QoL of male patients undergoing infertility treatment. We conducted a pilot study involving 41 infertile couples from September to October of 2021. We used a quasi-experimental design (pre-test and post-test with comparison) involving purposive sampling. A subgroup analysis was conducted to determine which demographics of the participants would benefit from the program.P = 0.03), the no medical history group (P = 0.032), and the with experience of changing hospital group (P = 0.027) showed a significant increase in the relational subscale scores of the QoL before and after the program. The majority of the participants  expressed satisfaction with the support program. Participation in the Web-based partnership support program may improve the QoL of some men undergoing infertility treatment.Thirty-four participants  were included in the final analysis (follow-up rate 82.9%). Although there was no significant increase in the participants’ QoL under the Web-based partnership support program, the assisted reproductive technology group . Most developed countries are currently facing serious social problems owing to its declining birth rate and aging population –3. In coTemporary social distance and closure of emergency medical services have been advocated to mitigate the adverse effects of the COVID-19 pandemic , 23. As α) = 0.05, and power (1-β) = 0.8. Before the study, the participants were informed both verbally and in writing about the research goals and the confidentiality of any disclosed information. Subsequently, written informed consent was obtained from all the participants.The present non-randomized controlled trial was conducted among 41 infertile couples in Japan from September to October of 2021 at a leading fertility clinic in Japan. We used a quasi-experimental design for the pre-test and the post-test with comparison using purposive sampling. Potential participants were couples referred to the clinic for infertility treatment. The inclusion criteria were as follows: (i) must be within 3 years of the infertility treatment, (ii) have an online communication environment using a personal computer or smartphone at their home, and (iii) participation in the program as a couple. The exclusion criterion was the presence of any sexual dysfunctions to avoid additional mental burden. The sample size was calculated to be 35 participants using the software G*Power 3.1.9.7 with a one group nonparametric test, effect size d = 0.5, significance level   was usedThe Japanese version of the distress scale developed by Asazawa and Mori (2015)  was usedP-value of < 0.05 indicated a statistically significant difference. The open-ended remarks were analyzed using constant comparative analysis.Data were analyzed using SPSS software (version 26.0). A pre-test and post-test comparison was carried out using a non-parametric test as the scales were not normally distributed according to the Shapiro–Wilk test. Pre-intervention and post-intervention changes in the participants were analyzed using the Wilcoxon signed-rank test. A subgroup analysis was conducted by attribute to determine which demographics of the participants would benefit from the program. A frequency distribution table was created from the 5 items of the process evaluation. A P = 0.03). The analyses were divided into 2 groups: with a medical history group and with no medical history group. In the with no medical history group, the post-test relational subscale score  was significantly higher than the pre-test relational subscale score . The analyses were divided into a group with experience of changing hospital and a group without experience of changing hospital. In the group with experience of changing hospital, the post-test relational subscale score  was significantly higher than the pre-test relational subscale score  . Based on the test results, there were no significant differences in the pre-test and post-test scores in the 2 scales   comprehended the program, 29 (85.3%) of whom were satisfied with the treatment methods, 31 (91.2%) were positive regarding program availability, 20 (58.8%) indicated a high match between their expectations and the implementation, and 32 (94.2%) felt the ease of viewing the site (Fig. This study demonstrated that the male patients undergoing infertility treatment in the ART with no medical history group and with experience in changing hospital group showed improvement in the relational subscale of QoL. Additionally, the high satisfaction for the program and the availability of the program were judged to be beneficial from the standpoint of the care recipient. However, this program for men undergoing infertility treatment showed no significant effect on QoL and distress. This indicates that participation in the program had modest beneficial effects. These findings should be considered as incremental benefits at this point and can be used as inclusion/exclusion criteria for the next study.The 2 factors underlying the lack of a significant effect of the program on QoL and distress were the wide range of selection criteria for participants and the lack of individual support. Patients with male factor and those on ART treatment reportedly have a lower QoL than other patients . ParticiThe responses of the participants indicated a high level of satisfaction for the program, as well as the availability and easy comprehension of the program. The participants also felt the ease of viewing the sites. Online psychoeducational support was reported to reduce stress and increase self-efficacy of women , 33. OurWe developed a Web-based partnership support program for men undergoing fertility treatment. Although this program showed no significant changes in the QoL and distress, the participants in the ART treatment group, with no medical history group, and with experience of changing hospital group showed a significant increase in the relational subscale of QoL. Moreover, most of the participants were satisfied with the content of the program, felt that some of the contents can be improved and used in the future, and acknowledged the need for such a Web-based intervention program. A further enhanced program modified based on the needs identified and participants’ feedback would provide cost-effective and beneficial support to men undergoing fertility treatment and the couple.As the couples were recruited from a high-level infertility treatment facility which was capable of addressing both male and female factors, the couples’ background may be different from that of the average Japanese couples. There is insufficient confirmation whether the couples have engaged in a helpful discussion.


The prevalence of obesity is increasing among men, and this population remains under-represented in lifestyle and weight management interventions. The current study aims to explore the experiences of men living with obesity (body fat ≥25 %) toward a 12-week supervised online exercise platform. Ten men were interviewed for this qualitative study. Semi-structured, open-ended phone interviews were conducted, and the transcripts were thematically coded using the qualitative data analysis Nvivo QSR software package. The research findings are illustrated using quotes from participants. The results were organized into two main themes: those that removed barriers to exercise and those that improved the enablers of exercise. Eliminating barriers included not purchasing specialized equipment or travelling to a gym facility. The enablers to their success with the program included the structured format of the circuit program and having supervised sessions. By removing barriers and enhancing enablers, the 12-week online exercise circuit program increased compliance to and success of the exercise program for men living with obesity. Future research should explore the long-term effects of an online program for men living with obesity and its appeal beyond COVID-19. For exaDespite the well-documented benefits associated with exercise , only abThe prevalence of obesity is high for both men and women and is rising particularly among men, along with more chronic health conditions . HoweverCOVID-19 forced people to engage in various online exercise programs that they may not have normally ,22. ThisThere is currently a lack of studies targeting males living with obesity and their personal experiences with an online exercise program . In the 2ClinicalTrials.gov (NCT04679090), which involved recruiting 60 men with obesity (body fat ≥25 %) for a 12-week intervention. Participants were eligible if they 1) characterized themselves as men; 2) 19 years or older, 3) living with obesity defined as a body fat % equal or above 25 kg/m2 , and, you know, get in your gym clothes and be there at 10 o'clock or something. That might be a bit more difficult to actually undertake, rather than having it available in your house.One of the interview questions asked if any of the men had ever participated in an online exercise program. All ten participants indicated they had never utilized an online exercise program before and that our study was advertised as exclusively online was an incentive to join. Shawn said that travelling to the gym would have been a barrier for him.If I had to travel to the [local gym] if I had to travel to anywhere to be in person, that probably would have become a barrier. I think the online thing was actually a benefit regardless of the state of the world; the online supervision and doing this at home was an absolute thumbs up from me.3.5Shawn also brought up an interesting point about being self-conscious and having to exercise in a gym and in front of or with others. Similar to Ref.  reportedBeing able to do it in my house, away from others, I may have been self-conscious about doing this in front of people, but online; nobody's looking at you. So, there's no, you know, there's no real scrutiny or judgement from others, or, you know, you're basically on your own, in a safe place without any barriers, from equipment to having to travel or anything like that, it's basically you just need to go into the room and do it. There's nothing really more to it. So it takes away all the things that somebody could use as an excuse.The male participants in our study had an 89.4% rate of compliance in attending all the sessions of their 12-week online circuit-style program. Each participant commented on the removal of barriers to exercise as a key contributor to their compliance and the overall success of the program. Shawn stated it well when he said;It's relatively easy to do, other than the hour of actually doing the work. It's, you know, it's exercise. You get breathing hard and that, but there's no, there are no other barriers to it. It's just going in the room before supper and do it for an hour. Or you could just sit in the chair and waste that hour away.3.6The second of the two primary themes pertained to enhancing the enabling factors to exercise compliance for our participants. For our male exercisers, having a well-structured program and one that was supervised was important to their success with the program.3.7“this program gave me the structure I was looking for. Because doing it on my own it was, well, ‘I'll do it tomorrow’ kind of thing. Which never happened.” Dane felt that having access to a structured program kept him more accountable than when he was left to work out on his own. He also liked the inclusive nature of the program; he continued by saying, "it's all you put into it is what you're going to get out of it. But that's what's good about the program is that everybody can participate in the class."The online circuit program was structured around four prescribed exercises  for 45 s each, then switched immediately (15 s) to the next exercise, followed by 1 min of rest at the end of each circuit. The circuit is repeated until the session is complete. For the men in our study, a structured program was one with a set start and end time and a simple program design. Dane said, Maverick also agreed regarding the inclusivity of the program.I always found that circuit training, you know, appeals to the mass because I could be working out with somebody who's much fitter than me, and both of us would get something out of the workout. So, I think that's what appealed to me.3.8The program was designed around functional strength exercises that were easy to execute in a small space with limited equipment. Beau, a 45-year-old engineer, had extensive past exercise experiences that indicated he was quite knowledgeable about creating his fitness routines but conceded that as he aged, his life became increasingly more complicated. He welcomed the opportunity to be supervised through a program he didn't create.I'm comfortable that I could design my programs, but it, it's nice to kind of have it handed to you or set up and scheduled for you.Sam, a 22-year-old full-time university student, also praised the design of the program and the benefits it gave him.It was good because it wasn't extremely long … it was only an hour or less. It was perfect for a tight schedule. It was good because it was easy to exercise and easy to follow, with just 10–15 s of rest in between. I felt how my heart rate increased, and I was sweating. I was losing weight. It was amazing.Aidan found both challenge and success with the program's repetitiveness and duration.It [the program] had a certain amount of repetitiveness to it … I think everybody was aware that it would get potentially repetitive. I thought it was appropriate. And I thought it was it was well-balanced. And it was challenging enough … because the duration of it is what made it especially challenging.3.9The participants had to schedule their workouts a week in advance to ensure a trainer would be available to them on the online platform. Matiss, a 33-year-old professor, stated that the trainers expecting him to attend his required sessions provided the right amount of motivation for him.There was a scheduled time, and I promised somebody I would be there. And they were expecting me. And, like, I was usually the only one that chose the eight o'clock in the morning one. So, I was the only participant, and if I didn't show up, then [the trainer] would be just sitting there. So, I felt bad about that. I liked knowing that someone was expecting me.Dane concurred that structured time was paramount for his continued attendance and completion of the program.I found that the structure was better because it was at a specific time, and I just made time to do that. But what I find with, like right now, like the other night, seven o'clock came, I was busy. So, well, ‘I'll do it later’ and you know, eight-thirty came. Okay, well, that was too late to do it. So, I ended up doing it the next morning. But you start to slide that way easily. With a structured one, you know when your time was, and it is easier to maintain. (Dane).3.10“when this [online program] came up, I said, 'that's what I'm looking for. I'm looking for something structured,’ because I'm trying to do stuff on my own and it wasn't working for me.” By structured, Maverick is referring to the scheduled times he had to commit to for his supervised exercise sessions.At the beginning of the intervention, each participant was required to declare their supervised workout sessions for the upcoming week, thus ensuring they got their circuit times booked and a trainer would be available for the allotted time on the online platform. Maverick, a 58-year-old retired military member, stated that For Dane, having the supervised portions of the program was the key to his success and his ability to create new habits.For me, that's exactly what I was looking for and it was really good. What I did like about it was the supervised parts. Because they were supervised, I did them three times a week and then after a while, I went down two times a week and one time a week, I missed the supervised portions of it. And I still did it, like three times a week and everything and I still do, but I missed the supervised portions of it.3.11“once I've committed to something, then I tend to stay at it. And it really helps that it's a group and that it is supervised.” In fact, Aidan, a 38-year-old project manager, found it increasingly difficult to complete the workouts on his own after the supervised sessions had ended.The participants expressed feeling accountable for showing up to their scheduled sessions because the trainers expected them to be there. Beau, a 44-year-old engineer, concurred that the commitment to the supervised portions was critical to his 100% attendance record by saying, The two supervised sessions was still fine, but the one supervised session, I started slipping a bit, and since the no-supervised sessions hit, I've slipped quite a bit for most of the summer. Like I am doing none of the program now. (Aidan).Rowen echoed similar sentiments that his compliance with the program dropped off after he was no longer accountable to anyone external.Those first few weeks were very good because, you know, it was three times a week … and I found myself starting to slip towards the end of the project because I wasn't accountable to anyone; it was just kind of expected that I would continue doing it and I didn't. (Rowen).3.12“Oh, it's good; I like the direction. If I'm not doing a particular exercise correctly, he'll [the trainer] tell me to bend a little lower, push that right knee lower. It was motivational and helpful."The participants talked about the trainers' adding motivation to their workouts by providing technique corrections and other encouraging cues. When asked what Jake thought of the trainers, he said, Tristan, a 32-year-old social worker, also liked the attention and support offered by the online trainers.I think because you had someone there counting down the timer, right? So that meant not having to do the extra paying attention to the time. Also, having someone there to talk to you through some exercises and be encouraging. So that was a bit of support to me.Beau summed up his thoughts on what the trainers brought to the online format and that although each participant worked out in their own home, the trainers made it feel like a group atmosphere.You still felt part of a group, and it was a group because there was a trainer there running your timings telling you when to start and stop, correcting you on things and not just correcting but boosting your morale along the way, too. And the feedback was, I found, really helpful.4The results from our interviews suggest that by removing the barriers and enhancing the enablers to exercise, it is possible for men living with obesity to succeed with an online exercise program specifically designed for them. This study also suggests that exercise programs specifically targeting men would also interest men living with obesity. Our study identified that barriers to participation are cost and access to the program, whereas the enablers are the structured and supervised sessions of the program. Our results add to the current literature on men living with obesity, as not enough is known about the barriers and the enablers to success with online exercise programs for these men.Our study identified novel ways to connect with men living with obesity who wanted to participate in an exercise program. Specifically, using an uncomplicated circuit-style program that required no equipment to purchase and very little space to execute the exercises was seen as desirable for these men. This format made it highly accessible to our participants. Jones and Nies (1996) also reported that increasing access to exercise increased compliance with an exercise program in their study of older African-American women .Men living with obesity in our study preferred to exercise in non-judgemental spaces. The notion of stigma-free zones was vital to help prevent obese individuals from feeling “uncomfortable” and “self-conscious” when engaging in activity . For our47.Our study also identified enhancing the enablers to exercise was critical for engagement with an exercise program. It was consistent with Elliot et al. (2020) previous research on the experiences of men living with obesity accessing weight loss services. A key finding from Elliot et al. (2020) is that men often see weight loss services as feminized spaces; thus, tailoring the program specifically to men is critical in men's adoption of the exercise intervention. Our findings echo the need for male-targeted health promotion and exercise programs similar to Coles et al. (2010) reported in the UK  also indFinally, our results also suggest that men living with obesity are motivated to continue with online exercise programs if they are externally accountable to others, specifically a motivating trainer who organizes the sessions. Strong support suggests that providing continuous sessions will further ensure greater and longer compliance with the exercise program.5Although the current study provides insight into removing barriers and enhancing the enablers to increase adherence to regular exercise in men living with obesity, some limitations need to be acknowledged. First, the initial sample size was calculated based on the study's main outcome, and therefore, the sample size for the qualitative portion was affected. We must also acknowledge the limitations in generalizability to other populations, such as women living with obesity or individuals facing chronic conditions associated with obesity. It is also important to note that participants had to live in the surroundings of the laboratory for baseline testing, needed internet access at home, be technology literate, and the space to exercise. Considering the study conducted during the COVID-19 pandemic, we acknowledge the research's unique circumstances. There might have been differences in outcomes if the study was conducted outside of the pandemic period. The urgency and health concerns associated with the COVID-19 pandemic may have motivated participants differently than in non-pandemic times. Participants may have been more committed to the program offered online during the pandemic due to the uncertainty and isolation it brought. Also, the pandemic introduced significant stressors into daily life. Participants may have viewed the online exercise program as a coping strategy during this stressful period.6We explored the experiences of ten men living with obesity who participated in a 12-week online exercise circuit program. Our study suggests that removing barriers and enhancing the enablers increased compliance to the exercise program for men living with obesity. Given that there is limited evidence to support interventions that lead to long-term sustained change in health and behaviour regarding obesity, future research would benefit from an empirical examination of the results of continued supervised sessions over a longer time frame if that intervention leads to physiological benefits, and if the intervention would attract as much interest outside of the COVID-19 period, or for women living with obesity. This study is important because it offers facilitators to implement a specific online exercise program suited for males that leads to adherence to the current physical activity guidelines.10.13039/100004411Heart and Stroke Foundation of New Brunswick funded this project.The The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Regular physical activity (PA) is a key lifestyle component for hypertension prevention. Previous studies have shown that mobile health  apps can be an effective tool for improving PA behaviors. However, adherence to and poor engagement with these apps is a challenge. A potential solution to overcome this challenge may be to combine financial incentives with innovative behavior theory, such as the Multiprocess Action Control (M-PAC) framework. Currently, there is a lack of PA financial incentive–driven M-PAC mHealth programs aimed at hypertension prevention.We aimed to describe the process of developing an 8-week mHealth PA and financial-incentive hypertension education program  and to evaluate usability of the Healthy Hearts program.The first 2 stages of the Integrate, Design, Assess, and Share framework were used to guide the development of the Healthy Hearts program. The development process consisted of 2 phases. In phase 1, the research team met to discuss implementing the M-PAC framework to adopt an existing web-based hypertension prevention program to a mobile app. The app was developed using a no-code app development platform, Pathverse (Pathverse Inc), to help decrease overall development time. In phase 2, we created a prototype and conducted usability testing to evaluate lesson 1 of the Healthy Hearts program to further enhance the user experience. We used semistructured interviews and the mHealth App Usability Questionnaire to evaluate program acceptability and usability.Intervention development among the research team successfully created an 8-week financial-incentive hypertension education program for adults aged 40-65 years who did not currently meet the Canadian Physical Activity Guidelines (<150 minutes of moderate to vigorous PA per week). This program lasted 8 weeks and comprised 25 lessons guided by the M-PAC framework. The program used various behavior change techniques to further support PA adherence. Usability testing of the first lesson was successful, with 6 participants recruited for 2 rounds of testing. Feedback was gathered to enhance the content, layout, and design of the Healthy Hearts program to prepare the mHealth program for feasibility testing. Results of round 1 of usability testing suggested that the content delivered in the lessons was long. Therefore, the content was divided into multiple lessons before round 2 of usability testing, where feedback was only on design preferences. A minimum viable product was created with these results.The iterative development process and the usability assessments suggested by the Integrate, Design, Assess, and Share framework enabled participants to provide valuable feedback on the content, design, and layout of the program before advancing to feasibility testing. Furthermore, the use of the “no-code” app development tool enabled our team to rapidly make changes to the app based on user feedback during the iterative design process. Hypertension is the leading risk factor for cardiovascular disease and death in the world . The preRegular physical activity (PA) is a key lifestyle factor to reduce resting blood pressure and prevent hypertension . HoweverWith the recent technological advancements and the adoption of smartphones, mobile health  apps have shown the potential to deliver scalable and personalized PA programs ,10. HoweFinancial incentives are a BCT that may assist in adding to the instrumental benefits of PA participation, as health benefits have been criticized as being too distal to an outcome to motivate short-term PA participation -20. ThusUsability testing is essential for app development to ensure success . To effiThus, the objectives of this study were to (1) describe the process of developing an 8-week self-guided PA financial-incentive hypertension program  using the IDEAS framework and (2) evaluate the usability of lesson 1 of the intervention. We hypothesized that the first lesson of the Healthy Hearts program would be usable and acceptable as it was based on a previous web-based intervention . FollowiThis study used the first 2 stages of the IDEAS framework  to guideEthics approval for this study was obtained from the Human Research Ethics Board at the University of Victoria (protocol number 17-361).All participants provided written informed consent. Each participant was given a unique identifier to maintain anonymity for data analysis. The participants were informed that they would receive a CAD $20 (US $14.57) Amazon gift card for their time.The first step in intervention planning and development was to address the needs of the target user. As the research team adapted an existing web-based 10-week hypertension management program  and prioThe second step of the integrate stage is to specify the target behavior to be used in the intervention. To do this, the research team reviewed the current public health guidelines ,34 and cThe final step in the integrate stage of the IDEAS framework is to ground the intervention in behavioral theory. The M-PAC framework was selected as it recognizes 3 stages of the formation of motivation to conduct PA while subsequently addressing the BCTs that assist in closing the gap between intention and behavior. An additional behavior strategy identified by the research team incorporated financial incentives into the M-PAC intervention. Furthermore, the research team explored evidence on past financial-incentive PA interventions and the type of PA to incentivize  to complement the M-PAC framework as a means of increasing the outcome expectation of performance ,36. WithThe fourth step of the IDEAS framework, in the design phase, is to ideate implementation strategies. Specifically, we drew on information from various sources, conducted iterative brainstorming sessions to determine the intervention dose, and focused these ideas on app feature implementation and user experience . The rescards in lessons, which contained educational material, surveys, and quizzes [The fifth step in the design phase is to prototype potential products. To do so, the research team used the Pathverse program. This program enabled the research team to create an 8-week program that consisted of a combination of  quizzes . FurtherUsability testing of lesson 1 was intended to determine how the target user interacts with a mobile-based program and whether the program meets its intended purpose. We used a mixed methods approach to gain an in-depth analysis of the ease of use and satisfaction and the system information arrangement of the Healthy Hearts program . OverallParticipants were recruited through paid Facebook posts on the Digital Health Lab at the University of Victoria’s Facebook page. The advertisements ran from January to March 2021. The advertisements were targeted at the intended inclusion criteria of Canadian adults aged 40 to 65 years. Participants were then screened to confirm that they met other inclusion criteria, including access to a smartphone (Apple or Android) with internet, and to confirm that they did not meet the Canadian Physical Activity Guidelines of 150 minutes of MVPA per week . As physIn total, 2 rounds of usability testing were conducted over video chat owing to the COVID-19 pandemic. Individuals were informed that they were allowed to use a nickname or a substitute name and that having their camera on was optional. The individual was informed that this meeting would take approximately 45 minutes and that they must take this meeting from their smartphone, and the individual was asked to download the mobile app, Pathverse, in advance. The meeting was recorded via Zoom local recording along with notetaking by the interviewer throughout the meeting. Audio files were saved to the University of Victoria’s Digital Health Lab secure drive once downloaded following the meeting. Participants were asked to share their phone screen during the video call, so the researchers could follow along with the experience in real time.Participants were asked to complete a series of goal-oriented tasks to gather user feedback. These tasks included app log-in, completing a lesson, connecting a fitness device to the app’s self-monitoring tools, and setting a personal goal on the app. Participants were asked to “think aloud” as they completed the tasks to further capture their ongoing thought processes while they are using the program as well as any difficulties they may experience . The parThe second task was to complete the first lesson of the Healthy Hearts program. This lesson addressed the content that would be covered in the 8-week PA program, including earning a financial incentive, and introduced the benefits of PA for overall heart health. This lesson also incorporated both survey and quiz cards so that the user could see the full potential of a lesson on Pathverse. After completing this lesson, individuals were invited to scroll through all lessons offered in the Healthy Hearts program and were invited to share feedback on lesson photos and names. The next task was to access the fitness page of the program and connect the individual’s phone to display selected fitness data. At the time of testing, only Apple iOS devices were able to display their fitness data. The final task was to go to the “Goal Setting” page and set a goal. After the participant completed the goal-oriented tasks, a semistructured interview was conducted to direct open-ended feedback and further understand the thoughts and emotions of the participants. Question 1. Can you tell me what you liked best about the app?Personalization: all participants who tested the personalized program (n=4) appreciated an introduction of the program creator.Question 2. Can you tell me what you liked least about the app?Display: Pathverse was not yet optimized to as many phones, including those with larger font sizes, so words were often cut out.Question 3. Can you tell me about how easy it was to navigate or our find your way around the app?Lesson progression: many participants (n=4) were not under the assumption to swipe left to progress through the lesson.Page access: while accessing 3 pages on the app was well responded to, the symbols representing different pages  on the menu bar were not clear.Question 4. Can you tell me about what you thought about the overall look of the app?Design: participants all liked the simple visual of the app, including layout and fonts.Question 5. Can you tell me what you thought about the information provided on the app?Heart health: one participant stated that the content was “very clear” and “didn’t use jargon.”Financial incentive: all participants were intrigued by a chance to earn money while simultaneously using the app.Following the semistructured interview, the participant was asked to complete the mHealth App Usability Questionnaire (MAUQ)  to quantThe end goal of step 7 was to create a functional smartphone app to be used for subsequent feasibility testing. Modifications recommended from step 6 were repeatedly reviewed to ensure accuracy and adherence. Functionality was repeatedly tested by our internal research team, and any technical issues were reported to the Pathverse development team. Any content issues  were reported to 1 researcher (AW) who made final changes to the program. In addition, during this step, the research team ensured that the Pathverse program could adequately support multiple research participants and that engagement data could be accurately downloaded from the Pathverse program.The 6-step process of thematic analysis was followed to analyze transcripts and conduct a meaningful evaluation of the qualitative data  collecteAs determined from the meetings with the research team, the mobile-based program will consist of 8 weeks of content structured around the M-PAC framework. The name of the program was chosen to be “Healthy Hearts.” The goal of the Healthy Hearts program was to prevent hypertension in Canadian adults (aged 40-65 years) who were not currently meeting the PA guidelines (150 minutes of MVPA per week). MVPA minutes were selected as the PA metric, as they are a better gauge of the intensity of the activity completed  and are The adapted program consisted of 25 lessons developed based on the M-PAC framework ,17. The In this phase, we additionally proposed 2 financial-incentive interventions to accompany the Healthy Hearts app. At the time of writing, the conversion rate was CAD $1 (US $0.73). The first financial-incentive design was a pay-per-minute payout, where participants were rewarded CAD $0.02 for each minute of MVPA tracked through the Fitbit (Fitbit Inc), up to CAD $2.50 per week, and CAD $20 for all 8 weeks. The second financial-incentive design was structured as a self-funded investment incentive where participants commit to invest CAD $400 into their health for the duration of the 8-week program. Participants received a percentage of return on this initial investment based on the number of weeks they successfully met the Canadian Physical Activity Guidelines, as recorded by their Fitbit. The maximum return on investment was 5% or CAD $20, and this was rewarded if the participant met the goal for all 8 weeks of the intervention, similar to the pay-per-minute arm. A 5% return rate of investment was chosen as the maximum to align with the annualized Standard and Poor's 500 stock based on the last 50 years . ConsensTo address implementation strategies for the mobile-based app, the research team iteratively designed various working prototypes to assess the content and layout of the mobile app. During this iterative process, we considered the features, user interaction, and experiences and combined our unique experiences before usability testing. When the research team assessed what we considered to be an adequate layout , we determined that the product was ready for user feedback.The first lesson was created as an overview of the program and titled “Getting Started.” This lesson included instructions on weekly topics, PA guidelines, information about the financial incentive, and how to use their Fitbit. Of the 11 potential participants contacted, 6 (55%) individuals were recruited. The participant sample was 67% (4/6) female, with half of the participants (n=3) being within the age range of 50 to 59 years and all participants being White. All participants had at least 5 years of experience using a smartphone and were experienced in using their smartphone for internet browsing. In total, 2 rounds of acceptability and usability tests were conducted. Comments made by participants 001 and 002 were from the first round of acceptability and usability testing, and comments from participants 003 through to 006 were from round 2 of testing. The acceptability and usability results from both rounds of testing were cumulated and summarized below.Acceptability questions and feedback determined how the target population used the app and the content they read in their daily life. Findings for the acceptability of the Healthy Hearts program on the Pathverse app were relatively consistent among participants. It was immediately clear after 2 participants completed the first round of usability testing (n=2) that the introductory lesson was too long. The lesson initially consisted of 28 cards , and participants 001 and 002 both commented on “losing interest near the end.” This resulted in dividing up the introductory lessons into 2 separate lessons for round 2 of usability testing. From both rounds of testing, 67% (4/6) of the participants “agreed” (score=2) with the following statements: “This app will be useful for my health and well-being,” “I enjoyed reading the content in the lessons,” “I enjoyed using the interactive tools in each lesson,” and “I found the content in each lesson useful.” These statements, along with other acceptability statements that were used in the quantitative analysis, are listed in There were mixed feelings regarding the self-monitoring fitness page on Pathverse. Overall, 50% (3/6) of participants commented that the star icon to indicate the fitness page “doesn’t mean fitness to me,” as participant 006 stated. On the fitness page, steps were displayed in an accumulation of the past 3 days, the past week, and the past month, a display that participant 002 said, “is simple” and “a good illustration for all abilities.” Participant 004 shared this sentiment, stating “steps is really common and good.” The simplicity of this page was not as positively received, with participant 006 stating “the visual doesn’t mean anything to me.” When probed about what they would like to see displayed on the fitness page, participant 006 stated they “value the distance” and would like to see “steps converted into kilometers.” Participant 004 would “like to see stairs.”Similar to the fitness page, the icon that depicted the Goal Setting page (a flag) “did not mean goals” to participant 006. All participants  commented on the location of the “Add a Goal” button on the page. Participant 005 mentioned that the button “is a little bit hidden away” and “kind of got lost” in the header image. Furthermore, “more pathway to setting a goal” was a suggestion from participant 004, and participant 003 suggested addressing “when goal setting will be taught in the program” so the user is aware. Ease of use and satisfaction examined the extent to which the app was an effective, efficient, and satisfactory platform for delivering a heart health education program to those at risk of developing hypertension. All data under the theme “usability” and positive-coded qualitative data were analyzed to supplement the quantitative scores from the MAUQ_Ease of use and satisfaction. Quantitative data analysis supplemented the qualitative data, with most “agreeing” (score=2) or “strongly agreeing” (score=1) with the items in this category. The statement “The amount of time involved in using this app has been fitting for me” was the most agreeable statement, with all participants (n=6) at least “agreeing” with the statement. The same results were obtained for the statement “I feel comfortable using this app on my own” with all participants (n=6) at least “agreeing” with the statement. One participant, participant 003, exclaimed, “Wow, I am impressed with how user friendly this is!” A list of means and SDs for all statements in MAUQ_Ease of use and satisfaction is reported in The system information arrangement referred to the user interface of the mobile app. Questions and comments relating to the navigation, content and feature arrangement, and general interaction with the Pathverse platform were analyzed under this category. The statement “The navigation was consistent when moving between screens” from the MAUQ_System information arrangement was the most agreed upon statement with 83% (5/6) of participants “agreeing” (score=2) with the statement. A suggestion from participant 005 was to “include a card or a tutorial to explain what each piece of the app does.”One participant “disagreed” (score=4) with the statement “Whenever I made a mistake using the app, I could recover quickly.” This participant brought forward that their fitness data were not displayed, and they were left with “no direction on how to fix it.” The issue has since been resolved. Means and SDs from the MAUQ_System information arrangement statements are presented in When participants were asked about the layout of the home page and the lesson details, comments were overall positive. All participants  were able to switch tabs from “Current Lessons” to “All Lessons” without difficulty. Comments relating to the lessons, including the lesson name, photo, and tags included in the lesson, participant 001 commented, “I like the pictures that have nature in them, it makes me want to go outside.” Participant 003 had the same sentiment and added that nature photos and those exercising “showed fitness” and they “liked that to feel inspired.” Half of the participants (n=3) commented on the colors of tags associated with the lesson. The tags at the time were blue, yellow, and red. The yellow tag with white text was “hard to read” according to participant 001. This statement was matched by 2 other participants, and participant 003 suggested “using orange instead of yellow to compliment the blue and make it easier to read.”On the basis of these results, along with the following breakdown of ease of use and satisfaction, system information arrangement, and acceptability, the content and the user interface underwent adjustments to improve (1) accessibility and readability of content and colors, (2) accessibility of the Goal Setting page, and (3) guidance for the Goal Setting page within the lessons. Other key changes included layout and esthetic design of the platform, such as adjusting paragraph spacing, adding images, and hyperlinks to access other health-related information .Through this iterative design process and the collection of user feedback, the research team was able to efficiently develop a minimum viable product to be tested in a feasibility study. The feedback provided by the end user from 2 rounds of usability testing was implemented in the working version of Healthy Hearts. This feedback included optimizing the content and various design changes to the Pathverse platform. Usability testing revealed minimal technical barriers or user interface challenges.This study was designed to highlight the development and usability of the Healthy Hearts mobile-based program. As such, the first objective of this study was to describe the development process of Healthy Hearts, an 8-week PA, and a financial-incentive mobile app grounded in the M-PAC framework. The second objective was to evaluate the usability of the first lesson of Healthy Hearts. The iterative design process used in the first 2 stages of the IDEAS framework involved in the program development promoted the ability to find and make changes quickly when any issues arose. The evaluation of the first lesson of the Healthy Hearts program used a mixed methods approach with quantitative data through an adapted MAUQ, which was described through qualitative semistructured interviews.By following the IDEAS framework  for deveUsability studies that have used only qualitative data typically only present how and why participants make decisions with mobile apps -54 and pTo our knowledge, the Healthy Hearts program is the first financial-incentive hypertension program designed for Canadian adults. The Healthy Hearts program was also grounded in the M-PAC theory to address intention formation and action control . The 2 rThe Healthy Hearts program is the first of its kind to be delivered through the no-code app development platform (Pathverse). This new platform was an efficient tool to adopt a web-based program into a mobile app deliverable and enhance the program with features, including surveys and quizzes. The use of Pathverse enables our team to rapidly prototype and change the design of the mobile app without previous knowledge of software coding. We believe that the use of such “no-code” app development can be useful for future health researchers developing mHealth interventions.Despite the overall positive feedback received from the study, it did not come without its limitations. One of the main limitations of this study was its sample size. However, previous research has shown that samples of 3 to 5 participants for usability testing are adequate ,43. OwinThis study described the development process and evaluated the usability of an 8-week financial-incentive hypertension education program delivered through a mobile app for Canadians aged 40 to 65 years who did not meet the PA guidelines. We used the IDEAS framework to guide the development of the mHealth intervention. The iterative development process and usability assessments helped our team to identify areas of improvement  before feasibility and efficacy evaluations. Furthermore, the use of the “no-code” app development platform, Pathverse, enabled our team to rapidly prototype and apply these changes to the app based on participants’ feedback. The use of the IDEAS framework and the “no-code” app development tool should be considered for future mHealth intervention development by researchers.


PRAME , a cancer-testis antigen expressed in normal and neoplastic tissues with several functions, proved to be a useful diagnostic tool in the differential diagnosis between benign and malignant melanocytic lesions. The current study aims to perform PRAME stain on a retrospective case series of mucosal melanocytic tumors of the head and neck region to compare 3 different scores and evaluate the most reliable one in this diagnostic set. Immunohistochemical analysis for PRAME was performed in 54 benign and malignant mucosal melanocytic tumors of the head and neck region collected from 41 patients. The best-performing cutoff of PRAME-positive cells (nuclear stain) to differentiate benign and malignant mucosal melanocytic tumors of the head and neck region is that proposed by Raghavan and colleagues (<60%/≥60% of PRAME-positive cells), with 100% and 77.8% of benign lesions and malignant tumors respectively correctly identified. Applying this score, PRAME stain showed the best results  for the diagnosis of head and neck melanocytic tumors. However, a subset of PRAME-negative malignant tumors was identified, especially located in the palatal area . Finally, high PRAME expression (≥60%) was associated with specific sites , nodular histotype, and female sex. TERT promoter mutations) and molecular tests (FISH) distinguishing MM-H&N from benign mucosal melanocytic lesions of the head and neck region (MBML-H&N), limits their adoption in this diagnostic field.The diagnosis of mucosal melanoma of the head and neck region (MM-H&N) is challenging due to the peculiar anatomic, histologic, and genetic features of these melanocytic tumors.2, lymphovascular invasion, perineural infiltration, and pT stage. For each MBML-H&N, only the following pathologic features were recorded: type of histologic specimen, site, and histologic diagnosis. All cases were reviewed and diagnosed according to the World Health Organization Blue Book on Classification of Head and Neck Tumours  by a panel of 4 pathologists with specific expertise in melanocytic pathology and/or head and neck pathology ; all MM-H&N were staged according to the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual.All MM-H&N and MBML-H&N excised between 2004 and 2022 were retrieved from the database of 3 pathology units: Bellaria Hospital of Bologna (37 cases), Maggiore Hospital of Bologna (9 cases), and IRCCS Azienda Ospedaliero-Universitaria Policlinico di Sant’Orsola, Bologna (8 cases). The patients were selected according to the following inclusion criteria: (1) primary mucosal melanocytic lesion ; (2) availability of formalin-fixed and paraffin-embedded samples with enough material to perform immunohistochemical analysis; (3) availability of clinical data. By contrast, cases judged as nonprimitive of the mucous membranes, and with no availability of formalin-fixed and paraffin-embedded samples for immunohistochemical analyses and/or clinical data were excluded. For each patient, the following clinical data were recorded: age at first diagnosis and sex. For each MM-H&N sample, the following pathologic features were recorded: type of histologic specimen , site, histologic subtype, pigmentation, prevalent cytotype, ulceration, bone and/or cartilage infiltration, number of mitoses/mmFor each case, a representative slide was selected, and from the corresponding paraffin-embedded tissue block one 3-μm-thick section was cut and stained with Melan A/PRAME  according to the previously described and published protocol.2 test. The statistical tests were performed using the IBM SPSS software, with a P-value <0.05 (2-sided) indicating statistical significance.For each score sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV), and accuracy (AC) were evaluated. The score with the highest values of these parameters has been chosen to dichotomize the cases (low and high expression of PRAME) and analyze the association with the other dichotomous/categorical clinicopathologic features using the χThe study has been approved by the Review Board of the Area Vasta Emilia Centro-AVEC (protocol n. 03-2022-OSS-AUSLBO).A total of 54 histologic samples from 41 patients were collected. Samples consisted of 37 (68.5%) excisions of the primary tumor, 10 (18.5%) excisions of residual tumor/relapse, and 7 (8.5%) incisional biopsies. Twenty-five (61%) patients were females and 16 (39%) were males; the age at diagnosis ranged from 10 to 96 years . Clinicopathologic features of the case series are summarized in Tables Eighteen histologic samples from 18 patients were collected: 13 72.2%) excisions of the primary lesion and 5 27.8%) incisional biopsies. Eleven (61.1%) patients were females and 7 (38.9%) males; the age at diagnosis ranged from 10 to 79 years . The most represented sites were gum  and palate . The cases were diagnosed as follows: 10 (55.6%) melanotic macules, 6 (33.3%) blue nevi, and 2 (11.1%) common nevi. All MBML-H&N resulted negative for nuclear PRAME expression according to the 3 tested scores. Thirteen (72.2%) cases were completely negative, 5 (27.8%) showed focal and weak immunoreactivity in a low percentage of cells (5% to 15%) with a random distribution and no intralesional intensity variation   excisions of the primary tumor, 10 (27.8%) excisions of residual tumor/relapse, and 2 5.5%) incisional biopsies, both followed by the surgical excision of the primary tumor. Fourteen (60.9%) patients were females and 9 (39.1%) males; the age at diagnosis ranged from 29 to 96 years . The most represented sites were the nasal cavity/nasal septum/turbinates  and palate 6, 16.7%). The most frequent histologic subtype was the nodular one , with a high percentage of cases showing ulceration  and prevalent epithelioid cytology . According to the eighth edition of the AJCC Cancer Staging Manual, the pT stages included 18 (78.3%) pT3, 4 (17.4%) pT4a, and 1 (4.3%) pT4b. MM-H&N were characterized by a diffuse (≥60% of neoplastic cells) nuclear expression of PRAME in 28 (77.8%) cases, and with a moderate/intense expression (intensity 2+ and 3+) in 20 (55.6%) . The overview of PRAME results in MM-H&N and MBML-H&N, and the comparison between the 3 different scores are provided in Table All the tested scoresP=0.016) and nodular histologic subtype . Notably, although the mucosal lentiginous histotype showed lower rates of positivity, PRAME highlighted the intraepithelial component/growth, so being potentially useful for the appropriate evaluation of the mucosal resection margins , as follows: MM-H&N of the nasal cavity/nasal septum/turbinates showed high expression of PRAME in 20/22 (90.9%) cases, whereas MM-H&N of the palate in 0/6 (0%). Albeit affected by much fewer cases, all the cases of the nasopharynx  and the maxillary sinus  exhibited high expression of PRAME. No statistically significant association was found between high expression of PRAME and the other clinicopathologic features   was chosen to dichotomize MM-H&N cases in high (≥60%) and low (<60%) expression, and analyze the association of PRAME expression with the other dichotomous/categorical clinicopathologic features. Accordingly, high expression of PRAME was associated with female sex . By contrast, the subsequent surgical excisions of both patients turned out positive with all 3 scores. This finding suggests that, due to the well-known heterogeneity of PRAME expression and the little amount of tissue often obtained with incisional biopsies, PRAME could be more appropriately scored in the incisional biopsies by adopting a lower threshold of PRAME-positive cells. Notably, all 5 incisional biopsies of MBML-H&N resulted negative with all 3 tested scores. Based on the present findings, it is proposed that positive PRAME stain strongly encourages a diagnosis of MM-H&N, since we did not find MBML-H&N positive for PRAME. By contrast, if the histopathologic features are strongly suggestive for MM-H&N but PRAME stain is negative, as it happens in a not negligible number of MM-H&N and especially in specific sites , the possibility of malignancy should not be discarded. Finally, if the histopathologic features are supportive for MBML-H&N, PRAME stain is expected to be negative and a positive result should lead to reconsidering the possibility of malignancy. An additional benefit of PRAME could be the assessment of the mucosal resection margins, especially in cases of lentiginous MM-H&N. This latter, although with a lower positivity  compared with the nodular one , exhibited an intraepithelial component nicely depicted by Melan A/PRAME , and adopted statistical test . A large amount of evidences emerging from recent literature data suggests that, although MM-H&N are grouped as such unique subgroup for classification purposes, they should be more appropriately considered as a heterogenous family of tumors, with marked differences from clinical, histologic, immunohistochemical, molecular, and pathogenetic sides.BRAF, NRAS, KIT, TERT, GNAQ/GNA11); besides, NRAS and TERT promoter mutation were significantly higher in sinonasal than in oral location. More recently, Chłopek et alBRAF/RAS mutants) and nasal (26/64 [41%] BRAF/RAS mutants) cases. The present results support the existence of differences among MM-H&N arising in different mucosal sites and suggest that PRAME, as a pivot molecule in the biology of melanoma, could be differently involved and expressed in distinct sites.After the introduction of immunohistochemistry for PRAME in the diagnosis of melanocytic lesions, many researchers focused on its application in several diagnostic scenarios  with encouraging but also partially discording results.AME Fig. . Althougtivity 9/6, 56.8% tivity 9/, 56.8% ctivity 9/6, 56.8% tivity 9/, 56.8% c


Mycoplasma pneumoniae infection in children. Serum samples from 165 children with acute community-acquired respiratory tract infections were examined using CLIA, PPA, and IFA, and consistency coefficient, specificity, and sensitivity were analyzed. Compared with the PPA (titer ≥ 1:160), the consistency coefficients of the immunoglobulin(Ig)M-CLIA, immunoglobulin(Ig)G-CLIA and IgM-IFA methods were 93.94%, 75.76%, and 83.64%, respectively. The positive likelihood ratio (PLR) and specificity of IgM-CLIA was 19.40 and 95.49%, respectively. The consistency coefficient of (IgM+IgG)-CLIA and PPA (titer ≥ 1:160) was 89.1%, and the sensitivity and negative predictive value of (IgM+IgG)-CLIA were 96.88% and 98.94%, respectively. CLIA MP-IgM has high concordance with PPA, and its specificity and sensitivity are higher than those of CLIA MP-IgG and IFA MP-IgM, suggesting its better diagnosis of early MP infection. The sensitivity and negative predictive value of CLIA MP (IgM+IgG) were higher than those of PPA or IFA, indicating that it should be considered as a priority in the diagnosis of MP infection.This study aimed to evaluate the diagnostic value of chemiluminescence immunoassay (CLIA), passive particle agglutination (PPA), and indirect immunofluorescence assay (IFA) in detecting  Mycoplasma pneumoniae is one of the most frequently detected pathogens of atypical pneumonia and is responsible for more than 20% of community-acquired pneumonia cases worldwide, especially in children and adolescents.,2 It usually causes respiratory tract infections, even leading to death in severe cases.–5 Early diagnosis is crucial for the immediate treatment of M pneumoniae infection. There are different diagnostic tests, but neither polymerase chain reaction (PCR) nor culture methods can distinguish colonization from symptomatic infections.,7 Serologic assays such as passive particle agglutination (PPA) and immunofluorescence assay (IFA) for detecting serum M pneumoniae-immunoglobulin (Ig) antibodies are still the main laboratory methods for the clinical diagnosis of M pneumoniae infection. Currently, no single test can accurately identify M pneumoniae infection, and the combination of multiple detection methods is the most reliable approach,,9 which can not only improve diagnostic specificity and sensitivity, but also reduce false-negative and false-positive rates. Serology tests are basic and important tools for the diagnosis of M pneumoniae infection. Chemiluminescence immunoassay (CLIA), a convenient operation and highly sensitive, has also been widely used for serological testing of M pneumoniae infections in recent years. In this retrospective study, 165 blood samples were collected to detect M pneumoniae infection using PPA, IFA, and CLIA, which were compared in their clinical application value for finding the most helpful method in the diagnosis and treatment of M pneumoniae infection. 3 cases of bronchiolitis, 76 cases of bronchopneumonia, 10 cases of pneumonia and 16 cases of upper respiratory tract infection, including 15 outpatients and 150 inpatients who were admitted to Qilu Hospital (Qingdao) from March 1, 2019 to April 20, 2019. The range of patients was less than 14 years . Children with fever, cough, and/or radiologically confirmed respiratory tract infection were included in the study. The children with immunosuppressive illness, bronchial asthma, noninfectious pulmonary disease or who have had respiratory tract infections within 3 months were excluded. This study was approved by the Ethics Committee of Shandong University Qilu Hospital .The study population consisted of 165 children  with fever, cough, and/or radiologically confirmed 60 cases of bronchitis,Blood sample in all children were collected for laboratory test including the PPA test, Chemiluminescent immunoassay and Immunofluorescence assay.Blood samples were collected from 1 day after the onset of the disease at the earliest and 30 days after the onset of the disease at the latest, with an average of 5.10 ± 4.21 days. Serum was separated by centrifugation following clot retraction and tested immediately. Samples were stored at 4°C for less than 48 hours if they were not detected on time.M pneumoniae antibodies, and the antigen labeled with manufactured artificial gelatin particles was the cell membrane component of M pneumoniae (Mac strain). Serum was diluted to a final reaction concentration of 1:40 to 1:20,480, and unsensitized particles were added to the control well with a final dilution of 1:20. The results were recorded after 3 hours. A definite compact button in the center of the well with a smooth round outer margin was read as a negative result, and a definite large ring with firmly agglutinated particles spread within the ring was read as a positive result. A titer of ≥1:40 was regarded as positive for M pneumoniae antibodies, and ≥1:160 was regarded as a recent or acute M. pneumoniae infection of M pneumoniae.The PPA test  was used to detect M. M pneumoniae antibodies were detected using 2-step direct chemiluminescence technique on YHLO iFlash 3000-A CLIA analyzer  according to the manufacturer’s instructions. IgG > 36 AU/mL and IgM > 1.1 Coi were judged as positive.Serum IgM and IgG specific M pneumoniae-specific serum IgM was performed using the indirect immunofluorescence method , according to the manufacturer’s instructions. Fluorescence was observed under a fluorescence microscope, M pneumoniae antigen on the biofilm combined with M pneumoniae IgM in the serum; net-like bright fluorescence was judged as positive. All samples were detected by experienced technicians.Serological testing for P ≤ .05. The proportions were compared using the χ2 test. The kappa coefficient was calculated to compare the consistency of different methods. Kappa ≥ 0.75 indicates good consistency, 0.75 > kappa ≥ 0.4 for medium consistency, and kappa < 0.4 for poor consistency. Spearman’s tests were used to determine the correlation between age and positive rates of the 3 methods. Rs > 0 indicated a positive correlation, Rs < 0 indicated a negative correlation, and Rs = 0 indicated no correlation. |Rs| between 0.8–1.0 indicates a high strong correlation, 0.6–0.8 for strong correlation, 0.4–0.6 for moderate correlation, 0.2–0.4 for weak correlation, and 0.0–0.2 for no correlation. The higher Likelihood ratio indicate the accuracy of the diagnosis and LR+ >10 usually can be used for the diagnosis.Statistical analyses were performed using SPSS 20.0 version . Quantitative data are presented as the mean ± SD and qualitative data are presented as percentages (cases). Chi-square tests were used to compare groups, M pneumoniae antibody IgG-CLIA and IgM-CLIA alone or simultaneously. Females had a higher positive rate of M pneumoniae antibodies than males using the 3 methods, but the difference was not significant , 32 of which had titers of ≥1:160. The IgM-IFA results were positive in 47 patients. 66 cases were IgG-CLIA-positive, 34 cases were IgM-CLIA-positive, 29 cases were (IgG+IgM)-CLIA positive and 71 patients were positive for :40, 32 o2 = 21.21, P < .01) (Tables In the PPA titer ≥ 1:40 group (59 cases), 32, 53, and 56 patients were positive for IgM-CLIA, IgG-CLIA, and (IgM+IgG)-CLIA, respectively. The consistency coefficients are 82.4%, 88.5%, and 89.1%, respectively. Kappa coefficients were 0.578, 0.756, and 0.773, respectively. In the titer ≥1:160 group (32 cases), 28, 29 and 31 cases were positive for IgM-CLIA, IgG-CLIA, and (IgM+IgG)-CLIA, respectively. The consistency coefficients were 93.94%, 75.76%, and 75.2%, respectively. Kappa coefficients were 0.754, 0.448 and 0.457 respectively, the consistency coefficients between IgG-CLIA and PPA was significantly different with that between IgM-CLIA and PPA . Twenty-eight cases were positive in the IgM-CLIA and IgM-IFA tests  (Tables Forty cases were positive for IgM-CLIA and PPA (titer ≥ 1:40) , 26 of which were positive for PPA ≥ 1:160 . The consistency coefficients of IgM-CLIA and PPA ≥ 1:160 were significantly different from those of IgM-IFA and PPA ≥ 1:160 -CLIA and PPA were 89.1% higher than 79.6% reported by Chen et al. This may be related to the differences in the enrolled patients and needs to be further verified by clinical application.The CLIA method uses an automatic instrument; it is fast and flexible, and detection can be completed in 30 min. The sensitivity and specificity were better than those of IFA and PPA, the repeatability of the detection results was good, and IgM-CLIA and IgG could be detected separately.pneumoniae infection. Additionally, we did not perform a PCR assay for comparison with the CLIA method in this study, which showed that more samples and populations need to be included in our next step.There were some limitations to our study, such as the small sample size and the fact that all samples were collected in less than 2 months from March to April in 2019, which may have resulted in the loss of the prevalence of Mycoplasma pneumoniae IgM-CLIA has a high concordance with PPA, and its specificity and sensitivity are higher than those of IgG-CLIA and IFA, suggesting that it can accurately indicate early infection and help doctors treat M pneumoniae infection in the early time. The sensitivity and negative predictive value of M pneumoniae (IgM+IgG)-CLIA were higher than those of PPA and IFA, which is useful for reducing the rate of misdiagnosis, especially in patients with a low concentration of M pneumoniae IgM. Compared with manual detection methods such as PPA and IFA, automated chemiluminescence with the advantages of higher sensitivity, specificity, and accuracy should be widely applied for the laboratory diagnosis of M pneumoniae infection.Conceptualization: Mengyang Liu, Ke Meng, Shiying Sun.Data curation: Mengyang Liu, Ke Meng, Jun Jiang.Methodology: Mengyang Liu, Ke Meng, Jun Jiang.Project administration: Mengyang Liu, Ke Meng, Jun Jiang, Shiying Sun.Software: Mengyang Liu.Supervision: Ke Meng, Jun Jiang.Writing – original draft: Mengyang Liu.Writing – review & editing: Mengyang Liu, Li Zhang, Shiying Sun.