Autism spectrum disorders (ASDs) include autistic disorder, Asperger’s disorder and pervasive developmental disorder. The manifestations of ASDs can have an important impact on learning and social functioning that may persist during adulthood. The aim here was to summarize the evidence from Cochrane systematic reviews on interventions for ASDs.  Review of systematic reviews, conducted within the Discipline of Evidence-Based Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo.  We included and summarized the results from Cochrane systematic reviews on interventions for ASDs.  Seventeen reviews were included. These found weak evidence of benefits from acupuncture, gluten and casein-free diets, early intensive behavioral interventions, music therapy, parent-mediated early interventions, social skill groups, Theory of Mind cognitive model, aripiprazole, risperidone, tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRI); this last only for adults. No benefits were found for sound therapies, chelating agents, hyperbaric oxygen therapy, omega-3, secretin, vitamin B6/magnesium and SSRI for children. Acupuncture, gluten and casein-free diets, early intensive behavioral interventions, music therapy, parent-mediated early interventions, social skill groups and the Theory of Mind cognitive model seem to have benefits for patients with autism spectrum disorders . Aripiprazole, risperidone, tricyclic antidepressants and SSRI (this last only for adults) also showed some benefits, although associated with higher risk of adverse events. Experimental studies to confirm a link between probable therapies and the disease, and then high-quality long-term clinical trials, are needed. Autism spectrum disorders (ASDs) have an estimated prevalence ranging from 3.3 to 116 children per 10,000 and it is widely accepted that autism affects approximately 1% of children worldwide.ASDs include autistic disorder, Asperger’s disorder and pervasive developmental disorder. Patients with these conditions present deficits in communication, social interaction and cognitive function; problems with feeding; hypo or hypersensitivity; and, sometimes, self-harmful behavior.Healthcare and social care for people with ASDs is frequently complex, since they are more likely to have mental health comorbidities and suicidal ideation.,Individual or parent-based psychosocial interventions are frequently used in clinical practice, with the aim of improving clinical features relating to communication, cognition, behavior and relationships, for instance. Pharmacological therapy is an option for mental health comorbidities. New therapeutic approaches include genetic and pharmacological strategies for reducing synthesis of specific proteins and inhibiting pleiotropic growth factors.Despite recent advances in this area, the evidence regarding autism remains limited and this condition continues to be a challenge for researchers, healthcare professionals, patients, parents and caregivers.To map out and summarize all Cochrane systematic reviews on interventions for autism spectrum disorders, and present the results on the basis of the quality of the evidence.Review of Cochrane systematic reviews on interventions for ASD.Discipline of Evidence-Based Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), Brazil.We included completed Cochrane systematic reviews, with no restriction regarding date of publication. Protocols for systematic reviews and reviews signaled as “withdrawn” in the Cochrane Database of Systematic Reviews (CDSR) were not considered. We included only the latest version of each review.Individuals with ASD.All types of interventions  aiming to treat ASD.Clinical and laboratory outcomes were considered, as presented by the systematic reviews.We conducted systematic searches in the Cochrane Database of Systematic Reviews (CDSR) (via Wiley) using a sensitive search strategy .Three reviewers evaluated the titles and abstracts of records that had initially been retrieved on the basis of the inclusion criteria. The full texts of records with the potential for inclusion were read to confirm whether they should be included (reasons for exclusions were recorded and presented). Divergences between reviewers were resolved through reaching a consensus.We used a narrative structure  to present the results from the systematic reviews included.,,,,,,,,,,,,,,,,The initial search retrieved 24 reviews. From these, we excluded six reviews addressing other clinical situations  and one that was a previous version of a review that we included. Thus, we included 17 systematic reviews.,,,,,,,,,,,,,,,,A summary of each systematic review is presented narratively below. In addition, The reviewNeedle acupuncture  versus sham: no difference in core autistic features. On the Ritvo-Freeman Real Life Rating Scale (RFRLRS): mean difference (MD) = 0.09; 95% confidence interval (CI) = -0.03 to 0.21; P = 0.16. Acupuncture seemed to result in better language comprehension, cognition, selfcare and social functioning, and a higher probability of improvement in overall functioning greater than or equal to 25%.Needle acupuncture  versus no treatment: acupuncture seemed to improve autistic features, speech, cognition, overall functioning, self-care, sensory functioning, imitation, repetitive behavior and physical health.Acupressure versus no treatment: acupressure might result in improvement in overall functioning, communication attitude, non-verbal communication and matching, and in language and social interaction.Most of the favorable outcomes were observed only in a single study and few outcomes were supported by pooled results from more than one study. The majority of the effect size estimates for the significant outcomes had imprecisions with a wide confidence interval. These limitations mean that the significant result was not sufficiently robust to enable a reliable conclusion. http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD007849.pub2/abstract.Therefore, the authors concluded that the current evidence did not support use of acupuncture for treating ASDs. For further details, the original abstract can be consulted, available from: Antipsychotics have been used as medications for ASD-related irritability. The reviewAberrant Behavior Checklist (ABC) - irritability subscale ;ABC hyperactivity subscale ;ABC stereotypy subscale .In terms of side effects, aripiprazole was associated with greater increase in weight , higher risk of sedation  and tremor . The discontinuation RCT study showed no difference in relapse rate, with regard to symptoms of irritability . http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009043.pub3/abstract.The authors concluded that aripiprazole might be effective as a short-term intervention for some behavioral aspects of ASDs among children/adolescents. However, they stated that notable side effects, including weight gain, sedation, drooling and tremor, needed to be considered. For further details, the original abstract can be consulted, available from: Auditory integration therapies (including Tomatis therapy and Samonas sound therapy) are techniques for improving abnormal sound sensitivity in individuals with behavioral disorders including ASDs. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003681.pub3/abstract.The authors concluded that, considering the methodological limitations of the existing RCTs, no evidence supporting the use of auditory integration therapy or other sound therapies was available up to that time. For further details, the original abstract can be consulted, available from: It has been suggested that the severity of autism spectrum disorder (ASD) symptoms is associated with the levels of serum or stored toxic metals, and that chelating agents improve symptoms. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD010766.pub2/abstract.The authors concluded that there was no evidence to suggest that multiple rounds of oral DMSA had any effect on ASD symptoms. Considering previous reports of serious adverse events, including hypocalcemia, renal impairment and reported death, they stated that the risks involved in chelation to treat ASDs currently outweighed the benefits. Evidence supporting a causal relation between heavy metals and autism would be needed before further trials were conducted. For further details, the original abstract can be consulted, available from: Early intensive behavioral intervention (EIBI) is based on the principles of applied behavioral analysis on interventions delivered over many years for 20 to 40 hours per week. For the review,Adaptive behavior ;Intelligence quotient, IQ ;Expressive language ;Receptive language ;Daily communication skills ; Socialization ;Daily living skills . http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009260.pub2/abstract.The authors concluded that there was some evidence that EIBI was effective for children with ASDs. However, they considered that the current state of the evidence was limited because the data came from non-randomized studies. For further details, the original abstract can be consulted, available from: It has been hypothesized that peptides from gluten and casein may participate in the origins of autism and that that physiology and psychology of autism might be explained in terms of excessive opioid activity linked to these peptides. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003498.pub3/abstract.The authors concluded that, despite the high rates of use of gluten and/or casein-free diets for children with ASDs, the current evidence was not robust enough to support these practices. For further details, the original abstract can be consulted, available from: It has been hypothesized that hyperbaric oxygen therapy might reduce the biochemical dysfunction and clinical symptoms of ASDs. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD010922.pub2/abstract.The authors concluded that up to that time, there was no evidence that hyperbaric oxygen therapy improved the core symptoms and associated symptoms of ASDs. They stated that it was important to consider that adverse events could occur. For further details, the original abstract can be consulted, available from: The reviewSocial interaction within the therapy context ;General social interaction outside of therapy context ;Nonverbal communicative skills within the therapy context ;Verbal communicative skills ;Initiating behavior ;Social-emotional reciprocity ;Social adaptation ;Joy ; andQuality of parent-child relationships .http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD004381.pub3/abstract.There was no difference in nonverbal communicative skills outside of the therapy context . None of the studies reported any adverse events. For further details, the original abstract can be consulted, available from: The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD007992.pub2/abstract.The authors concluded that, up to that time, there was no high-quality evidence that omega-3 fatty acid supplementation was effective for children with ASDs. For further details, the original abstract can be consulted, available from: Approaches that help parents develop strategies for interactions and management of behavior can be useful in cases of ASDs. The reviewNo benefit for most of the primary outcomes ;Strong and statistically significant benefits for patterns of parent-child interaction: shared attention  and parent synchrony ;Evidence suggestive of improvement of child vocabulary comprehension reported by parents  and reduction in the severity of children’s autism characteristics .http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009774.pub2/abstract.For further details, the original abstract can be consulted, available from: The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD005040.pub2/abstract.The authors concluded that risperidone might be beneficial in relation to some features of autism, but that the evidence was limited because the existing studies had small sample sizes with short follow-ups, and because of the lack of a single standardized outcome measurement. For further details, the original abstract can be consulted, available from: Secretin, a gastrointestinal hormone, has been suggested as an effective treatment for ASDs based on anecdotal evidence. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003495.pub3/abstract.The authors concluded that there was no evidence that secretin was effective and that therefore currently it could not be recommended and should not be administered as a treatment for ASDs. They stated that further RCTs would only be justifiable if there was any new high-quality replicable scientific evidence proving a link between secretin and ASDs. For further details, the original abstract can be consulted, available from: The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD004677.pub3/abstract.The authors concluded that there was no evidence of effects from SSRIs on children and that the evidence relating to adults was limited due to the small size of the studies, with unclear risk of bias. For further details, the original abstract can be consulted, available from: Major difficulties in social interactions have been found to be a defining feature of individuals with ASDs. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008511.pub2/abstract.The authors concluded that there was some evidence that social skill groups might improve social competence for some children and adolescents with ASDs. They stated that further research was needed in order to reach conclusions that would be more robust, especially with regard to improvements in quality of life. For further details, the original abstract can be consulted, available from: The Theory of Mind (ToM) model suggests that people with ASDs have difficulty in understanding the minds  of other people. Interventions to teach ToM for patients could provide some benefits for ASDs. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008785.pub2/abstract.The authors concluded that there was some evidence that teaching ToM to people with ASD seemed to present benefits. However, inconsistencies in the findings and measurements meant that that the evidence was of very low or low quality, which reduced the confidence in these findings. For further details, the original abstract can be consulted, available from: Because of the impact of tricyclic antidepressants (TCAs) on serotonins, they have been used to treat ASDs. The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD008372.pub2/abstract.The authors concluded that since there was limited and conflicting evidence regarding the effects and side effects of TCAs for this purpose, further research would be required before TCAs could be recommended for ASDs. For further details, the original abstract can be consulted, available from: The reviewhttp://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003497.pub2/abstract.The authors concluded that due to the small number of studies, the methodological quality of studies and the small sample sizes, no recommendations could be made regarding use of vitamin B6 plus magnesium for ASDs. For further details, the original abstract can be consulted, available from: This review found that despite increasing prevalence of ASDs, there are still few systematic reviews and there is a scarcity of high-quality randomized trials addressing interventions for improving the main clinical features of ASDs. None of the reviews included here provided high-quality evidence for any outcome. Even more surprisingly, some of the interventions studied do not have any proven pathophysiological link with the disease or any well-known potential therapeutic mechanism . Furthermore, many of the off-label interventions used present no benefit for patients and could also increase the risk of adverse events.Overall, the primary studies from each of the systematic reviews included had limited methodological quality, small sample sizes, short-term measurements of outcomes and poor reporting of adverse events. All of these facts increase the uncertainty surrounding the effects of the pharmacological and non-pharmacological interventions that are frequently used in clinical practice as options for patients with ASDs.Regarding the implications for practice, some non-pharmacological interventions seem to be useful, given that they present some beneficial effects with few adverse events . These include gluten and casein-free diets, acupuncture, early intensive behavioral intervention, music therapy, parent-mediated early intervention, social skill groups and the Theory of Mind cognitive model. Their use needs to be discussed with patients, parents and caregivers in order to clarify the uncertainties regarding the results, time taken and costs.Among the pharmacological options, aripiprazole, risperidone tricyclic antidepressants (clomipramine and tianeptine) and selective serotonin reuptake inhibitors (this last category only for adults) seem to have some benefits for specific symptoms, but the adverse events that have been reported need to be carefully considered before making decisions.Regarding the implications for further research, this review of reviews makes it clear that much needs to be done on the therapeutics of ASDs. Firstly, experimental research exploring physiopathological mechanisms needs to be conducted in order to support further clinical studies. Subsequently, good-quality long-term clinical trials are required.This review included 17 Cochrane systematic reviews. None of them provided high-quality evidence for any autism-related outcome. Acupuncture, early intensive behavioral intervention, gluten and casein-free diets, music therapy, parent-mediated early intervention, social skill groups and the Theory of Mind cognitive model seem to have benefits for patients with autism spectrum disorders . Aripiprazole, risperidone, clomipramine, tianeptine and selective serotonin reuptake inhibitors are pharmacological options that seem to have some benefits (this last one only for adults), but all of them are associated with high risks of important adverse events. Experimental research to confirm the links between therapeutic options and the disease is needed, followed by high-quality long-term clinical trials.


Open proficiency tests meet accreditation requirements and measure examiner competence but may not represent actual casework. In December 2015, the Houston Forensic Science Center began a blind quality control program in firearms examination. Mock cases are created to mimic routine casework so that examiners are unaware they are being tested. Once the blind case is assigned to an examiner, the evidence undergoes microscopic examination and comparison to determine whether the fired evidence submitted was fired in the same firearm. Fifty‐one firearms blind cases resulting in 570 analysis and comparison determinations were reported between December 2015 and June 2021. No unsatisfactory results were obtained; however, 40.3% of comparisons in which the ground truth was either elimination or identification resulted in inconclusive conclusions. Due to the quality of some of the evidence submitted, inconclusive results were not unexpected. A ground truth of elimination and comparison result of inconclusive was observed at a rate of 74%, while a ground truth of identification and comparison result of inconclusive was observed at a rate of 31%. Bullets (61.8%) were the main contributors to inconclusive conclusions; variables such as the assigned examiners, training program, examiner experience, and the intended complexity of the case did not significantly contribute to the results. The program demonstrates that the quality management system and firearms section procedures can obtain accurate and reliable results and provides examiners added confidence in court. Additionally, the program can be tailored to target specific research questions and provide opportunities for collaboration with other laboratories and researchers. Initial findings from a blind testing program in firearms examination are presented.No identifications declared for nonmatching pairs; no eliminations declared for matching pairs.40.3% of comparisons (ground truth identification or elimination) were determined to be inconclusive.Bullets were the main contributors to inconclusive results (61.8%).Benefits and limitations of a blind testing program in firearms examination are discussed.1Proficiency testing is a requirement for accredited forensic science service providers and serves an important role in the ability to confirm adequate competence among individual analysts and across laboratories. Most proficiency tests are prepared by a vendor, and the results are unknown to the participant but are “open,” meaning the forensic practitioners are aware that they are being tested. Open proficiency tests are tools for assessing the performance of analytical steps and providing a means by which to conduct interlaboratory comparisons. However, proficiency tests do not mimic routine casework of the laboratory in packaging, paperwork, or distribution. Despite these differences, analysts are asked to work these proficiency tests as routine casework, which may inflate accuracy rates , 2, 3. SIn 2009, the National Academy of Sciences (NAS) published a report that described the current state of forensic science practice and outlined recommendations for many forensic science disciplines . The repThe Houston Forensic Science Center (HFSC) is a local government corporation that operates independently from law enforcement. In September 2015, HFSC implemented a blind quality control (blind QC) program. The program was initiated in the toxicology section and has expanded over the years to include the seized drugs, latent prints, forensic biology, multimedia, and firearms sections. The intent of the blind QC program is to supplement open proficiency tests required for accreditation; HFSC is accredited to the International Organization for Standardization/International Electrotechnical Commission (ISO/IEC) 17,025:2017 standard by the American National Standards Institute (ANSI) National Accreditation Board (ANAB). The program is facilitated and maintained by HFSC's quality division, which is organizationally separate from the laboratory sections; as such, blind QC cases are prepared and introduced into the workflow by personnel who are not associated with the testing. Blind QC cases are created to mimic real casework with the intent that the analysts will be unaware that the cases are mock cases and give the cases no special treatment.Blind testing was introduced in the firearms section in December 2015. Firearms blind QC cases are intended to be submitted and packaged in a similar manner to the casework seen by the firearms section. Blind QC cases are submitted at a rate that equals approximately 5% of the monthly firearms examination case output average from the previous year. The goal was implemented in the Firearms section in mid‐2018, equating to one blind QC submission per month.Describes preliminary results from a blind testing program in firearms examination.Examines the prevalence of examiner conclusions and explores the extent to which there are trends related to examiners and examiner conclusions.Discusses the benefits the firearms section garners from the blind QC program.This manuscript:22.1The firearms section conducts casework on a request basis from stakeholders. The majority of requests are for microscopic examination and comparison of fired bullets and/or cartridge cases to determine whether the fired evidence was fired in the same firearm. Fired evidence also includes shotshells, shot pellets, shot carriers, and fragments. Unfired cartridges, or live rounds, may be submitted as evidence. Since live rounds are not fired in a firearm, they are not typically submitted for comparison purposes; however, unfired cartridges may be used to create test fires when the submitted unfired cartridges match the ammunition type of the submitted fired evidence. The firearms section also operates a program for the National Integrated Ballistic Information Network (NIBIN). This program is not request based; instead, firearms suitable for NIBIN processing are automatically submitted. NIBIN technicians test fire firearms for NIBIN entry. The fired bullets and cartridge cases created from test firing may be used as known samples for examination in casework.If a firearm is submitted, an examiner fires the firearm to test functionality and create a set of test fires. The test fires, which are a set of cartridge cases and bullets known to have been fired in a firearm , can be compared to the fired evidence submitted in the case . Firearms examiners use comparison microscopes to compare two items  side by side. Items are examined for markings made during the firing process, and conclusions are rendered based on the level of agreement or disagreement of these marks. Additional microscopic examinations on fired evidence include determining what types of firearms may have fired the evidence.Identification, Elimination, Inconclusive, and Unsuitable. Additionally, firearms examiners at HFSC may conclude that items of evidence are Insufficient for analysis. The HFSC firearms section interprets unsuitable to mean that an item has no markings created by a firearm. The non‐standard conclusion of insufficient is used to record more information regarding an item and/or provide additional information to the stakeholder. Unsuitable and insufficient conclusions are suitability determinations, whereas identification, elimination, and inconclusive are comparison conclusions. Conclusions of identification are based on individual characteristics, while conclusions of elimination can be based on class or individual characteristics. An inconclusive conclusion indicates an inadequate correspondence of individual and/or class characteristics needed to make an identification or elimination decision. Table Based on the Association of Firearms and Tool Mark Examiners (AFTE) range of conclusions , the firClass characteristics refer to features of firearms that are under the control of the manufacturer of the firearm . Subclass characteristics are features that may be produced during manufacture that are consistent among items fabricated by the same tool in the same approximate state of wear . These fSome case requests, such as firearm functionality testing and single items submitted for rifling characteristic analysis, do not require a second examiner, but are technically and administratively reviewed by two additional firearms examiners. Every case in which comparisons are conducted or in which the item(s) is deemed unsuitable or insufficient for comparison is examined by a secondary examiner in a process called verification. When a case requires a second examiner, the second examiner conducts an administrative and technical review before a third examiner also technically and administratively reviews the case. Should the primary and second examiner reach different conclusions during examination or verification, the examiners would follow the section's consultation and conflict resolution policy, which was put into practice in 2018 .2.2The firearms blind QC cases are designed and submitted in a manner consistent with the evidence items and offense types that the section observes in routine casework. Most of the casework received by HFSC is submitted by the Houston Police Department (HPD), so understanding what HPD submits on a regular basis is integral for creating blind QC cases that most closely mimic HPD submissions in packaging, submission process, and offense type. In routine casework, HPD enters cartridge case evidence into NIBIN prior to the evidence being submitted to HFSC for examination. The blind QC program must bypass this process in order to keep mock evidence from being uploaded to NIBIN. Instead, blind QC evidence is packaged in a way that mimics HPD’s NIBIN procedures without going through this process. See Hundl et al. (2019)  for moreFired evidence is created using firearms slated by HPD for destruction, HFSC staff's personally owned firearms, or firearms from HFSC's reference collection (a library of firearms used for parts and training). The firearm(s) used to create the fired evidence may or may not be submitted as an item of evidence. When more than one firearm is used to create fired evidence, bullets and cartridge cases are marked with an ultraviolet (UV) pen or otherwise made identifiable by documenting unique features. Marking the evidence that was created from one firearm allows the firearms section manager and/or the quality division to review the evidence after analysis and determine whether ground truth was reached in the case. Since determining how the analyst will itemize the evidence is not possible, marking the fired evidence with a UV pen is a way to keep track of which item was fired from a particular firearm. The markings from the UV pen will remain invisible to the examiner through the course of examination. If a piece of fired evidence has distinguishing features that the firearms manager or the quality division can use to identify the evidence after analysis, then marking the evidence with a UV pen may be unnecessary.Since the blind QC evidence must mimic normal casework to appear authentic to the examiner, a variety of samples are submitted. Not all items submitted are intended to be suitable for comparison, such as bullet fragments, bullet cores, and other items which the examiner may conclude are insufficient or unsuitable for comparison due to quality. Additionally, some items are intentionally submitted to make comparisons challenging. For example, Glocks, which were used to create evidence for four blind QC cases, are known in the firearms community to poorly mark bullets due to the method Glock uses to rifle their barrels . Another way in which the examiners can be challenged is by submitting fired evidence created using more than one firearm with the same class characteristics. Two hundred and ninety (51%) comparisons were created with two different firearms of the same class. These comparisons are challenging because class characteristics will be the same, but individual characteristics will not; thus, the ground truth will be elimination despite class characteristic similarities. Open proficiency test consensus results are typically either identification or elimination conclusions, providing few circumstances in which examiners might determine inconclusive. HFSC can mitigate the lack of inconclusive consensus results in proficiency tests by submitting blind QC items with a range of complexity to further test the firearms workflow.Firearms section management evaluates the created evidence prior to submission to determine the expected results and reviews the results of the completed blind QC cases to determine satisfactory completion. A satisfactory result may include: (1) a result that conforms to the known ground truth, or (2) a result that does not necessarily conform to the known ground truth but is technically sound  . A fireaFragments and bullet cores are submitted as ground truths of unsuitable or insufficient regardless of which firearm was used to create the evidence. Because all suitability and comparison conclusions are verified by an additional examiner, all conclusions are the consensus opinion of two examiners. Similar to proficiency test procedures, the final decision agreed upon by the primary and second examiners is reviewed for satisfaction. Documentation of consultations is maintained in the case record.During this study, 11 firearms examiners were actively participating in casework at HFSC. Six (55%) of the examiners were trained by the Houston Police Department Crime Laboratory prior to the formal inception of the Houston Forensic Science Center in April 2014. The remaining five examiners were primarily trained by other agencies or programs. During this time frame, six examiners were certified by AFTE. In addition, all examiners were required to be licensed by the Texas Forensic Science Commission as of January 1, 2019. Examiner experience ranged from 5.5 to 23 years, with a median of 11.5 years. Table 2.3p < 0.05.Statistical analysis was performed using JMP version 13.2.1. Categorical data for the satisfactory rating were converted to continuous data on a numeric scale, so the means could be analyzed with a one‐way analysis of variance (ANOVA). A one‐way ANOVA test was performed to determine whether factors such as complexity of the case or evidence type had a statistically significant difference in the means of these reported results. The data were considered significant for The data set yielded two results overall. The reported results either matched the ground truth or resulted in an inconclusive decision. All comparisons were deemed satisfactory; however, the results were further analyzed to gain more insight into the inconclusive decisions. To do this, a numeric value of 100 was assigned to the satisfactory rating when the ground truth was the reported result and a numeric value of 0 was assigned when the reported result was inconclusive. Unsatisfactory results would have been assigned a numerical value as well, but because no unsatisfactory results were observed, additional values were not requisite. Once the data were in a continuous format, the numeric results of the conclusions in the data set could be used to obtain the average and allowed for the application of statistical analysis. With the numeric scale used, one can expect that a lower mean indicates that there were more inconclusive results in the data set.3The results of 51 blind QC cases were reported between December 21, 2015, and June 22, 2021. Most cases contained a handful of evidence items, while some contained as little as two items or as many as 41 items (median = 9). Five hundred and fourteen evidence items were submitted; however, not all items, like unfired evidence or magazines, were examined as a part of routine casework. Four hundred and sixty items, including test fires created as part of routine casework, were examined. A total of 570 sufficiency determinations and comparison conclusions were made. Because the data were examined at the comparison level, an item of evidence can appear in the data set in multiple comparisons and be represented by multiple comparison conclusions. For example, Item 1 may have been compared to Item 2 and Item 3 with comparison conclusions of elimination and identification, respectively. Thus, a case may contain more comparisons than reported conclusions.n = 386) identification, 25.0% (n = 143) elimination, 2.0% (n = 11) insufficient, and 3.2% (n = 18) unsuitable. Due to the small sample size, insufficient and unsuitable conclusions will be grouped together for the purposes of this study. Ground truth was unable to be determined for 2.1% (n = 12) of the conclusions that were reported as inconclusive. The items were bullet items that were either not marked with a UV pen prior to submission or the pen markings rubbed off during examination. The firearms section manager conducted a post‐analysis comparison on the items but was unable to reach a conclusion of identification or elimination, supporting the examiners' conclusions of inconclusive. The 12 unknown ground truth bullet item comparisons were excluded from analysis. Table The mock evidence items were created with ground truth being 67.7% n = 86 identin = 333) of the comparisons. In 40.3% (n = 225) of the comparisons, an inconclusive conclusion was made when the ground truth was either elimination or identification. A ground truth of elimination and comparison result of inconclusive was observed more frequently at 74% (n = 106), while the ground truth of identification and comparison result of inconclusive was observed at a rate of 31% (n = 119). All items submitted as ground truth insufficient or unsuitable were satisfactorily determined as such. Furthermore, no ground truth submissions of identification or elimination were determined to be unsuitable or insufficient. Inconclusive decisions were only reported for items with a known ground truth of identification or elimination; however, due to the quality of the evidence submitted, inconclusive results were not unexpected. Table n = 1) and shot carriers (n = 2) were excluded from the data set because this evidence was not compared.Satisfactory results were obtained for all items evaluated, or, by the “hard error” definition , no hardThe data were examined at the comparison level, so the number of inconclusive conclusions may appear to be inflated when compared to casework rates. One item of evidence may have been determined to be inconclusive to multiple items of evidence, consequently appearing in the data set more than once. The inconclusive conclusions rendered were evaluated for trends. Data factors such as evidence type and complexity of the comparison were evaluated to determine whether these factors contributed to a higher rate of inconclusive results.n = 168) were the main contributor and then cartridge cases . When comparing the means between the bullet items (38.235) and cartridge cases (78.491), bullet items had a lower mean, which indicates more inconclusive conclusions since these conclusions were assigned a 0 in the data set. The difference between these means was statistically significant. Table n = 1) and shot carriers (n = 2).The data showed that evidence type significantly contributed to inconclusive conclusions. Specifically, bullet items  were submitted for this blind QC, all fired from the same firearm. The ground truth for all items was identification, with the exception of the two fragments, which were correctly concluded to be insufficient for examination.4The results presented here represent preliminary outcomes from a blind testing program in firearms examination over c. five and a half years. HFSC's blind QC program inserts mock firearms cases into the sectional workflow to mimic real casework, and the outcomes offer a glimpse into the complete process of firearms examination, from evidence submission to reporting of results. Notably, the results reflect outcomes when examiners were truly blind .Firearms examiners determined the correct ground truth result in over half (~60%) of comparisons. Inconclusive determinations were reached for 40% of comparisons; ground truth eliminations resulted in a higher rate of inconclusive responses (74%) than ground truth identifications (31%). This could be because identifications are generally considered easier to make based on individual characteristics alone. When an examiner arrives at an elimination conclusion, the examiner has determined that the examined items being fired in the same firearm would be, in their opinion, a practical impossibility. When class characteristics differ, elimination is the appropriate choice, by definition. However, when observable class characteristics do agree, the examiner must consider how variables such as differences in ammunition and condition of the bearing surfaces of the firearm  impact the individual characteristics imparted on fired bullets and casings.Lack of sufficient agreement of individual characteristics could be due to the previously mentioned factors  or to the items being fired in different firearms. Given that the burden of proof for an elimination conclusion is greater than that of identification, a more prudent conclusion could be inconclusive when the examiner cannot rule out factors that contribute to insufficient agreement of individual characteristics. HFSC firearms examiners can make conclusions of elimination on individual characteristics without having a firearm submitted; however, when a firearm is submitted, the examiners can use the firearm to create additional test fires and examine the bearing surfaces of the firearm. An elimination decision may be easier to conclude when a firearm is submitted.Inconclusive conclusions did not appear to be related to primary and second examiner pairings, examiner experience level, or the examiners' primary training locations. Most examiners are long‐stay examiners and have been working under the same procedures for years; thus, these results are not surprising. A little over half (51%) of the cases were created using two different firearms of the same class with the intent of making comparisons more challenging; however, this variable was shown to be an insignificant source of inconclusive results.Breaking down the inconclusive conclusions by evidence type showed that comparisons of bullet items resulted in inconclusive conclusions more often than cartridge cases . When conducting microscopic comparisons on bullets, there are fewer areas of interest at which to look  because only one part of the firearm contacts the bullet (barrel). Cartridge cases are contacted by more parts of the firearm, therefore providing more surfaces  and more information with which to make comparison conclusions. The brand of ammunition used can contribute significantly to differences in individual marks. Comparisons can be easier if the same brand of ammunition was used. Since the brand is typically discernable on cartridge cases, examiners can use the same brand of ammunition to create test fires, making comparisons easier. However, bullet comparisons can be more challenging because the brand of bullet is typically not discernable. Similar to Smith et al. , these rAs the results show, not all comparison conclusions are simply either identification or elimination. Neither HFSC nor AFTE considers inconclusive decisions to be errors or “hard errors” as defined by Smith et al. . Rather,Equating inconclusive determinations to not being able to exclude is inappropriate. The results suggest that examiners more easily identify cartridge cases than bullets, but an inconclusive result for comparison of either evidence type may be a ground truth of identification or elimination. Alternatively, an inconclusive decision means that not enough information is available to allow the examiner to conclude either identification or elimination. The distinction of the simple false positive or false negative as “hard errors,” as is done in many studies, does not account for all the possibilities in which the known ground truth might not be concluded. An inconclusive decision when a ground truth result is intended must also be examined as this could indicate a form of error in the analytical process. However, inconclusive conclusions must remain a viable result because the limits of currently used technology and test methods are not specific or sensitive enough to render a ground truth result in every situation. The results suggest that inconclusive results in firearms examination could be considered as analogous to a detection limit in a quantitative toxicology analysis.If a primary examiner makes an inconclusive decision, the reason for the conclusion must be explained in the case record. A second examiner will verify and agree or disagree with the decision. If the second examiner disagrees, the firearms section's consultation and conflict resolution policy  will be While blind QC cases are intended to mimic actual casework in packaging and offense type, the program does provide a unique opportunity to submit challenging cases that test the examiners' abilities. Philosophically, HFSC has chosen to specifically target challenging case scenarios in an effort to better define the limitations of analysis. Submitting “easy” comparisons in which the examiners are more likely to conclude elimination or identification instead of inconclusive would not make blind QC cases distinct from open proficiency tests. Focusing on challenging cases may result in blind QC data that does not accurately represent inconclusive rates in real casework. Rather, the intent is to have blinds be more sensitive than casework to sources of variance.4.1In the initial stages of the program, record keeping was inconsistent until the submission process became more comfortable. Which firearm was used to create the fired evidence was not consistently recorded, thus leaving gaps in understanding if the fired evidence was created using a firearm that produced robust or indistinct marks. In addition, while most items have a ground truth of identification or elimination, the manager preparing the evidence items has an idea about whether the examiner will conclude the ground truth or make an inconclusive determination. However, the ground truths for potential inconclusive determinations were not consistently documented, which made data analysis challenging. Moving forward, potentially inconclusive items should be documented or marked with a UV pen to better evaluate the data in future.Limitations also exist in resource availability. The most easily accessible source of firearms that can be used to create fired evidence is HFSC's reference collection; however, the collection does not contain all the firearms that are commonly seen in real casework. Firearms that are slated by HPD for destruction and HFSC personnel‐owned firearms can also be used to create fired evidence, but the firearms available through either source is limited in diversity and quality. One advantage to having access to HPD‐destruction firearms is being able to submit in blind QC cases firearms that examiners will not recognize, which would be the case if a reference collection firearm was submitted.4.2The firearms section at HFSC benefits from the blind QC program in several ways. Because the program is an extension of the open proficiency testing program, the section can more fully gauge their processes in the areas that the proficiency tests do not cover while still providing a ground truth. For example, proficiency tests typically consist of only bullets or only cartridge cases for microscopic comparisons, but blind QC cases can include both bullets and cartridge cases, as well as fragments and firearms. Blind QC cases also have the potential to test caliber determinations and trigger pull measurements. This allows the laboratory to be tested in areas on the scope of accreditation that proficiency tests cannot meet. Compared with proficiency tests, more challenging blind QC cases can be created to test the examiners' thresholds for determinations. Since blind QC cases mimic actual casework and are conducted blind, the cases allow for a more accurate and effective measure for how examiners and processes and procedures are operating. Measuring the entire workflow could provide the section with a way to potentially discover bottlenecks or areas for improvement in their processes and procedures, which is more difficult to do with proficiency tests and real casework. Furthermore, regular participation in the program gives the examiners the opportunity to bolster their credibility in court when testifying on real cases.4.3In November 2015, the firearms section implemented a blind verification procedure for select cases. In a typical case, the primary examiner's conclusions are visible to the second examiner. In a blind verification, the primary examiner's conclusions are masked from the second examiner, allowing the second examiner to conduct an independent examination with minimized bias from the primary examiner's conclusions. Blind verifications are selected by section management at a rate of one case per month and can be performed on real casework or blind QC cases. In future, HFSC would like to examine trends in blind verification cases as well the rates of inconclusive conclusions and consultations and conflicts in real casework.The firearms examination community will benefit from a more focused and narrowed experimental design. For example, formal research on the ability of firearms to leave distinct marks on bullets and casings could be used in court when testifying to inconclusive results. Deliberately creating and submitting evidence from firearms that do not mark well could be an advantageous and more specific route for the blind QC program to take next. Blind QC cases could be constructed well ahead of time and submitted with intention, depending on the research question being asked.Blind QC cases can be utilized for training new examiners or determining the efficacy of new technology. HFSC is in the early stages of exploring the use of a 3D imaging instrument in firearms examination. 3D imaging may make visible previously imperceptible details, which can be usable data points in comparisons. Using blind QC cases for this study will help determine whether 3D imaging technology will impact the firearms comparison practice. If firearms procedures or methods change, the blind QC program will inevitably have to change as well; thus, the program and possible research evolve naturally with the discipline.The Houston Forensic Science Center would also like to collaborate with other laboratories to expand the availability of firearms and ammunition to submit as blind QC cases. Not only would collaborating with other laboratories provide a bigger selection of evidence, but multiple laboratories could use test fires created from the same firearms providing opportunities for between‐laboratory comparisons of blind testing results. Another future direction is to collaborate with researchers to study rates of inconclusive decisions. Further studies could help address criticisms aimed at inconclusive decisions as well as provide a current standard in the field to determine whether new technologies  assist with inconclusive decisions. Such studies could help better define and improve sensitivity and specificity of firearms examination.


Bile acids play a significant role in the digestion of nutrients. In addition, bile acids perform a signaling function through their blood-circulating fraction. They regulate the activity of nuclear and membrane receptors, located in many tissues. The gut microbiota is an important factor influencing the effects of bile acids via enzymatic modification. Depending on the rate of healthy and pathogenic microbiota, a number of bile acids may support lipid and glucose homeostasis as well as shift to more toxic compounds participating in many pathological conditions. Thus, bile acids can be possible biomarkers of human pathology. However, the chemical structure of bile acids is similar and their analysis requires sensitive and specific methods of analysis. In this review, we provide information on the chemical structure and the biosynthesis of bile acids, their regulation, and their physiological role. In addition, the review describes the involvement of bile acids in various diseases of the digestive system, the approaches and challenges in the analysis of bile acids, and the prospects of their use in omics technologies. There has been an increased interest in omics technologies in the fields of biology and medicine in recent years. The term “omics” commonly refers to technologies that aim toward the achievement of genomics, transcriptomics, proteomics, and metabolomics. According to M. Bennett, metabolomics is “a systematic study of unique chemical ‘fingerprints’ specific to processes occurring in living cells”, that is, the study of their low-molecular metabolic profiles . The foc−9–10−11 M), and the introduction of the aforementioned method of analysis into routine practice is required for their systematic content. Because of this, the development of metabolomics was made possible using highly sensitive and specific methods of analysis, such as high-performance liquid chromatography (HPLC), nuclear magnetic resonance spectrometry (NMR), mass spectrometry, etc. [Many metabolites contained in the human body exist in very low concentrations , which are typically known for emulsifying lipids during digestion, have recently captured the interest of a number of researchers. However, with the development of modern methods of analysis, their other functions, connection with other body systems, and participation in metabolic pathways have also been identified . It has The review has a traditional structure and contains seven main sections, in addition to the Introduction. The first section reviews bile acids’ structure and their biosynthesis, along with their regulation. The second section focuses on the physiological functions of bile acids, with their emphasized signaling function, as well as relationships between bile acids and gut microbiota. The third section reviews the pathological conditions and diseases of the gastrointestinal tract which are associated with bile acids according to published studies. The “Approaches to the analysis of bile acids” section is about modern methods of analyzing bile acids in diagnostics, especially using high throughput chromatography and mass spectrometry as selective and sensitive methods for many bioactive molecules. “The metabolomics of bile acids” (earlier “Bile acids in omics technologies”) section describes previously published studies of the whole bile acids metabolome to assess the condition of the human organism with various diseases and during therapy. The section “The perspectives and limitations in medicine” reviews several limitations and difficulties attached to recent studies of the metabolome as well as an outlook of adaptation of actual data to clinical diagnostics. The traditional Conclusions section summarizes all outlined reviewed data and provides a conclusion about the growing role of the bile acids metabolome in clinical and diagnostical medicine.Bile acids (BAs) are a group of cholesterol derivatives which are produced during its catabolism. The structure of BAs includes a cholane ring of 24 carbon atoms condensed into 4 rings, a pentanoic chain with COOH-group in the C24 position, and 1–3 hydroxyl groups  5,6,7,8,87,8,9. Cis-trans isomerism is a characteristic for BAs due to the different orientation of the substituents in the C3-, C7-, and C12-positions, as well as methyl radicals in the C18- and C19-positions. In most cases, hydroxyl substituents have an α-orientation (“under the plane” of the molecule) and methyl radicals have a β-orientation (“above the plane” of the molecule). For this reason, the upper surface of BA molecules has hydrophobic properties while the lower surface is hydrophilic [rophilic .In the human body, BAs are in an ionized (deprotonated) state and have amphiphilic properties due to their different polarity at the molecular level. This determines their surface-active properties and fat-emulsifying ability. Depending on the composition of substituents in the cholane core, the amphiphilic balance of BAs may be different . For exaThe total amount of BAs synthesized in human de novo is about 5 g per day. Approximately 0.05 g of this is excreted both in urine and feces .The general scheme of BAs biosynthesis is shown in There are two pathways involved in BAs biosynthesis, namely “neutral” and “acidic”. A “neutral” pathway is more important for humans, producing more than 90% of free BAs ,15,16. I2+ ions. As a result, glycocholic (GCA), taurocholic (TCA), glycochenodeoxycholic (GCDCA), and tuarochenodeoxycholic (TCDCA) acids are produced [In the human body, primary BAs are poorly represented. Their conjugates in the C24 position with glycine and taurine are much more common because they are more polar and easier to excrete through the membrane of hepatocytes followed by biliary secretion . Conjugaproduced .Primary BAs are also conjugated with sulfonic acids. This metabolic pathway is associated with a pathological imbalance of BAs ,18. GeneThe “acidic” pathway of BAs synthesis is characteristic for neonatal development. In this pathway, the steroid ring is first hydroxylated in the C27 position by CYP2, followed by C7-hydroxylation mediated by oxysterol-7α-hydroxylase CYP27B1. In the liver, lung, and brain, some levels of 24 and 25-hydroxycholeterol are involved, and they also participate in the “acidic” pathway. Produced CDCA and CA are conjugated with glycine and taurine that is mediated by bile acid-CoA:amino acid N-acyltransferase ,11,14,192+ concentrations than free BAs [After conjugation with amino acids, primary BAs are transported across the hepatocyte membrane via BAs salts pump into bile ,17,20,21free BAs .Upon completion of the emulsification in the intestine, BAs are reabsorbed by apical sodium-dependent bile acid transporter (ASBT) located in intestinal microfibers. They are then transported by ileal bile acid-binding protein  to the basal membrane and then to the bloodstream via organic solute transporters (OSTα/OSTβ) ,22,23.Some BAs  also undergo enzymatic deconjugation by bile salts hydrolase (BSH) contained in the intestinal anaerobic microflora, resulting in the initial production of free, unconjugated CA and CDCA and the subsequent production of secondary BAs via epimerization and dehydroxylation reactions ,17. ThesSecondary BAs hydroxylated in two positions are intensively reabsorbed through the portal system of the liver. This process is known as “enterohepatic circulation” and repeats about six times per day ,16. It iMost of the BAs are then excreted with bile into the intestinal lumen, while some part circulates in the bloodstream ,11.BAs synthesis is regulated by nuclear transcription mechanisms. Most BAs interact with the farnesoid X receptor (FXR) in the liver through a negative feedback mechanism ,28. ActiDuring intestinal digestion, BAs first facilitate the anchoring of lipase and colipase on the surface of emulsion droplets, and then interact with the formed monoglycerides and free fatty acids, forming micelles which are then transferred through the intestinal wall ,31,32,33Hydrophobic BAs, due to their emulsifying ability, play an important role in digestion. They stimulate the production of cholesterol and phospholipids via sodium taurocholate-conveying peptide (NTCP) and cassette ATP-binding ABC transporters ,36,37. MHydrophilic BAs  are also involved in the emulsification of food fats due to their detergent activity. In addition, they reduce the absorption and synthesis of cholesterol and its entry into bile and, in contrast to hydrophobic BAs, stimulate the production of α-interferon in liver hepatocytes. Hydrophilic BAs also have a cytoprotective and anti-inflammatory effect ,43, i.e.The signal function of BAs is performed by their circulating fraction. The human blood contains about 28 BAs and their derivatives. The most abundant of them are polar compounds. i.e., CA, CDCA, and DCA .The BAs’ receptors might be divided on nuclear and membrane. The binding of BAs with nuclear receptors activates FXR, PXR, and vitamin D receptor systems, as well as xenobiotic sensors ,47,48. TThere are a number of tissues in which BAs’ receptors are located, i.e., hepatocytes, epithelium, cells of immunity, adipose tissue, etc. Some of them are listed and described in The most known pathway mediated with BAs is the FXR receptor system. Through this mechanism, BAs regulate both their own synthesis through FXR activation and homeostasis of lipid and glucose ,8,9,29.VDR is another pathway regulating the lipid–glucose homeostasis and influencing gut microbiota ,61. It iBile acid receptors have also been reported to be involved in a signaling role in other tissues and organs. Thus, multidrug resistance protein 4 located in proximal renal tubules and the ependyma of the choroid plexus is involved in bile acid and glutathione transportation ,79.Transporters such as the bile salt exporting pump, ABST, multidrug resistance protein 3/4, and the organic anion-transporting polypeptide were found to be expressed in the blood–brain barrier. They transport bile acids produced by gut bacteria ,81, whilEnterococcus faecium and E. faecalis are common species represented in the intestine. Moreover, E. hirae, E. durans, E. gallinarum, and E. casseliflavus have also been reported.The tight association of BAs with the intestinal microbiota is well known ,25,38,82The main method of nutrition for these microorganisms is enzymatic glycolysis, and the substrate for growth is plant polysaccharides, exfoliating epithelium of the intestinal mucosa, cellulose, and bile components.The condition of the intestinal microbiota is strongly influenced by factors such as diet, medication , and age. Recently, the impact on the intestinal microbiota from other organs of the digestive system has also been established. Kakiyama et al. have shown that the composition of the microbiota is associated with liver cirrhosis and the spectrum of bile acids produced by the liver ,84. ThusBAs are also closely related to the functioning of gut microbiota. Firstly, bacteria contribute to the formation of secondary BAs from primary ones. Secondly, BAs themselves have antimicrobial activity, inhibiting the growth of pathogenic microflora. An antimicrobial activity is related to the detergent properties of BAs, especially in unconjugated form . Under t7α-hydroxylating intestinal bacteria were shown to be responsible for the conversion of primary BAs into secondary ones and they synthesize proline and tryptophan-based antibiotics, inhibiting the growth of pathogenic microflora .Commensal bacteria can prevent the development of liver cancer by stimulating the synthesis of T-killers in hepatocytes with primary BAs . At the A key factor in the selection of probiotic bacteria seems to be the development of their resistance to the toxic effects of bile . Hence, A number of studies revealed the relationship of gastrointestinal diseases with changes in the level of BAs and their conjugates. The decrease of the BAs’ level was shown to be associated with to various metabolic diseases, i.e., dysbacteriosis , cholestSimultaneously, an increase in the level of BAs in urine and saliva might also be associated with pathological conditions, e.g., Barrett’s disease ,96 and gThus, the level of BAs and their conjugates is closely related to the state of the digestive system. It determines their potential value in clinical diagnostics; however, due to the large number of interactions in these relationships, their level also varies greatly. This has been shown for CA, GCA, TCA, and LCA . DetermiBesides their diagnostic utility, BAs and their derivatives can be used as therapeutic agents for treatment of various metabolic diseases . A classFirmicutes, Bacteroidetes, and Actinobacteria are healthy gut microbiota and express BSH lysing the amide bond and liberating free BAs. The BSH enzyme deconjugates the conjugated BAs  and thus regulates the detergent and antimicrobial action of BAs, preventing the microbiota from destruction [The close relationship between gut microbiota and BAs were confirmed by a number of scientific groups in recent years ,104,105.truction . Simultatruction .Meanwhile, in pathological statements and during antibiotics therapy, a healthy microbiota is suppressed, and the number of host bacterial flora is increased. Subsequently, the homeostasis of BAs is altered towards a high primary BAs/secondary BAs ratio . PrimaryIn a number of cases, a high fatty diet followed by an imbalance of BAs was shown to be the main pathological mechanism of dysbiosis ,111,112.Vdr gene expression [Lactobacillus and enrichment of Clostridium and Bacteroides in feces [ΔIEC) leads to microbial dysbiosis due to a decrease in the butyrate-producing bacteria [ΔIEC and VDR∆lyz mice in a gender-specific manner [Thus, one of the signaling systems regulating BAs homeostasis is vitamin D receptors, which are produced via pression . It was in feces . Moreovebacteria . LCA, DCc manner .Acetatifactor and Bacteroides [In addition to improving glucose and lipid sensitivity, the FXR pathway is also involved in gut microbiota metabolism. The activation of intestinal FXR induces the growth of lithocholic acid-producing bacteria teroides . The altteroides .One of the most serious dysbiotic statements is clostridial infection. BAs have shown to provide high antibacterial activity against Clostridiae .Crohn’s disease, or non-specific ulcerative colitis, is a chronic inflammatory bowel condition, which may affect any part of the gastrointestinal tract, and can be accompanied by abdominal pain, diarrhea, bowel obstruction, and weight loss. It is considered as immune-competent, though there were no antibodies to be detected. Furthermore, the symptoms of Crohn’s disease suggest that it arises due to dysregulation of intestinal immune cells. There are a number of triggering factors providing this dysregulation .A high fatty acids diet with a lack of fruit and vegetables is one such triggering factor contributing to pathogenesis . In thisThere is a clear shift of the BAs’ level in Crohn’s disease correlating with a chronical inflammation . Simulta+/+ organoids. The activation of the IFN I type pathway by DCA also contributes to Paneth cells dysfunction and inflammation [BAs do not only have a direct toxic action on immune cells, but they also lead to excess activation of the FXR pathway linked to them. The overexpression of FXR causes the reduction of Paneth cells in Fxrammation ,118.IL1B gene and subsequent secretion of another antimicrobial agent, IL-1β, is also induced by 1,25-dihydroxyvitamin D via VDR [An activation of VDR also was shown to be one of the Crohn’s disease mechanisms . Liu et  via VDR . Thus, iThe digestive and emulsifying activities of BAs towards fats have been described earlier. However, their highly possessive biological activities are the mediators and signaling pathways in glucose and lipid metabolism pathways see . Now, thVdr gene expression  is a well-known pre-cancer state of esophageal mucosa when squamous epithelium tends to be replaced with intestinal-type columnar epithelium . The totUnconjugated BAs are the crucial components of gastroduodenal reflux that might be attributed to chronic injury and BE inducing IL-6, Cdx2, or Notch1 gene expression activation and accelerating dysplasia . It is iAlong with the proinflammatory activity of free BAs, their glycine conjugates, i.e., GCA, GDCA, and GCDCA, were shown to be the major components in the esophageal aspirate of patients suffering gastroduodenal reflux disease ,133. TheIn the past 10 years, BAs have been shown to promote intestinal metaplasia of gastric mucosa ,108,134.Fgf15−/− mice which are the model of FXR knockout have been shown to be enriched by 7α-dehydroxylating bacteria in feces followed by an increased level of BAs in feces. The most increased BA for the mice is DCA [+ cells were observed in wild type mice receiving a high fatty acids diet dysregulating FXR function [Other widespread oncopathological conditions of the gastrointestinal tract are colorectal and liver cancer. According to studies, colorectal cancer is a diet-related disease, especially expressed in the case of high fatty acids western diets ,130,136.e is DCA . Such mifunction . The redfunction ,53,124 afunction . At the function . All theCXCL16 gene, whereas secondary BAs, in contrast, decrease it. The activity of Gram-positive bacteria, which conversed primary BAs to secondary, correlated in an inverse manner with the level of NKT in the liver and the number of tumors. The suppression of bacteria with antibiotics was sufficient to induce NKT cells and to inhibit tumor cells in mice. This treatment was accompanied by an increase in the CDCA level and a decrease in the GLCA level [Activity of BAs is also one of the main mechanisms of liver cancer ,124. TheCA level .One of the most urgent problems of oncology is the untimely diagnosis of tumor diseases at early stages. For a number of these, there are no unambiguous markers, for example, gastric carcinoma , and forThe ability of HPLC-M/MS to detect compounds at very low concentrations makes it possible to screen for oncological diseases at an early stage and in conjunction with other routine laboratory studies, as well as to track the dynamics and response to ongoing therapy.One of the biggest challenges of BAs’ mass-chromatographic analysis is the large number of compounds. They include up to 25 substances, with varying quantities within several orders of magnitude. Additionally, they have similar chemical structures and the same molecular weight. In this regard, the optimization of chromatographic conditions, ionization conditions, and mass spectrometric detection is important for the reliable identification and accuracy of the determination of BAs ,9,44,151Gas chromatography methods are less common due to the low volatility of BAs. Therefore, high-performance liquid chromatography combined with mass spectrometric detection (HPLC-MS) is most widely used for a number of different analytes ,95,106.The use of HPLC-MS/MS allows the creation of a fast way to assess the content of bile acids in objects of study with a complex matrix with simple sample preparation ,44.Recently, the determination of BAs in the blood plasma of patients has also become relevant. It is a more difficult task due to much lower concentrations of target compounds in comparison with other biological samples . Successful detection also requires the use of HPLC with MS/MS detection ,134.Mass spectrometric analysis of BAs in negative mode is more sensitive than analysis with positive ionization, which allows a reduction of the number of samples and aliquots.Our studies allowed us to choose necessary transitions for the selected reaction monitoring of 25 compounds, BAs, and their derivatives, which are listed in In short, the aim of metabolomics is to conclude whether metabolism interacts with various biological factors and its dependency on them. The data obtained using HPLC-MS/MS undergo bioinformatic analysis to constrain clinically useful information. Usually, the data are normalized and logarithmically transformed to make them more compact followed by classification. Principal component analysis (PCA) and independent component analysis (ICA) methods are used to classify the specimens ,155.In clinical practice, obtained data are usually compared to datasets with known parameters to evaluate their sensitivity and selectivity. One of the most frequently used methods is ROC (Receiver Operation Characteristic) analysis. Moreover, the risk of disease can be assessed by odds ratio, OD .Due to increasing use of mass-spectrometric analysis in biological and clinical practice, special databases were created. They allow the analysis and evaluation of obtained data more easily and adequately. They include Human Metabolome Database (HMDB), Madison Metabolomics Consortium Database (MMCD), Golm Metabolome Database, dbGaP database, and others .Being the products of lipid metabolism, which are dependent on lipid homeostasis, gut microbiota statement, cancer progression, and other conditions, BAs can be regarded as useful object of metabolomics. One of the most widespread approaches using BAs in omics technologies is their spectrum detection comparing to a sample of healthy humans. The use of BAs metabolomics for the assessment of organism health, i.e., liver injury, is not exclusive in recent years ,159,160.per os in rats. By using it, the temporal change of the BAs’ pool after UDCA administration was revealed, as well as its beneficial effect towards BAs correlating to cholestasis [We have already described the role of BAs as metabolic markers of liver diseases in the light of their role in human physiology. Liver injuries are widespread and include infectious hepatitis, alcohol liver disease ,157, nonlestasis .The BAs metabolome was used for the screening of alcohol liver disease in a mice model. Among various signaling pathways, the most statistically significant was FXR regulating BAs homeostasis. Further, selected BAs to assess the alcohol liver disease were measured. The level of CA and TCA was significantly higher in ethanol-consuming mice than in the control group. A slight, though non-significant, increase of DCA, TDCA, and THDCA was detected in the experimental group. No differences between EtOH-consuming and control mice were found for liver levels of HDCA, CDCA, UDCA, and TLCA .In addition, the BAs metabolome can be assessed in other pathologies, i.e., breast cancer  and chroOncology is an area of medicine where metabolomic studies are useful and provide perspective. In this light, the BAs’ profile was also studied to elucidate the potential biomarkers of breast cancer and differentiate it from benign breast diseases. UHPLC with quadrupole/time-of-flight mass spectrometry was used to measure the concentrations of BAs in the serum of 29 patients with benign breast diseases and 47 patients with breast cancer. The profile of BAs in patients with cancer differs from that of non-cancer patients. The cancer group had an increased level of CDCA and other free BAs, while the levels of conjugated BAs were decreased, especially for taurine-conjugated BAs. The most effective biomarker, the level of CDCA together with taurine-conjugated BAs, had a sensitivity of 95.0% and specificity of 92.3% .BAs metabolomics is also an area of interest for toxicologists. This approach was applied to cadmium (Cd), with its accumulation in the liver leading to dysfunction and fibrosis. The use of BAs in toxicology seems to be a better predictor of Cd-mediated liver disease than histopathological studies ,163.In this review, the metabolomics of BAs has been shown to provide great promise for clinical medicine and diagnostics. Still, there are some limitations to the mass-spectrometric detection of BAs coupled with HPLC despite the high specificity of the method. These should be taken into consideration when a metabolomic study is planned.The first important moment is the type of biological sample. The most abundant samples are biofluids, i.e., blood serum ,41,127, There has been a growing interest in non-invasive diagnostics in recent times ,165. ThiThe matrix effect is also crucial, given the fact that the target substances or metabolites are not inert concerning other substances contained in biological samples, for example, in blood serum ,167. IgnOne of the technical difficulties is a good enrollment of volunteers and patients in the study. As can be seen, BAs are involved in many biological signaling ways ,119,124,There are challenges related to the analysis of metabolomic data. Thus, the levels of many metabolites  differ by orders of magnitude, and MS/MS detection does not allow measuring them simultaneously with equal precision . InterprLast but not least, one factor limiting the metabolomic approach to BAs is the relatively high cost of mass spectrometry devices. At the same time, commercially available solutions for HPLC and HPLC-MS/MS  have permanently entered the market.In total, we can conclude that BAs are multipotent and informative biomolecules that potentially allow us to use them in diagnostics. Their chemical structure requires specific and selective methods of analysis, but it is for a reason, because it helps obtain the information that is crucial for a patient’s health. The metabolomic research is multistage, starting from the right choice of a biological sample and its pretreatment. The type of biological sample must be selected depending on the concentration of the target compound in it and possible interaction with a biological matrix. For a long time, blood serum and urine have seemed to be actual objects to assess BAs in humans due to the high amount of information, but non-invasive methods of biological sampling  are areas of interest for metabolomic research. In recent years, many effective automatized solutions for sample pretreatment have been developed. They significantly facilitate the following analysis of metabolites. At the end of the study, the proper choice of bioinformatic statistics is crucial to big data analysis.Despite the fact that metabolomics is the youngest of the “omics” technologies, it has also been the fastest moving in recent years. Among its advantages are its big information load about biochemical pathways, high selectivity and specificity, and broad spectrum of detected substances. For this reason, metabolomics is useful to study environmental pollution, the food industry, and medicine. Metabolomic approaches in medicine are designed to search the new biomarkers of pathological alignments, e.g., diabetes, heart diseases, and cancer. Moreover, the optimization of drug therapy and therapeutic drug monitoring are also possible with the development of metabolomics.In the light of metabolomics, the interest in BAs is increasing. This group of functionally similar compounds are involved in various physiological processes in the human organism. These processes include both the well-known digestive function and signaling function related to endocrine, immune, muscular systems, and whole lipid and glucose metabolism. Furthermore, BAs were shown to be informative markers of various metabolic diseases. Our recent studies have confirmed a number of published data that indicate metabolic conditions such as diabetes are accompanied by a change in the BAs’ profile. In part, the UDCA level in serum and feces can reflect the efficacy of the treatment as well as the state of gut microbiota.The approaches to the analysis of BAs usually focus on gas and high-performance liquid chromatography coupled with tandem mass spectrometry. They provide high sensitivity and specificity. At the same time, like any analytical method, GC/HPLC-MS/MS has its restrictions and challenges. These include adequate sample pretreatment intended to preserve key compounds and trial-and-error adjustment of the analysis conditions.Despite several complexities in the analysis of the whole BAs metabolome and the biological interpretations of results, the expanding knowledge about the role of bile acids in the human body has helped to develop new high-throughput methods of diagnostics of various diseases as well as their treatment.In summary, the role of bile acids in human physiology and pathological processes is very important. However, the role is still not completely clear and will be the subject of further research. As knowledge about the physiological role of bile acids in the human body expands, new concepts will appear that explain the reasons for the emergence and formation of various pathological processes that, to date, are unclear.