Four of five frequently used orthopedic PROMs possess inadequate content validity: a COSMIN evaluation of the mHHS, HAGOS, IKDC-SKF, KOOS and KNEES-ACL.

PURPOSE: Content validity is the most important property of PROMs. The COSMIN initiative has published guidelines for evaluating the content validity of PROMs, but they have only sparsely been applied to relevant PROMs for musculoskeletal conditions. The aim of this study was to use the COSMIN Risk of Bias checklist to evaluate the content validity of five PROMs, that are highly relevant in musculoskeletal research and used by the arthroscopic surgery community: the modified Harris' Hip Score (mHHS), the Copenhagen Hip and Groin Outcome Score (HAGOS), the International Knee Documentation Committee Subjective Knee evaluation Form (IKDC-SKF), the Knee injury and Osteoarthritis Outcome Score (KOOS) and the Knee Numeric-Entity Evaluation Score ACL (KNEES-ACL). METHODS: The development articles for the five PROMs were identified through searches in PubMed and SCOPUS. A literature search was performed to identify additional studies assessing content validity of the PROMs. Additional information, necessary for the assessments, was obtained from the PROM developers after direct request. To evaluate the quality of the development studies and rate the content validity, the COSMIN Risk of Bias checklist was applied to all studies. RESULTS: All five development studies were identified. Three subsequent content validity studies were identified, all evaluating KOOS and one also IKDC. One content validity study was of inadequate quality and excluded from further analysis. The development of mHHS, IKDC-SKF, and KOOS was rated inadequate and possess insufficient content validity for their target populations. Due to the irrelevance of multiple items, KOOS was in particular inappropriate to evaluate patients with an ACL injury. The development of HAGOS was also rated inadequate, although the insufficiency aspects can be regarded as minor. KNEES-ACL possessed sufficient content validity. CONCLUSION: Out of five PROMs, only KNEES-ACL possessed sufficient content validity. Particularly, KOOS should not be used as an outcome for patients with an ACL injury. There is an urgent need for condition-specific PROMs for musculoskeletal conditions, developed with adequate methods. LEVEL OF EVIDENCE: III.

Isolation and characterization of muscle stem cells, fibro-adipogenic progenitors, and macrophages from human skeletal muscle biopsies.

Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c+/-) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.

A catalogue of PROMs in sports science: Quality assessment of PROM development and validation.

Choosing the most adequate PROM for a study is a non-trivial process. The aim of this study was to provide a catalogue with analyses of content and construct validity of PROMs relevant to research in sports science, including all published local translations. The most commonly used PROMs in sports research were selected from a PubMed search "patient reported outcome measures sports", identifying 439 articles and 194 different PROMs. Articles describing development of the 61 selected PROMs were assessed for content validity, and all articles regarding construct validity of each PROM and all published translations (in total 622 articles) were analyzed. A catalogue with assessments of the 61 PROMs was produced. The majority were of inferior validity, with few exceptions. The most common reason for this was that the PROM had not been developed by methods that ensure high content validity. Another major reason for inferior validity was that construct validity had not been secured by adequate statistical methods. In conclusion, this catalogue provides a tool for researchers to facilitate choosing the most valid PROM for studies in sports research. Furthermore, it shows for popular PROMs where further validation is needed, and for fields in musculoskeletal medicine where valid PROMs are lacking. It is suggested that a targeted effort is made to develop valid PROMs for major conditions in musculoskeletal research. The current method is easier to practice compared with assessment after COSMIN guidelines.

Responsiveness, minimal important difference, minimal relevant difference, and optimal number of patients for a study.

Several terms are used to describe changes in PROM scores in relation to treatments. Whether the change is small, large, or relevant is defined in different ways, yet these change scores are used to recommend or oppose treatments. They are also used to calculate the necessary number of patients for a study. This article offers a theoretical explanation behind the terms responsiveness, minimal important difference (MID), minimal important change (MIC), minimal relevant difference (MIREDIF), and threshold of clinical importance. It also gives instructions on how these and the optimal number of patients for a study are calculated. Responses to two domains of the Knee Injury and Osteoarthritis Outcome Score (KOOS), before and 1 year after reconstruction of the anterior cruciate ligament of 164 patients, are used to illustrate the calculations. This paper presents the most common methods used to calculate and interpret MID. Results vary substantially across domains, patient location on the scale, and health conditions. The optimal number of patients depends on the minimal relevant difference (MIREDIF), the standard error of the measure (SEM), the desired statistical power for the measurement, and the responsiveness of the measurement instrument (the PROM). There is often uncertainty surrounding the calculation and interpretation of responsiveness, MID, and MIREDIF, as these concepts are complex. When MID is used to evaluate research results, authors should specify how the MID was calculated, and its relevance for the study population. These measures should only be used after thorough consideration to justify healthcare decisions.

How to develop a condition-specific PROM.

Developing new patient-reported outcome measures (PROMs) for application in clinical studies can be necessary if an adequate PROM does not exist. For adequate measurement, it is essential that the PROM has face validity (ie, is perceived to be relevant by clinicians and researchers) and has high content validity (ie, content relevance and content coverage for the targeted patient group). The steps needed to create PROMs that possess face and content validity for a specific condition are described in this paper. Face validity is achieved by item identification and generation through literature review. Content validity is confirmed through repetitive cognitive interviews of patients from the targeted patient group in order to generate a consensus-based pilot-version of the new PROM. This qualitative process ensures that items are appropriately worded, understandable, and minimizes doubts about how items should be answered. A practical example of this process is presented, which shows the development of the Knee Numeric-Entity Evaluation Score (KNEES-ACL), a condition-specific PROM for patients with deficiency of the anterior cruciate ligament (ACL).

Choosing the most appropriate PROM for clinical studies in sports medicine.

Choosing the most appropriate patient-reported outcome measure (PROM) for a clinical study is essential in order to achieve trustworthy results. This choice will depend on (a) the objective of the study and hence the research question; (b) the choice of a theoretical framework, such as the World Health Organization's International Classification of Functioning, Disability, and Health (ICF); (c) whether there currently is a PROM that possesses high content validity and high construct validity for the specific patient group and objective, and if not; (d) the decision on whether to use a suboptimal PROM or develop and validate a new PROM. This paper presents the steps that should be followed in order to assess the relevance of PROMs and suggests ways to enhance the choice depending on the goal of the study.

Are adequate PROMs used as outcomes in randomized controlled trials? an analysis of 54 trials.

Results by patient-reported outcome measures (PROMs) from randomized controlled trials (RCTs) in musculoskeletal research often influence healthcare strategies. We aimed to evaluate to which extent these RCTs use adequate PROMs, and how this influences the results and conclusions. We identified RCTs of sports research relevance with PROMs as primary outcomes published in 13 preselected journals between January 1, 2008, and November 1, 2019; all journals regularly publish results from musculoskeletal research. Five journals have a high impact factor (>15), and eight with lower impact factors are widely read journals. It was assessed whether the RCTs had used PROMs with high content validity and whether the most adequate PROMs were used (ie, the most well developed and well validated for the patients enrolled in the study). We registered journal impact factor, year of publication, existence of a registered protocol, and whether the study showed significant difference between interventions. A total of 54 RCTs with 56 primary outcomes comprising 26 different PROMs were identified. For 13 RCTs (24%), a protocol was not published. In only 24 of RCTs (44%), the most appropriate PROM had been used as primary outcome, independent of a registered protocol, ranking of the journal, and year of publication. In seven cases, PROMs were used to evaluate a condition that they had not been developed for. RCTs that used the most adequate PROM showed significantly more often (46%) difference in outcomes in contrast to RCTs that used inadequate PROMs (22%) (P = 0.0483). In conclusion, in the majority of RCTs, the most adequate PROM had not been used. Studies, in which the most adequate PROM had been used as outcome, were significantly more likely to show significant difference between interventions. The extent to which protocols were not available was surprisingly high. Journals should request that adequate PROMs are used in RCTs, and if this is not the case that it is discussed how it might influence the results and conclusions. Likewise, it should be requested that a protocol is published or registered.

Potential problems in the use of patient reported outcome measures (PROMs) and reporting of PROM data in sports science.

To use an inadequate patient reported outcome measure (PROM) or use a PROM in an inappropriate way potentially influences the quality of measurement. The objectives of this study were to define potential inadequate uses of PROMs in sports research studies and estimate how often they occur. A consensus group consisting of medical researchers, statisticians, and psychometricians identified and defined potentially irregular applications of PROMs. Occurrence of these in 349 consecutive articles in sports medicine in which PROMs were used as primary outcomes was reviewed. In all, 14 different potential problems were defined, and one or several occurred in 172 of the articles (49%). These were as follows: using a PROM that was developed for a different patient group (100 cases), using two or more PROMs with identical questions (94), aggregation of domain sum scores (82), combinations of subjective and objective measures (27), using a PROM to diagnose or evaluate the individual patient (7), using a PROM for a single limb (3), recall bias (3), exclusion of domains or items (3), construction of a PROM for a specific occasion (2), categorization of the scale (2), and mixing different versions of a PROM (1). Adaption of scale scores (e. g., to percentage) when results are reported (144) carries a risk of miscalculation and distorted impression of results. Data related to uncertainty about completing the PROM and the handling of missing data were not provided in the manuscripts. In conclusion, potential problems in the use and reporting of PROMs are common in sports research, and this can influence the validity of reported results.

Are PROMs used adequately in sports research? An analysis of 54 randomized controlled trials with PROMs as endpoint.

Deviations from adequate use and reporting of PROMs may be problematic and misleading. The aim of this study was to investigate the extent of such problems in randomized clinical trials (RCTs). RCTs involving sports medicine research that used PROMs as primary outcomes were identified in 13 preselected journals. The articles were reviewed for nine potential problems related to how the PROM was used and how the data had been reported. The potential problems were as follows: aggregating subscale scores; combining patient-reported scores with physical, clinical, or para-clinical measures; using a PROM to diagnose or evaluate the individual patient; using a PROM for one leg or arm; selectively excluding domains or items; constructing a PROM for the specific occasion; mixing PROM formats (ie, digital, paper, telephone, e-mail, in person); ambiguous instructions for how the PROM should be completed; and recall bias. As covariates, we registered journal impact factor, year of publication, and existence of a registered protocol. In 29 (53.7%) of 54 identified RCTs, at least one potential problem was identified, the most common being aggregation of domain scores. This was not different with a published protocol or dependent on journal rankings, except for exclusion of domains, which was most common in high-ranking journals. Aggregation of domain scores was significantly less common in recently published articles compared with older articles (P = .03). Potential problematic use of PROMs and reporting of PROM data are common in RCTs, also in high-ranking journals, but less so in more recent articles.

What is a PROM and why do we need it?

The purpose of this article was to introduce the reader to the nature of patient-reported outcome measures (PROMs) and pitfalls in their use. PROMs collect subjective information directly from the patient regarding specific or general conditions and add to clinical and functional outcomes, and turn unmeasurable subjective qualities into quantitative measures. PROMs are questionnaires consisting of items: questions or statements with predefined response options. The items in an adequate PROM have been developed by involvement of patients with the condition in focus, and the PROM has been validated for these patients using suitable statistical methods. An adequate well-targeted PROM is more responsive than an inadequate PROM. Unfortunately, many studies use inadequate PROMs as outcomes. The methods used to generate PROMs should be described as thoroughly as those used to develop any other types of measurement instruments, and the choice of PROM should always be explained and thereby justified. If the PROM used is not adequate, the consequences for the interpretation of the results should be discussed. In many cases, an adequate PROM does not exist. If the best available PROM is chosen, there are methods to validate the adequacy of the chosen PROM, which make an interpretation of the study results possible.

Psychometric validation of PROM instruments.

The aim was to provide an overview of the different statistical methods for validation of patient-reported outcome measures, ranging from simple statistical methods available in all software packages to advanced statistical models that require specialized software. A non-technical summary of classical test theory (CTT) and modern test theory (MTT) is provided. Specifically, confirmatory factor analysis, item response theory, and Rasch analysis is outlined. One CTT and three MTT methods were used to validate the two subscales (Symptoms and Quality of Life) from the Knee Injury and Osteoarthritis Outcome Score (KOOS). For each methodology, two analyses were considered: (i) a unidimensional analysis ignoring the pre-specified dimensionality, and (ii) a two-dimensional analysis using the pre-specified dimensionality. While CTT did not adequately address central issues regarding the validity of the KOOS subscales, the three MTT methods yielded very similar results. In conclusion, MTT methods offer analysis of all relevant properties related to the validity of patient-reported outcome measures, while this is not the case for CTT. Claims about sufficient validity based on CTT methods are inadequate and should not be trusted.

How to translate and locally adapt a PROM. Assessment of cross-cultural differential item functioning.

Translating patient-reported outcome measures (PROMs) can alter the meaning of items and undermine the PROM's psychometric properties (quantified as cross-cultural differential item functioning [DIF]). The aim of this paper was to present the theoretical background for PROM translation, adaptation, and cross-cultural validation, and assess how PROMs used in sports medicine research have been translated and adapted. We also assessed DIF for the Knee Injury and Osteoarthritis Outcome Score (KOOS) across Danish, Norwegian, and Swedish versions. We conducted a search in PubMed and Scopus to identify the method of translation, adaptation, and validation of PROMs relevant to musculoskeletal research. Additionally, 150 preoperative KOOS questionnaires were obtained from the Scandinavian knee ligament reconstruction registries, and cross-cultural DIF was evaluated using confirmatory factor analysis and Rasch analysis. There were 392 studies identified, describing the translation of 61 PROMs. Ninety-four percent were performed with forward-backward technique. Forty-nine percent used cognitive interviews to ensure appropriate wording, understandability, and adaptation to the target culture. Only two percent were validated according to modern test theory. No study assessed cross-cultural DIF. One KOOS subscale showed no cross-cultural DIF, two had DIF with respect to some (but not all) items, and thus conversion tables could be constructed, and two KOOS subscales could not be pooled. Most PROM translations are of undocumented quality, despite the common conclusion that they are valid and reliable. Scores from three of five KOOS subscales can be pooled across the Danish, Norwegian, and Swedish versions, but two of these must be adjusted for DIF.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

Global identification of hnRNP A1 binding sites for SSO-based splicing modulation.

BACKGROUND: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. RESULTS: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5' splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5' splice site can be blocked by SSOs to activate the exon. CONCLUSIONS: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy. Moreover, together with the hnRNP A1 consensus binding motif, the binding map may be used to predict whether disease-associated mutations and SNPs affect hnRNP A1 binding and eventually mRNA splicing.

Poor osteochondral repair by a biomimetic collagen scaffold: 1- to 3-year clinical and radiological follow-up.

INTRODUCTION: Treatment of osteochondral injuries is challenging, and no gold standard has been established. Layered cell-free scaffolds are a new treatment option for these defects. The aim of this study was to evaluate the osteochondral repair in patients treated with the MaioRegen(®) scaffold, a cell-free biomimetic scaffold consisting of type I collagen and hydroxyapatite. Treatment using this scaffold has previously shown promising clinical results. METHODS: Ten patients with osteochondral lesions in the knee (n = 6) or in the talus (n = 4) were enrolled. The patients underwent pre-operative MRI and CT scans and were assessed at 1- and 2.5-year timescales post-operatively. The cartilage and bone formations were evaluated semi-quantitatively using the MOCART score. Knee patients were clinically evaluated using KOOS, subjective IKDC and Tegner scores, whereas ankle patients were evaluated using AOFAS Hindfoot and Tegner scores. RESULTS: Two patients were re-operated and excluded from further follow-up due to treatment failure. None of the patients had complete regeneration of the subchondral bone evaluated using CT. At 2.5 years, 6/8 patients had no or very limited (<10 %) bone formation in the defects and 2/8 had 50-75 % bone formation in the treated defect. MRI showed no improvement in the MOCART score at any time point. The IKDC score improved from 41.3 to 80.7, and the KOOS pain subscale improved from 63.8 to 90.8 at 2.5-year follow-up. No improvement was found with the remaining KOOS subscales, the Tegner or AOFAS Ankle-Hindfoot score. CONCLUSION: Treatment of osteochondral defects in the ankle and knee joint with a biomimetic scaffold resulted in incomplete cartilage repair and poor subchondral bone repair at 1- and 2.5-year follow-up. Clinical significant improvements were observed. These results raise serious concerns about the biological repair potential of the MaioRegen(®) scaffold, and we advise to use the MaioRegen(®) scaffold with caution. LEVEL OF EVIDENCE: Prospective therapeutic study, Level IV.

Infliximab treatment reduces tensile strength in intestinal anastomosis.

BACKGROUND: The antitumor necrosis factor (infliximab [IFX]) has gained widespread use in the treatment of inflammatory bowel disease. However, several patients must undergo surgical treatment due to treatment failure and there is a potential risk that preoperative IFX treatment may have a negative effect on the healing process in intestinal anastomosis. The objective of this study was to examine the effect of repeated IFX treatment on anastomotic strength and degree of inflammation in the anastomotic line in the small intestine of rabbits. METHODS: Thirty-two rabbits were randomized (2:1) to receive either repeated IFX treatment or placebo. On day 15, three separate end-to-end anastomoses were performed on the jejunum. On postoperative day 5, tensile strength and bursting pressure for the anastomoses were tested and histologic changes examined. RESULTS: We found a significantly reduced tensile strength in the IFX group (1.94 ± 0.44 N) compared with the placebo group (3.33 ± 0.39 N), (P < 0.001). Calculation of Spearman correlation coefficients showed a positive significant correlation between minimal tensile strength and serum values of IFX (coefficient = -0.63; P = 0.003) as well as number of sutures in the tested anastomosis (coefficient = 0.51; P = 0.024). The general histologic score was significantly higher in the placebo group (5.00 ± 1.26 versus 3.31 ± 1.65, P = 0.03). CONCLUSIONS: Repeated high-dose IFX treatment reduces tensile strength significantly in rabbits and should be investigated further as a potential risk factor of anastomotic dehiscence in inflammatory bowel disease surgery.

The multicopy sRNA LhrC controls expression of the oligopeptide-binding protein OppA in Listeria monocytogenes.

Listeria monocytogenes is the causative agent of the foodborne disease listeriosis. During infection, L. monocytogenes produces an array of non-coding RNAs, including the multicopy sRNA LhrC. These five, nearly identical sRNAs are highly induced in response to cell envelope stress and target the virulence adhesin lapB at the post-transcriptional level. Here, we demonstrate that LhrC controls expression of additional genes encoding cell envelope-associated proteins with virulence function. Using transcriptomics and proteomics, we identified a set of genes affected by LhrC in response to cell envelope stress. Three targets were significantly down-regulated by LhrC at both the RNA and protein level: lmo2349, tcsA and oppA. All three genes encode membrane-associated proteins: A putative substrate binding protein of an amino acid ABC transporter (Lmo2349); the CD4+ T cell-stimulating antigen TcsA, and the oligopeptide binding protein OppA, of which the latter 2 are required for full virulence of L. monocytogenes. For OppA, we show that LhrC acts by direct base paring to the ribosome binding site of the oppA mRNA, leading to an impediment of its translation and a decreased mRNA level. The sRNA-mRNA interaction depends on 2 of 3 CU-rich regions in LhrC allowing binding of 2 oppA mRNAs to a single LhrC molecule. Finally, we found that LhrC contributes to infection in macrophage-like cells. These findings demonstrate a central role for LhrC in controlling the level of OppA and other virulence-associated cell envelope proteins in response to cell envelope stress.

A single topical dose of erythropoietin applied on a collagen carrier enhances calvarial bone healing in pigs.

BACKGROUND AND PURPOSE: The osteogenic potency of erythropoietin (EPO) has been documented. However, its efficacy in a large-animal model has not yet been investigated; nor has a clinically safe dosage. The purpose of this study was to overcome such limitations of previous studies and thereby pave the way for possible clinical application. Our hypothesis was that EPO increases calvarial bone healing compared to a saline control in the same subject. METHODS: We used a porcine calvarial defect model. In each of 18 pigs, 6 cylindrical defects (diameter: 1 cm; height: 1 cm) were drilled, allowing 3 pairwise comparisons. Treatment consisted of either 900 IU/mL EPO or an equal volume of saline in combination with either autograft, a collagen carrier, or a polycaprolactone (PCL) scaffold. After an observation time of 5 weeks, the primary outcome (bone volume fraction (BV/TV)) was assessed with high-resolution quantitative computed tomography. Secondary outcome measures were histomorphometry and blood samples. RESULTS: The median BV/TV ratio of the EPO-treated collagen group was 1.06 (CI: 1.02-1.11) relative to the saline-treated collagen group. Histomorphometry showed a similar median effect size, but it did not reach statistical significance. Autograft treatment had excellent healing potential and was able to completely regenerate the bone defect independently of EPO treatment. Bony ingrowth into the PCL scaffold was sparse, both with and without EPO. Neither a substantial systemic effect nor adverse events were observed. The number of blood vessels was similar in EPO-treated defects and saline-treated defects. INTERPRETATION: Topical administration of EPO on a collagen carrier moderately increased bone healing. The dosing regime was safe, and could have possible application in the clinical setting. However, in order to increase the clinical relevance, a more potent but still clinically safe dose should be investigated.

Hemodynamic effects of low versus high dialysate bicarbonate concentration in hemodialysis patients.

INTRODUCTION: Hemodialysis treatment using standard dialysate bicarbonate concentrations cause transient metabolic alkalosis possibly associated with hemodynamic instability. The aim of this study was to perform a detailed comparison of high and low dialysate bicarbonate in terms of blood pressure, intradialytic hemodynamic parameters, orthostatic blood pressure, and electrolytes. METHODS: Fifteen hemodialysis patients were examined in a single-blind, randomized, controlled, crossover study. Participants underwent a 4-h hemodialysis session with dialysate bicarbonate concentration of 30 or 38 mmol/L with 1 week between interventions. Blood pressure was monitored throughout hemodialysis, while cardiac output, total peripheral resistance, stroke volume, and central blood volume were assessed with ultrasound dilution technique (Transonic). Orthostatic blood pressure was measured pre- and post-hemodialysis. FINDINGS: With similar ultrafiltration (UF) volume (2.6 L), systolic blood pressure (SBP) tended to decrease more during high dialysate bicarbonate compared to low dialysate bicarbonate; the mean (95% confidence interval) between treatment differences in SBP were: 8 (-4; 20) mmHg (end of hemodialysis) and 7 (0; 15) mmHg (post-hemodialysis). Stroke volume decreased whereas total peripheral resistance increased significantly more during high dialysate bicarbonate compared to low dialysate bicarbonate with mean between treatment differences: Stroke volume: 12 (1; 23) mL; Total peripheral resistance: -2.9 (-5.3; -0.5) mmHg/(L/min). Cardiac output tended to decrease more with high dialysate bicarbonate compared to low dialysate bicarbonate with mean between treatment difference 0.7 (0.0; 1.4) L/min. High dialysate bicarbonate caused alkalosis, hypocalcemia, and lower plasma potassium, whereas patients remained normocalcemic with normal pH during low dialysate bicarbonate. Orthostatic blood pressure response after dialysis did not differ significantly. DISCUSSION: The use of high dialysate bicarbonate compared to low dialysate bicarbonate was associated with hypocalcemia, alkalosis, and a more pronounced hypokalemia. During hemodialysis with UF, a better preservation of blood pressure, stroke volume, and cardiac output may be achieved with low dialysate bicarbonate compared to high dialysate bicarbonate.

Fluorescence-activated cell sorting and phenotypic characterization of human fibro-adipogenic progenitors.

The ability of stem cells to activate and differentiate is critical for maintaining the regenerative capacity of skeletal muscle. Here, we detail steps for specific quantification and isolation of primary human fibro-adipogenic progenitors and skeletal muscle stem cells using fluorescence-activated cell sorting. We describe important phenotypic traits such as time to enter the cell cycle and assessment of cell differentiation for the isolated cell populations. The technique has been applied on tissue obtained from surgery and needle biopsies. For complete details on the use and execution of this protocol, please refer to Farup et al. (2021).1.