Lysosomal-associated protein transmembrane-4 beta (LAPTM4B), a novel oncogene, promotes tumorigenesis and may be a potential prognostic biomarker in several cancers. This study was to determine the clinical significance and biological roles of LAPTM4B in lung adenocarcinoma (LAC).n = 216), chemotherapy group (n = 29), chemotherapy efficacy group (n = 179), EGFR-TKIs group (n = 57) and 68 healthy controls. Statistical analysis was performed to explore the correlation between LAPTM4B expression and clinicopathological parameters in LAC. Kaplan-Meier analysis was performed to assess the prognostic significance of LAPTM4B in LAC. In vitro assays were performed to assess the biological roles of LAPTM4B in LAC cells. Western blotting assays were examined to identify the underlying pathways involved in the tumor-promoting role of LAPTM4B.LAPTM4B expression was analyzed by immunohistochemistry (IHC) of 63 LAC tumors. Serum levels of LAPTM4B were measured by enzyme-linked immuosorbent assays (ELISA). The study included untreated group  gene mutations. In addition, LAPTM4B plays important roles in EGFR-promoted cell proliferation, migration and invasion and gefitinib-induced apoptosis.Collectively, our data propose that LAPTM4B may be a cancer biomarker for LAC and a potential therapeutic target which facilitates the development of a novel therapeutic strategy against LAC.The online version of this article (10.1186/s12885-019-5506-7) contains supplementary material, which is available to authorized users. Lung cancer is the leading cause cancer-related death worldwide and there are approximately 85% of lung cancer being classified as non-small cell lung cancer (NSCLC) . Lung adLysosome-associated protein transmembrane-4 beta (LAPTM4B), a novel oncogene, was first identified in human hepatocellular carcinoma (HCC). LAPTM4B is tetratransmembrane lysosomal protein that is overexpressed and associated with poor prognosis in various malignancies including breast cancer, gallbladder cancer, ovarian cancer, HCC, gastric cancer and cervical cancer –15. In aIn this study, we first demonstrated that LAPTM4B level was significantly elevated in LAC tissues as wells as serum samples, and indicated poor survival. Further functional assays showed that LAPTM4B promoted the oncogenic phenotypes of LAC cells in vitro via PI3K/AKT and EMT signals. In addition, LAPTM4B expression level was found to be associated with EGFR gene mutations and could be influenced by mutant EGFR in LAC. Taken together, we suggest that LAPTM4B is related to LAC progression and might be a potential biomarker and theraputic target for LAC.2.Human bronchial epithelial BEAS-2B (CRL-9609) cells and lung adenocarcinoma cell lines A549 (CCL-185), H1975 (CRL-5908), HCC827 (CRL-2868) and H1299 (CRL-5803) were obtained from American Type Culture Collection . The PC9 cell line was preserved by our laboratory. These cell lines were authenticated by short tandem repeat analysis (STR) by Beijing Microread Gene Technology Co., Ltd.  in August, 2017. All the cell lines were confirmed without mycoplasma contamination. All the cell lines were maintained in RPMI-1640 supplemented with 10% FBS  and antibiotics (100 μg/mL streptomycin and 100 units/mL penicillin) and cultured at 37 °C in a humidified incubator with 5% COThe total 63 LAC tissue samples used in this study were histopathologically diagnosed and surgically treated in Beijing Cancer Hospital between 2013 and 2014. Survival information was available for all patients until March 4, 2018. This study was approved by the Ethics Committee of Beijing Cancer Hospital and written informed consent was obtained from all participants.Paraffin-embedded samples were cut into 4 μm and stained with H&E for tumor confirmation. Selected sections were immersed in 0.01 mol/L citrate buffer (PH 6.0) and incubated with and anti-LAPTM4B polyclonal antibody  at 4 °C overnight. Stained tissue sections were evaluated separately by two pathologists without any knowledge of the clinical parameters. Staining was scored according to the previous criteria .Between September 2017 and January 2018, 216 serum specimens with histopathologically confirmed lung adenocarcinoma were enrolled as the LAC group. None of the patients had received adjuvant chemotherapy, immunotherapy, or radiotherapy before. In addition, 68 healthy controls including 28 males and 40 females, with a mean age of 40 years (range from 23 to 61), were chosen at the Medical Examination Center of Beijing Cancer Hospital. In addition, 29 LAC patients having received chemotherapy were enrolled in the chemotherapy group and their blood samples were collected before and after treatment, respectively. 179 LAC patients who were undergoing their chemotherapy cycles in Beijing Cancer Hospital were selected. Moreover, 57 LAC patients harboring EGFR mutations and having received EGFR-TKI treatment were enrolled. A CT scan was performed to assess the tumor size prior to initiating treatment and was repeated every 2 months. The treatment effect was assessed based on response evaluation criteria in solid tumors (RECIST) guidelines [A total of 5 mL peripheral venous blood was obtained and then centrifuged to collect serum. Serum samples were stored at − 80 °C for further use. The serum LAPTM4B was detected using quantitative human LAPTM4B sandwich enzyme immunoassay kits  following manufacturer’s instructions. The ELISA readings were measured at 450 nm in a microplate reader.The western blot assay was performed according to the previous description . All theStable LAPTM4B overexpression  and the corresponding negative control cells were created through lentivirus infection. They were constructed by GeneChem . The specific LAPTM4B and EGFR siRNAs were synthesized by RiboBio  and the sequences are shown in Additional file The Cell Counting Kit-8  was used to access cell viability. Cells (1 × 10^3 /well) were seeded into 96-well plates and each condition was repeated in triplicate. All cells were incubate for 5 days and 10 μL CCK-8 solution was added to each well at each indicated time point. Then the results were measured at 450 nm using a microplate reader according to the manufacturer’s instructions.Cells were plated in 60-mm dishes (500 cells/well). After incubation for 14 days, the colonies were fixed in 4% formaldehyde and stained with 1% crystal violet. Finally, positive colony formation (> 50 cells/colony) was counted under a microscope. All experiments were repeated in triplicate wells.In vitro cell migration and invasion assay were performed following the previous study . For theThe HCC827 cells were incubated with 0.01 μM Gefitinib or DMSO and collected after 48-h incubation. Apoptosis was analyzed using an PE-Annexin V Apoptosis Detection Kit (BD Biosciences) with FACSCalibur flow cytometer (BD Biosciences).P < 0.05 was considered statistically significant. Measurement data was expressed as median  when the data did not meet the normal distribution. Associations between LAPTM4B expression and clinicopathological characteristics of LAC tissues were analyzed using the chi-squared test. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Mann-Whitney U test was used for comparisons of two independent groups. Analysis of variance (ANOVA) was used to compare the differences among three groups.The statistical analyses were evaluated using the Statistical Software Package for the Social Sciences  and We first analyzed LAPTM4B mRNA expression levels in LAC tissue from TCGA (The Cancer Genome Atlas) database and revealed that LAPTM4B was upregulated in LAC tissues compared with normal tissue samples Fig. a .Fig. 1In addition, we sought to characterize LAPTM4B expression in 63 LAC specimens in the context of various clinicopathological variables including patients outcome Fig. b. The IHIn order to test the serum LAPTM4B as the potential serological marker for LAC, we first examined the LAPTM4B expression in the serum of a large set of patients with LAC and found that the serum LAPTM4B levels were dramatically increased compared with healthy controls Table , Fig. 2aIn addition, we evaluated the treatment effects on the serum LAPTM4B levels of LAC patients. The serum LAPTM4B level was significantly decreased after chemotherapy in 29 LAC patients Table , Fig. 2cIn order to investigate the clinical significance of LAPTM4B in EGFR-TKIs sensitivity, we analyzed a cohort of 57 LAC patients with EGFR mutations. These patients had received EGFR-TKIs treatment, either as front line or salvage treatment. As shown in Table P < 0.001), at a cut off value of 2.761 ng/mL  curve assessing serum LAPTM4B as a diagnostic tool for detection of LAC against normal controls was 0.838 . BEAS-2B exhibited the lowest expression level of LAPTM4B. A549 showed relatively lower LAPTM4B expression than the other cell lines Fig. a, b. TheTo investigate the effects of LAPTM4B on the metastatic ability of LAC cells, we performed transwell assays and found that overexpression of LAPTM4B increased migration and invasion ability of A549 cells compared to the controls Fig. f, g. In To explore the molecular mechanisms underlying LAPTM4B functions as a tumor promoter in LAC, it was overexpressed or knocked down in A549 and HCC827 cells. Western blotting revealed that upregulating of LAPTM4B increased, while silencing LAPTM4B decreased the phosphorylation levels of AKT and GSK3β. However, the total AKT and GSK3β levels were unchanged . Moreover, our data also identified that OS and PFS of LAC patients were significantly worse in the LAPTM4B high expression group compared to the low expression group and the result was consistent with the previous study reporting that LAPTM4B was associated with poor survival in LAC but not in all NSCLC patients .n = 216) than those in the healthy controls. Furthermore, serum LAPTM4B levels were positively correlated with smoking, advanced clinical stages, lymph node metastasis, ALK rearrangements and EGFR mutations. In addition, we evaluated the value of serum LAPTM4B levels in chemotherapy response of LAC patients. The results showed that serum LAPTM4B levels were significantly decreased after chemotherapy. We also compared LAPTM4B levels in different chemotherapy efficacy groups and found that serum LAPTM4B levels of chemotherapy patients with PD and SD were much higher than those with PR and CR, suggesting LAPTM4B have the potential to predict chemotherapy response of LAC patients. EGFR gene mutations are detected in 30 to 40% of patients with NSCLC in China and associated with poor prognosis [LAPTM4B is a type III transmembrane protein with four putative transmembrane regions and highly expressed in LAC tissues, so we test whether serum levels of LAPTM4B could be used as a tumor marker in LAC. This is the first report investigating the value of serum LAPTM4B levels as a biomarker for the patients with LAC. Our results demonstrated that serum levels of LAPTM4B were much higher in a relatively large series of LAC samples  with EGFR mutations. These observations led us to investigate the association between LAPTM4B expression and EGFR-activating mutations. Of note, activating mutations within the EGFR tyrosine kinase domain including in-frame deletions in exon 19 and L858R point mutations in exon 21 were found to be predictors of clinical response to EGFR-TKIs . Our res-mutated NSCLC, treatment with EGFR-TKIs  is associated with response rates of 56 to 74% and a median progression-free survival of 10 to 14 months. In addition, the majority of patients will have disease progression within 1 to 2 years after treatment initiation due to the acquired resistance [As we known, activation of the PI3K-AKT-mTOR axis is more robustly induced by mutant EGFR than by wild-type EGFR. Phosphorylated EGFR being the docking site of PI3K stimulates the activation of AKT , and thesistance –49. Our There are several potential limitations in our study. The relative small size population were enrolled in the study, and larger collaborative studies needed to validate our report. In addition, the roles of LAPTM4B on the efficacy of EGFR-TKIs treatment in LAC should be further elucidated.In summary, our results revealed that LAPTM4B was overexpressed in LAC and high LAPTM4B expression indicated poor prognosis in patients with LAC. The expression level of LAPTM4B in the serum of patients with LAC is significantly associated with the tumor progression and treatment effects, suggesting that detection of serum LAPTM4B will facilitate the diagnosis and better predict treatment response. Further functional assay indicated LAPTM4B plays an important role in tumor proliferation and metastasis in LAC. In addition, LAPTM4B may take part in EGFR-mediated cell pro-survival and gefitinib resistance of LAC cells with EGFR mutations. These fruitful works indicate that LAPTM4B might be an important biomarker and a novel therapeutic target for LAC.Additional file 1:Table S1. Antibodies used in Western blot assay. (DOCX 13 kb)Additional file 2:Table S2. The sequence for RT-PCR primers and siRNAs. (DOCX 15 kb)


Post-hospital residential brain injury rehabilitation outcomes research is a complicated undertaking because of the custom-tailoring of interventions needed to meet the complex and unique need of each individual. As such, there tends to be great variability across program settings, which generally limits large-scale intervention studies. Growing literature demonstrates that post-hospital residential programs are beneficial. The main criticisms of this work include the absence of randomized-controlled studies, lack of clear definition of treatment types/settings, and small sample sizes. This study is a retrospective analysis of program evaluation data for a large, multi-site, national provider of post-hospital residential brain injury rehabilitation services. Specifically, outcome of participants completing Intensive Residential Rehabilitation (IRR) were compared to participants in the Residential Supported Living (RSL) program. Results demonstrate that participants in the IRR program improve and that participants in the RSL group preserve functional ability over time, suggesting that each program is effective in achieving its intended outcome. The IRR treatment group achieved significantly better outcomes than those in the same setting not receiving the intervention. To isolate treatment effects of IRR, a subsample of participants across program types were matched on time post-injury, age, and sex. The treatment effect of IRR was strengthened in this analysis, suggesting that chronicity alone does not account for the variance between the two groups. An estimated 9 million Americans currently live with a brain-injury–related disability.Given that hospital stays in recent years have shortened, individuals with moderate-to-severe brain injuries are being discharged needing more assistance, care, and intervention because of more significant medical, cognitive, physical, and behavioral problems at discharge than observed in earlier decades. This has resulted in a heavy demand for an effective continuum of post-hospital services for individuals with these injuries. The burden of care is often too excessive for successful management in the home or community setting and has increased the demand for developing effective post-hospital residential rehabilitation programs to maximize functional outcomes for these individuals. Simultaneously, coverage and reimbursement for needed services after hospital discharge is becoming increasingly sparse, yielding a gap in our healthcare that ultimately leads to both increased disability and additional financial impact on society.Demonstration of effective rehabilitation after brain injury is paramount for restoring function and productivity of the individuals impacted by an individualized array of chronic physical, cognitive, psychological, behavioral, and functional impairments. It is also important to justify the necessity of treatment and reimbursement for such treatment from third-party payers.Outcomes research in post-hospital brain injury rehabilitation is complex and often a complicated undertaking for a variety of reasons. The nature of brain injury rehabilitation requires the custom-tailoring of interventions to meet the complex and unique need of each individual. As such, there tends to be great variability across program settings, which generally limits large-scale intervention studies containing a viable control group.5–7 The combined results of these reviews establish evidence that post-hospital brain injury services are beneficial to the individuals served. Significant pre-post gains in functional ability have been demonstrated across program setting and type.8–14 Past studies have also demonstrated that individuals who received post-hospital rehabilitation services within the first year of injury saw the greatest gains,15–18 and that even those who are exposed to post-hospital rehabilitation services when they are a year or more post-injury still experience significant functional improvement as a result of participation.16–22 This is likely attributed to the fact that in the post-hospital phase, treatments target both the recovery of lost function/ability  and the teaching of compensatory techniques and skills that can improve function despite residual deficit. Whereas those receiving treatment earlier may benefit from both aspects of treatment, those receiving treatment after the first year may benefit more from the compensatory training components of rehabilitation.The current body of literature on effectiveness of post-hospital brain injury rehabilitation programs has established an optimistic base for continued study. Several review articles examining the effectiveness of the post-hospital rehabilitation programs have been completed to date.The main criticisms of this body of work include: 1) the overall absence of randomized-controlled studies (RCTs), which have unwittingly become the perceived gold standard for establishing reimbursement guidelines for healthcare, 2) lack of clear definition of treatment, and 3) small sample sizes.The present study is a retrospective analysis of program evaluation data for a large, multi-site, national provider of post-hospital brain injury rehabilitation services. Specifically, outcome of participants completing Intensive Residential Rehabilitation (IRR) were compared to participants in the Residential Supported Living (RSL) program.Programs are defined as follows.IRR is an outcome-oriented, goal-directed residential program designed to maximize functional outcomes for adults with acquired brain injury. Intensive skilled individual therapies are provided between 10 and 20 h per week with the goals of maximizing physical ability, cognitive ability, psychosocial/behavioral adjustment, independent living skills, and family/community reintegration, in addition to daily group interventions. The focus of the program is functional improvement over time and generalizing skills from the therapy office to application in real-life community settings.RSL is an activity-based residential program designed to preserve current/optimal level of independent functioning by providing customized activity to preserve physical ability, cognitive ability, psychosocial/behavioral adjustment, independent living skills, and family/community reintegration. Focus of the program is on maintaining functional stability over time and improving overall quality of life.8 this study is focused on the comparison of two residential treatment programs, one focused on intensive rehabilitation and the other focused on maintenance of current status, within the same framework of programs in order to assess the specific effectiveness of IRR. Simultaneously, this study is unique in design that the RSL group is a natural control group, which allows for a comparison between individuals in the same residential treatment programs who are not receiving the IRR intervention to those who are receiving the IRR intervention.Where many other studies have treatment groups that are comprised of both residential and community-based interventions,The aim of this study is to 1) examine the characteristics of participants in a national network of post-inpatient brain injury services, 2) compare outcomes of participants across treatment-type groups, and 3) utilize a matched subset comparison of outcomes between treatment groups in order to further isolate treatment effect.Our hypotheses are centered on the idea that intensive post-acute residential rehabilitation does positively impact outcome for participants, independent of chronicity. Specific hypotheses were as follows: 1) Participation in IRR program is associated with improvement in outcomes, 2) participation in the IRR program results in significant functional improvement relative to participation in RSL program, and 3) the effect of IRR treatment is strengthened when chronicity is more rigorously controlled.A retrospective before/after observational study was conducted. Data were collected from all participants served between 2010 and 2015 within a national network of eight residential and outpatient programs across six states. Upon admission to the programs, participants consented to having their progress and outcome data available for analysis. The Indiana University Institutional Review Board provided exemption for analysis of de-identified data.The initial sample included 462 cases. Cases with atypical lengths of stay who were in the Intensive Rehabilitation programs were excluded. This included 2 cases with less than 7 days and 35 cases with more than 365 days in the program.n = 161); 2) Neurobehavioral Rehabilitation (n = 57); 3) Day Treatment (n = 38); 4) Supported Living (n = 131); 5) Neurobehavioral Supported Living (n = 25); and 6) Day Activity (n = 13). These six program types were further grouped into two major program categories: 1) IRR (program types 1–3), in which the overarching goal was to achieve significant gains through an intensive rehabilitation process in the residential setting, and 2) RSL (program types 4–6), in which the goal was to assist the individual in maintaining current status over the long term and enhance quality of life. Characteristics of participants in each program category on admission are described in The final sample included 425 individuals in six treatment tracks within the residential rehabilitation setting: 1) Neurorehabilitation  website (www.tbims.org).The primary outcome measure analyzed was the Mayo-Portland Adaptability Inventory-4 (MPAI-4). The MPAI-4Demographic information, injury-related characteristics, and MPAI-4 consensus ratings were recorded at admission for all 425 participants. MPAI-4 consensus ratings were recorded for the IRR group at discharge  to compare pre-treatment and post-treatment scores. MPAI-4 ratings for the RSL group were recorded on admission and annually to assess change over time. For the purpose of this analysis, we compared the admission MPAI ratings to the first annual reassessment ratings for the RSL group.t-tests or chi square tests, as appropriate. Differences on these variables among the program types in the IRR category were also examined using the same procedures. Differences among program types in the RSL category were not analyzed because of small numbers in two of the program types in this category. Because chronicity (days post-injury) was highly skewed, the log10 conversion of this variable was used in all computations. T-scores for the MPAI-4 Total and Index scores were derived with reference to a large sample used to compute T-scores in a previous study5 to make results comparable to those obtained in the earlier study. Algorithms for T-score derivation were provided by Inventive Software Solutions. Differences among the two major program categories on MPAI-4 Index and Total scores at discharge were evaluated using analysis of covariance (ANCOVA) with MPAI-4 score on admission, sex, age at injury, and log chronicity as covariates. Using similar procedures, ANCOVA analysis of discharge MPAI-4 scores were also conducted contrasting the program types within the IRR category.Differences in demographic, injury-related, and MPAI-4 variables on admission between the two major program categories were examined using 33 The MCID, defined as the smallest change in score that indicates a clinical significant change in functional status, was determined to be 5 T-score points or 0.5 SD. The RCID, defined as a change in score that indicates a clearly substantial change in functional status, was determined to be 9 T-score points. We compared the proportion achieving an MCID and an RCID between program categories and types.A previous publication also identified the minimal clinically important difference (MCID) and the robust clinically important difference (RCID) for the MPAI-4.Time post-injury (chronicity) was much greater for the RSL group than for the IRR group, and this variable was significantly related to outcome. In order to evaluate the degree to which chronicity explained the differences between the two major program categories more rigorously than can be accomplished through covariate analysis, we matched a group of RSL participants to a group of the IRR participants on chronicity. We then compared these two matched groups on the discharge/second assessment MPAI-4 T-scores.t = 5.12; p < 0.001), with those in the IRR group being slightly older at the time of injury. The two groups did not differ significantly on sex, age at admission, or ethnicity. Both groups were predominantly comprised of white male participants. Ninety percent of the IRR participants were male, and 85% of the RSL group was male. The IRR group was 69% Caucasian, and the RSL group was 74% Caucasian.Group comparisons were made between the IRR and RSL groups across demographic, injury-related, and funding source variables. With regard to demographic variables measured at the time of admission, the IRR and RSL groups differed significantly on age at injury , with the RSL group demonstrating higher chronicity (measured as mean days post-injury at time of admission). On average, IRR participants were 1.8 years post-injury compared to 7.5 years for the RSL groups. Both groups were similar in distribution across injury types. Closed traumatic brain injuries were the most prevalent across groups (IRR = 84% and RSL = 76%). Open brain injuries occurred at a higher proportion in the RSL group (7.1%) compared to the IRR group (2%). There were no other significant differences between the two groups on injury-type categories.With regard to injury-related variables, the IRR and RSL groups were significantly different on logBoth groups were similar, in that slightly over half of the participants were injured in work-related accidents and treatment was funded through Workers' Compensation insurance. The IRR group had a significantly higher proportion of participants covered by both other insurance (14%) and military/VA (12%) funding than the RSL group did. The RSL group had a significantly higher proportion of private pay or lien cases (23%) than the IRR group (17%).The two groups were compared on mean MPAI-4 T Index and Total scores at admission. There were no significant differences between the IRR and RSL group on mean Ability Index, Adjustment Index, Participation Index, or Total T-scores.F = 56.97; p < 0.001) with a large effect size . Admission MPAI-4 Total T-score was highly associated with outcome . Other covariates were also significantly associated with outcome: log chronicity  and age at injury . Effect size for change in the program for the IRR group without reference to the RSL group was very large, that is, around 1 SD , Adjustment Index , and Participation Index . Change over time for these variables is illustrated in MPAI-4 Index T-scores were subsequently analyzed using similar ANCOVA procedures with similar results. After covarying admission MPAI-4 Index T-score, age at injury, and log chronicity, program category was significantly associated with MPAI-4 discharge/second assessment T-score for the Ability Index  than in the Neurorehabilitation (64.0%) or Day Treatment (68.8%) programs relative to other racial/ethnic groups . IRR program types also differed on admission MPAI-4 Ability Index , Adjustment Index , Participation Index , and Total Score . Post-hoc least significant difference comparisons among the three program types indicated that greater disability for the Neurobehavioral group was recorded on the Ability Index, Participation Index, and Total Score than for the Neurorehabilitation group, which, in turn, showed greater disability on these measures than the Day Treatment group. The Neurobehavioral group showed greater disability on the Adjustment Index on admission than either of the other two program types. Admission characteristics of each IRR program type are detailed in Analysis of variance revealed no differences among IRR program types in length of stay (LOS), age at injury, age on admission, or logF = 2.95; p < 0.054); however, when correction for multiple comparisons was applied, this difference was no longer significant. Discharge T-scores by program type are displayed in ANCOVAs controlling for admission MPAI-4 T-score, age at injury, and log chronicity showed no significant differences among the IRR program types on discharge MPAI-4 Index and Total T-scores. The ANCOVA on the Ability Index was marginal . Similarly, a small proportion of those in Day Treatment (39.5%) achieved an RCID or better than in Neurorehabilitation (54.0%) or Neurobehavioral (61.4%) programs, but this difference was also not statistically significant . In both cases, the relatively small number of Day Treatment participants (n = 38) makes the estimate of the percentage of successful cases less statistically reliable.A smaller proportion of Day Treatment program participants (60.5%) achieved an MCID or better at discharge than participants in the Neurorehabilitation (77.0%) or Neurobehavioral (77.2%) programs. However, this difference was not statistically significant . In the RSL group, 75.8% were funded by Workers' Compensation and 3.5% by military, commercial, or no-fault insurance, whereas in the IRR group 37.9% were funded by Workers' Compensation and with 38.0% by military, commercial, or no-fault insurance. t-tests revealed no significant difference between groups on age on admission, age at injury, or log chronicity. LOS was longer in the RSL programs (mean = 5080.72 days) than in IRR programs . Admission T-score on the Adjustment Index was higher for participants in IRR programs than for those in RSL programs . There were no significant differences between groups on admission on the Ability Index, Participation Index, or Total T-score.Chronicity differed between participants in IRR and RSL programs and was significantly related to outcome. In order to control for this variable more rigorously than by using it as a covariate, we identified a sample of participants in both program categories matched on chronicity as well as age and sex. This sample consisted of 58 individuals (29 in each program category). There were no significant differences between IRR and RSL participants on sex, injury type, or ethnicity. Funding source did differ between the two groups , Adjustment Index , Participation Index , and Total T-score . Probably because error variance was more constrained in this matched sample, effect sizes are notably large. Admission and discharge/second assessment MPAI-4 T-scores are displayed in ANCOVA, covarying initial T-score, age at injury, and log chronicity, as in previous analyses, revealed significant differences between IRR and RSL participants at discharge for MPAI-4 Ability Index . Similarly, an RCID or better was achieved by larger proportion of IRR participants (69.0%) than RSL participants  (see An MCID or better was achieved by a larger proportion of those in IRR programs (79.3%) than in RSL programs  see .Results of this study contribute to the current brain injury rehabilitation effectiveness literature in several important ways. First, results demonstrate that participants in the IRR program significantly improve over the course of treatment and that participants in the RSL group preserved functional ability over the course of 1 year, suggesting that although each program is effective in achieving its intended outcome, the IRR treatment group achieved significantly better outcomes than those in the same setting not receiving the intervention. In order to more rigorously isolate treatment effects of IRR, a subsample of participants matched on time post-injury, age, and sex were analyzed. By controlling for time post-injury, the treatment effect of IRR was strengthened, suggesting that chronicity alone does not account for the variance between the two groups.Past literature has emphasized the role that chronicity is thought to play in the ability of IRR to produce a significant impact on functional improvement after acquired brain injury. It is important to note that in this study, the mean number of days post-injury at admission for the IRR group was 1.8 years, with a range of 14 days to 39.1 years. For the RSL group, the mean time post-injury at admission was 7.6 years with a range of 43 days to 36.1 years. The overlap between the two groups on time post-injury suggests that other factors besides time-post injury determine program entry, including funding and target outcome of the admission. The results of the matched sample comparisons speaks to the fact that IRR improves functional outcomes for individuals regardless of timing post-injury. Overall, these findings suggest that individuals do benefit from the range of services provided and underscores the need to understand the individualized nature of post-hospital treatment planning and timing of that treatment.The MPAI-4 as an outcome measure for post-acute rehabilitation is widely accepted. However, it is insensitive to more subtle changes, changes in very low functioning individuals, or changes that occur in higher-functioning individuals with very specific goals across a limited number of functions. It does not directly measure objective or subjective burden-of-care experiences.Information about the longer-term outcomes of these programs cannot be provided, thus limiting the ability to make assumptions about the durability of these program outcomes.Although participants in each program type received the same type of intervention framework with customized components based on individual need, this study did not examine the number of specific services received by each participant or the duration of intervention. Thus, we are not able to glean any information about potential dose-response impacts on outcomes. Similarly, predictors of outcome for each group are not addressed. Future research endeavors will address these limitations.One final limitation that is important to note is that although we attempted to more closely approximate an experimental design than past studies, the retrospective analysis clearly lacks the critical features of a true experiment, for example, prospective random assignment to control and experimental conditions.Chronic disability as a result of acquired brain injury is an expensive burden on society. Rehabilitation that is effective in restoring independence and productivity and reimbursement of such rehabilitation is of critical importance in reducing overall burden on the individual, the family, and society.Intensive rehabilitation programs result in significant functional improvements whereas supported living programs support stable functioning over time. The effectiveness of IRR is more clearly demonstrated when treatment sample is homogenously residential versus studies that have mixed residential and community-based IRR samples. Improvement in functional ability is significant for those who participate in residential IRR programs, regardless of chronicity.There is a continued need for collaboration across providers to collectively explore impact and expected outcomes associated with various post-inpatient rehabilitation programs.


The purpose of this study was to fill a gap in the existing research by exploring policies and practices of domestic violence shelters in one US state pertaining to clients with opioid use disorders (OUDs), as well as documenting some of their experiences providing services to those clients. We used semi-structured key informant interviews to gather information about Rhode Island shelter practices and policies pertaining to OUD-relevant topics and make meaning of shelter workers’ experiences with clients with OUDs. One researcher conducted all 30-min, telephone interviews. Qualitative data were analyzed using a content-based analysis approach. The open-ended interview questions yielded information that clustered in the following three main themes: (1) existing shelter policies and practices; (2) staff training on OUDs; and (3) ideas for improvement. Intimate partner violence (IPV) service providers reported that OUD is an issue that affects their clientele, creates problems for both IPV survivors and for staff who are helping them heal from IPV, and causes concerns about the safety of children and other shelter residents who may be housed with individuals with OUDs. Participants reported a range of policies and practices related to how IPV survivors with OUDs are served by their programs. They also offered multiple possible improvements that could be made to IPV survivor programming. Among their suggestions were the establishment of long-term housing, hiring substance use disorder specialists to work in IPV shelters, and improving interagency relationships between IPV programs, child protection services, and substance use disorder treatment providers. Some close-ended interview questions permitted calculations about the percentage of programs that had particular policies in place. For example, of the six programs, 50% (n = 3) reported that they keep naloxone on site. Only one of the six programs (18%) reported that they have a protocol for disposing of unused opioids, medications for OUD, or drug paraphernalia if it is found at the shelter. Additional data about the prevalence of OUDs among the IPV shelter population is needed, as are in-depth analyses of barriers and facilitators to OUD treatment for IPV survivors. In 2016, there were 42 249 opioid-related overdose deaths in the United States, or 13.3 per 100 000 persons.5 More than 5% (5.4%) of US women have these events occur in a 12-month period. Intimate partner violence has numerous health consequences. Affected women are three to five times more likely than other women to exhibit post-traumatic stress disorder (PTSD), depression, suicidal ideation, and substance use.6 Around 42% of women who have experienced IPV sustain injuries.7Intimate partner violence (IPV) is also a major public health concern. According to the U.S. Centers for Disease Control and Prevention (CDC), approximately 36% or 43.6 million US women have experienced sexual or physical violence, or stalking, by an intimate partner during their lifetimes.5 and that female survivors of IPV were at three times the risk for OUD as compared with male IPV survivors.8 There are several reasons why IPV survivors may be at increased risk for OUD. Between 51% and 75% of IPV survivors experience PTSD, and people with PTSD have 60% increased odds of developing an OUD than those without PTSD.9 Substance use is a common coping mechanism for IPV survivors.11 Some IPV survivors are encouraged or forced to take opioids by abusive partners who want to keep them docile, dependent, or want them to appear irresponsible to the court in custody cases.11 Not only is IPV associated with the risk for subsequent OUD, but drug use has also been associated with increased risk of IPV victimization.6 This suggests that the relationship between OUD and IPV may be bidirectional and reciprocal. Furthermore, partners’ tactics of controlling access to drugs,12 as well as the stigma associated with drug use, can increase relationship dependencies and complicate efforts to leave the relationship.15There is substantial overlap in IPV victimization and OUD morbidity; women who experience IPV are at increased risk of OUD. A recent nationally representative study found that IPV survivors were at 24 times the risk for OUD as those with no IPV history (2.4% vs 0.1%)14 However, a previous survey of IPV shelters in one state found that only half of the programs had policies related to clients who use substances, and only one-fourth had memoranda of agreement with substance use treatment providers.16 Research in both rural and urban communities has indicated that women face significant affordability, availability, and accessibility barriers to health and mental health service use and that women with victimization histories face additional barriers more than women without such histories.7 Working with IPV survivors with OUDs requires specific knowledge about responding to overdoses and facilitating access to OUD treatment, so focusing on improving collaborations between OUD treatment providers and IPV programs, specifically, makes sense. Despite recognition that IPV programs could, and should, be addressing the substance use related needs of survivors more comprehensively, there has been little research on this topic to help guide shelter workers in developing better policies. Dr Carole Washaw, the Director of the National Center on Domestic Violence, Trauma & Mental Health, has provided webinars and other trainings to IPV services providers on the intersection between IPV and OUDs that, anecdotally, have been very helpful to the community of IPV service providers, but too few published, peer-reviewed articles are available on the topic.8There have been calls for better collaboration between domestic violence and substance use treatment providers in general for more than a decade.The purpose of this study was to fill a gap in the existing research by exploring policies and practices of domestic violence shelters in one US state pertaining to clients with OUDs, as well as documenting some of their experiences providing services to those clients. Note that although the term “intimate partner violence” is more commonly used by researchers in research reports, the term “domestic violence” is still in use in the field . Therefore, both terms are used interchangeably in this article.The study design was exploratory and descriptive. We used semi-structured key informant interviews to gather information about Rhode Island shelter practices and policies pertaining to OUD-relevant topics, and make meaning of shelter workers’ experiences with clients with OUDs.The first author attended an in-person meeting of the executive directors (EDs) of RICADV programs in February 2017. The five EDs of the five programs that are full members of the RICADV were present, as well as one ED of one of the four partial member programs. All EDs present at the meeting were asked if they would consider participating in a study about IPV survivor clients and OUDs. All six EDs expressed support for the research project during the meeting with no deliberation. Subsequently, the research team submitted a protocol to the institutional review board (IRB) of the first and third authors. The research was determined to be exempt. Next, the first author emailed the EDs who had been present at the February meeting to ask them if they would schedule interviews. All six EDs responded that telephone interviews could be arranged with them or a representative of their program. Therefore, the inclusion criteria were either (1) being the ED of one of the five full member programs of the RICADV, or one of the partial member programs, or (2) being the ED’s designee. In two out of six cases, EDs nominated designees because they felt that a different program staff person had more knowledge about the topic of OUDs among program clients that they did themselves. The sample size was considered appropriate because it was comprehensive of RICADV full member programs, the study was not funded (ie resources for recruitment and interviews were limited), and because this was an exploratory, qualitative study.The first author conducted all six interviews over the telephone. Each interview lasted approximately 30 min. Five interviews were one-on-one, and one interview included one ED and two additional staff members of an IPV program. The interviewer took detailed notes during the interviews on a computer and typed quick enough to capture word-for-word quotations for most responses. In instances when the key informant made a comment that the interviewer anticipated might be particularly germane, given the research questions of interest (eg telling a story about an opioid-related overdose or describing a policy), the key informant was asked to repeat the comment slowly so that the interviewer could capture it precisely. The comment was then read back to the key informant to verify that their statement had been captured accurately.9 Sample interview protocol questions included: “What anecdotes or stories can you share about clients and opioid use as ‘lessons learned’ for the field?” “What are your personal feelings about substance using-survivors remaining in shelter and/or continuing to receive services?” and “If you could have all the money in the world, and do anything you wanted to improve conditions for your program clients in terms of opioid use problems, what would you do?”The interviewer used the same interview protocol (ie set of interview questions) for each interview and asked the questions in the same order. Most questions were open ended, although there were five closed-ended questions asked to obtain some factual, programmatic information  reported that they keep naloxone on site. Only one of the six programs (18%) reported that they have a protocol for disposing of unused opioids, medications for OUD or drug paraphernalia if it is found at the shelter. None of the programs reported that they track the number of their clients who have substance use disorders.The open-ended interview questions yielded information that clustered into that following three main themes: (1) existing shelter policies and practices; (2) staff training on OUDs; and (3) ideas for improvement. Each theme is presented in greater detail below.The main sub-topics related to shelter policies and practices pertained to (1) whether or not programs assessed prospective clients’ substance use disorder needs at program intake, (2) why policies about barring people with OUDs from shelters had evolved over time; (3) whether programs made referrals to inpatient or outpatient programs for clients with OUDs; and (4) how programs confronted challenges related to rural isolation and transportation on behalf of clients who needed to get to OUD treatment.I’m 30 years into this movement, and in the past 10 years this has become a big problem. It had stopped for a minute and now we’re seeing it again. It is so difficult for [the clients] to stay sober. I’ve had many sleepless nights trying to get them through.Key informants prefaced their comments about program policies and practices with some discussion of whether OUD were prevalent among their service population, and most noted that they had observed an increase in OUD among clients recently. A common theme was that there had been a recent increase in the number of clients who needed or deserved more support for OUD and that they were anxious and upset by the extent to which client need for quality substance use disorder services outweighed what was available. In the words of one program director,A particular concern about under-attention to the issue of OUD in IPV survivor clients was that Black women receive disproportionately less support. As one program staff person explained, she was concerned that staff could be quicker to identify substance use disorder problems in White and Latina clients, but overlook the needs of Black women clients because “there is a stereotype that Black women are strong so [they] don’t get offered the services that they need.”One recurring theme was that programs did not take a uniform approach to the question of whether or not to ask about substance use during a shelter intake interview, and the programs had clearly weighed pros and cons of their practices. Two shelters reported no screening policy at intake. This was the case even for one shelter that explained that they are “technically a dry shelter, but we don’t have substance abuse as a screening question when they check in.” The other shelter commented: “We do not have a policy to screen new clients [for substance use], because policies can create an unhealthy behavior where there is no wiggle room.” This program, like most others, reported that they handle cases “situation by situation.” In part, decisions about whether or not to shelter, or to continue to shelter, clients who are actively using substances depend on whether there are minor children also residing in the shelter and if the client’s substance use could conceivably cause harm to others (ie leaving needles in a room).NB: “clean” is a slang term for substance-free that is not used by treatment providers, but is used here to preserve the integrity of the data). Other programs had gone through similar evolutions in their approach, and as one staff person explained,It used to be that we took a real hard line on it, that you had to be clean for six months, and I think that’s a really unrealistic approach because the people we serve are in crisis. So we’ve relaxed our rules … That approach was unrealistic because it encourages clients to lie to us and we want to get them the help that they need … about 10-15 years ago was the beginning of starting to relax rules a little bit. We went from [a requirement of having been] six months clean to three months clean and then down from there. It’s mostly about safety for other clients living there.Other shelter programs reported a more flexible approach that had evolved over time. For example, one stated that at one time, they had a policy that clients “had to be clean,” but that they now allow clients with OUD and other substance use disorders to remain in their shelter if they were enrolled in an outpatient treatment program and not actively or obviously using . One additional staff person said that her agency would be participating in a Narcan training in the near future and that they did not presently keep Narcan on site. Of note, two of the six programs had been able to have staff participate in a Narcan training through a state Department of Health program, and the fact that the trainings were free to the IPV programs was a major factor in their decision to participate. As one program staff person said: “Anything free regarding stuff going on in the world, I’m there.”Two programs do not keep Narcan on site and have not yet trained staff to administer it. They had reasons why this was the case. One program said that staff were encouraged to call 911 for all emergencies, including opioid-related overdoses, for safety reasons. She was worried that they might administer Narcan incorrectly even after training and that there could be liability issues or other problems because, in her words: “We aren’t medical professionals.” This sentiment was echoed by another research participant, who explained “We are not supposed to dispense aspirin, so talk about assisting someone who is in overdose! I wonder about the legality of that? We’re not nurses.”It can be dangerous for women who have to leave the shelter to get methadone or go to a clinic because those types of places are limited nearby, and so if she was using with her abusive partner, she could run into him there. One woman told me, “he saw me, he chased me.” I had to take one survivor, she was so scared, to get her methadone. So even if they are trying to get well, there is this other piece of danger.Key informants were asked to reflect on their experiences providing direct service to IPV survivors who had OUDs. Each expressed deep empathy for program clients and were quick to point out the myriad ways in which coping with an OUD could present additional challenges and/or dangers. One key informant located in a non-urban area reported that the relative lack of OUD treatment providers was a particular concern because of the potential for the survivor to see the perpetrator of IPV at the treatment provider agency. In her words,Thus, a primary recommendation for improving services for IPV survivors with OUDs was to facilitate transportation to OUD treatment agencies, and to do so in a way that took into account the potential danger that IPV survivors face when they need to go in-person to a treatment location.It is challenging when you have children in the shelter and [you are] running a harm reduction [model]. Sometimes the actively abusing person is high or drunk at the shelter and acts in a way that is not adult-like, not causing harm but swearing and yelling and engaging in behavior that is disruptive, and that does seem more complicated when there are small children.A second recurring theme was that program staff worried about children’s safety in shelters where opioid-using clients were permitted to stay, and wished that there were separate transitional housing programs for IPV survivors in recovery. In the words of one key informant,I would have more housing. I am not a big fan of “shelter” because everyone needs permanent housing in the long run, so affordable housing should be the goal.Another solution informants proposed was building more interagency relationships, and as a community ensuring a greater variety of services to help “meet clients where they are.” Several stressed that permanent housing rather than short-term shelters would be beneficial. In the words of one informant,I would eliminate the concept of shelter if I could. I would look at someone on the road to recovery … and give her permanent supportive housing with support services including relapse prevention, [IPV] education and knowledge, all those components. When you are an addict, it’s severe and shelter is temporary.Another expressed an almost identical sentiment:The piece that is really concerning is that there is still a breakdown in communication with [child protective services] and the [intimate partner violence] community and the substance abuse community.Multiple informants also said that having trained substance use disorder counselors on staff at the IPV program would be ideal. Finally, all of the informants reported that they saw stronger interagency relationships as essential to improved responses to IPV survivors with OUDs in the future. There have been many gains in recent decades in terms of how IPV programs, substance use treatment programs, and state agencies work together, but informants felt that there was still a need to work on creating even stronger partnerships. In the words of one informant,This research resulted in several novel findings for both the IPV and OUD prevention fields. First, IPV shelter providers reported that OUD is an issue that affects their clientele, creates challenges for both IPV survivors and for staff who are helping them heal from IPV, and causes concerns about the safety of children and other shelter residents who may be housed with individuals with OUD. Second, participants reported a range of policies and practices related to how IPV survivors with OUD are served by their programs. Uniformity across programs may not be a necessary or worthwhile goal, but understanding the rationales for particular approaches and potentially best practices with regard to several aspects of IPV survivor services would benefit the field. Specifically, information about the effect of screening or not screening prospective clients for OUD at program intake would be useful, as well as information about whether and how referrals to OUD treatment are made, and assessments of the impact of training IPV program staff about OUD and related topics such as the use of naloxone or safe disposal of opioids and drug paraphernalia are urgently needed. Third, participants in this research offered multiple insightful possible improvements that could be made to IPV survivor programming. Among their suggestions were the establishment of long-term housing; hiring substance use disorder specialists to work in IPV shelters; and improving interagency relationships between IPV programs, child protection services, and substance use disorder treatment providers.Additional observations about the findings are that all the IPV program providers expressed respect for individuals with OUD, and each one articulated reasons why recovering from IPV can be more complicated for those with OUD (and vice versa). In cases where some reluctance about additional staff training on OUD was detected, there was a sense that it was because IPV program staff are already overburdened, overwhelmed and have lengthy lists of types of additional training that would be beneficial . When EDs need to be make choices about how to prioritize topics for training staff or forging new community-based interagency alliances, they are forced to rank-order the most pressing impediments to the health, safety, and welfare of their clients—whether OUD will be at the top of the list in the years ahead is unclear, in part because we lack data about how prevalent OUD is among the IPV shelter client population.Shelter workers’ estimates about the prevalence of OUD among their clients are unlikely to be sufficiently accurate for intervention planning or staff training purposes. To our knowledge, there are no existing estimates of opioid use among the IPV shelter population for any state, let alone the nation. Studies of the prevalence of IPV among women who are using methadone or are in substance use disorder treatment do not answer the questions that the IPV service providers need answered: is OUD a large or small problem in the IPV program client population? Which IPV program clients are most at risk for OUDs? And which policies, practices, and service models tend to result in the best outcomes for those individuals and, if relevant, their children? These are critically important research questions that remain to be answered. Given the nature and scope of the opioid epidemic in the United States, ideally resources will be devoted to research that provides answers to these questions without delay.12 Nevertheless, this study included representatives of every full member program of the RICADV to gather the spectrum of opinions and concerns from every type of Rhode Island IPV shelter program. These results may not be representative of IPV programs in other US states, nor in other countries. Additional research that attempts to expand upon these findings in other states is needed. Second, qualitative research is inherently subjective. In this case, the questions that were asked of the IPV programs were relatively straightforward so the risk that the perspectives and opinions of the research team influenced findings adversely is relatively low. Third, not every staff member of every IPV program was interviewed. It is possible that the memories, opinions, experiences, and ideas of these four EDs and their two designees do not reflect the thoughts of other IPV program staff people. This possibility could be investigated through a larger-scale study that incorporates data from more than one person per program. An additional limitation of this study is that our focus is on violence against women and shelters that serve predominantly women and children. As described in the introduction, women who experience both IPV and OUD are underserved and face many barriers to accessing the few resources that do exist. It is also true that male survivors of IPV are drastically underserved in most communities and that there are even fewer shelters and other service providers for this population. There is a great need for further research into the needs of male IPV survivors with substance use disorders.This study was subject to several limitations. Qualitative research is not intended to be broadly generalizable beyond a specific context.In conclusion, this exploratory and descriptive study is intended to break new ground in the intersection between IPV program service provision and OUD treatment services research. To our knowledge, it is among the first to explore themes related to IPV program policies and practices pertaining to clients with OUDs, and it lays a foundation upon which additional, larger-scale, and more complex research studies should be built.


Persistent antiphospholipid antibodies (aPL) constitute the serological hallmark of the antiphospholipid syndrome (APS). Recently, various new assay technologies for the detection of aPL better suited to multiplex reaction environments than ELISAs emerged. We evaluated the diagnostic performance of such a novel line immunoassay (LIA) for the simultaneous detection of 10 different aPL.Fifty-three APS patients and 34 healthy controls were investigated for criteria  and non-criteria aPL  IgG and IgM by LIA. Criteria aPL were additionally determined with the established Alegria (ALE), AcuStar (ACU), UniCap (UNI), and AESKULISA (AES) systems and non-criteria aPL with the AES system. Diagnostic performance was evaluated with a gold standard for criteria aPL derived from the results of the four established assays via latent class analysis and with the clinical diagnosis as gold standard for non-criteria aPL.Assay performance of the LIA for criteria aPL was comparable to that of ALE, ACU, UNI, and AES. For non-criteria aPL, sensitivities of the LIA for aPA-, aPI-, aPS-IgG and aPA-IgM were significantly higher and for aPC-, aPE-, aAnnV-IgG and aPC- and aPE-IgM significantly lower than AES. Specificities did not differ significantly.The LIA constitutes a valuable diagnostic tool for aPL profiling. It offers increased sensitivity for the detection of aPL against anionic phospholipids. In contrast, ELISAs exhibit strengths for the sensitive detection of aPL against neutral phospholipids. Antiphospholipid syndrome (APS) is clinically characterized by recurrent venous or arterial thromboses or pregnancy complications . To ensuBeyond the criteria anti-phospholipid antibodies (aPL) aCL and aβ2-GPI, aPL against several other anionic phospholipids (PLs) , neutral PL , and PL-binding proteins  have been observed in APS , 5. TheiEvaluation of diagnostic performances of novel technologies for aPL detection is challenging. Generally, methods for measurement of aPL still lack standardization –16. RefeThe aim of the study was to investigate the diagnostic performance of a novel commercially available LIA enabling simultaneous detection of 10 different aPL. Results for the non-criteria aPL were evaluated with respect to the clinical diagnosis. Sensitivities and specificities for criteria-aPL were assessed using latent class analysis (LCA). We recently demonstrated , that LCFifty-three sera of APS patients seen at the University Hospitals of Würzburg, Frankfurt, and the TU München were included in the study. All patients fulfilled the Sydney-criteria  as conclThe investigated samples constitute a subgroup of the samples employed in a recent study , i.e. onThe study was approved by the local Ethics Committee . All subjects gave written informed consent prior to inclusion. No financial compensation was provided.Sera were analysed for aCL, anti-phosphatidic acid (aPA), -phosphatidyl-choline (aPC), -phosphatidyl-ethanolamine (aPE), -phosphatidyl-glycerol (aPG), -phosphatidyl-inositol (aPI), -phosphatidyl-serine (aPS), -annexin V (aAnnV), aβ2-GPI, and anti-prothrombin (aPT) IgG and IgM with the novel aPL LIA (GA Generic Assays (GA), Dahlewitz, Germany). The immobilized cofactors β2-GPI, prothrombin, and Annexin V are of human origin. PLs are immobilized on the polyvinylidene fluoride membrane of the LIA without any cofactor. Thus, the patient sample was the only possible source of β2-GPI for immobilized, negatively charged PLs. Processed strips were analyzed qualitatively, i.e. samples were classified as positive or negative by visually comparing  the obtained intensities of the respective bands to those of an interpretation template. Intensities of the reference bands on the interpretation template were established employing a scanner with the evaluation software Dr. DotLine Analyzer (GA). Optical density values equal to or above 50 score positive. This cut-off was determined by calculating the 99% percentile of 150 apparently healthy individuals as recommended by the international classification criteria for aPL testing  and the In addition, criteria aPL (aCL and aβ2-GPI IgG and IgM) were determined with four well-established immunoassay systems in all included sera: ALE, ACU, UniCAP 250 (UNI) from Thermo Fisher Scientific , and AESKULISA (AES) from Aesku.diagnostics . All commercial aβ2-GPI assays employed the human protein as antigen. Detection of aCL was performed with human β2-GPI in the ALE, ACU, and AES systems and bovine β2-GPI in the AES ELISA. IgG and IgM isotypes of the non-criteria aPL  were additionally measured with the AES system in all sera with sufficient sample material left after performing the analyses mentioned above. The wells of the AES assays for aPA, aPC, aPE, aPG, aPI, and aPS contained human β2-GPI as cofactor in addition to the respective PL. AES assays for aAnnV and aPT employed the human protein as antigen. Detailed information on the interpretation of the results obtained by the comparative immunoassay systems is given in Supporting Information Samples were stored at -80°C in separate aliquots. For each measurement, a fresh aliquot was thawed. An explicit quality control for sample stability, however, was not performed. The presented results from the ALE, ACU, UNI, and AES for the criteria aPL are the same results also employed in  and hencDiagnostic performance of the LIA for criteria aPL was assessed by employing a LCA-derived “gold standard” calculated as described previously . BrieflyLIA results for non-criteria aPL were compared to the AES ELISAs. Inter-rater agreement was assessed via Cohen’s κ. Agreements with Cohens’s κ of ≥ 0.81, 0.61–0.80, 0.41–0.60, 0.21–0.40, and ≤ 0.20 were considered as very good, good, moderate, fair, and poor, respectively . For calSensitivities and specificities were compared in the McNemar test (significance level 0.05). “R” version 3.2.0  was employed for statistical evaluations. The statistical approach is illustrated in Supporting Information A comprehensive overview on the positivity of the APS patients for criteria and non-criteria aPL in the various assays is given in The aPL IgG combination found most frequently in the patient group was a concurrent positivity for criteria aPL (aCL and aβ2-GPI) and non-criteria aPL against anionic PLs  with or without additional aPT (n = 18 (34.0%) and 14 (26.4%), respectively). Regarding IgM, the combination of positive criteria aPL and positive non-criteria aPL against anionic PL was observed frequently, too (n = 5 (9.4%)). The most frequent pattern for this isotype, however, was single positivity for aPA (n = 8 (15.1%)). All other observed aPL combinations were detected only in a minority of samples. An overview on the frequencies of the investigated aPL and their respective combinations is given in Controls did not measure positive for aCL IgG and IgM and aβ2-GPI IgM. Three (8.8%) control sera were positive for aβ2-GPI IgG. When investigating the controls for non-criteria aPL, 4 (11.8%) positive reactions were found for aPT IgG, 3 (8.8%) for aAnnV IgM, 2 (5.9%) for aPA IgG, aPI IgG, and aPT IgM and 1 (2.9%) for aAnnV IgG and aPA IgM. Controls did not show positive staining for any of the other tested aPL.Data on the detection rates for criteria aPL employing the LIA for sera with low, medium, and high positive titers in the comparative assays are given in Considering non-criteria aPL , the LIAData on the diagnostic performances for criteria aPL are given in Results of the comparison of sensitivities and specificities for the criteria aPL are depicted in Thus, the LIA performed significantly better in the sensitive detection of aCL IgG than the UNI and of aβ2-GPI IgG than the AES. Specificity for aCL IgG was significantly lower than the ALE and for aβ2-GPI IgG than the UNI and AES. No further significant differences in the sensitivities and specificities for criteria aPL between the LIA and ALE, ACU, UNI, and AES were observed.With Cohen’s κ of 0.61–0.75, inter-rater agreement of LIA and AES for aPG and aPI IgG and IgM can be considered as good. For aPS, agreement was good for the IgM and moderate for the IgG isotype . Similarly, a slightly lower Cohen’s κ of 0.34 indicating fair agreement was found for aPA IgG as compared to a Cohen’s κ of 0.43 for the IgM isotype suggesting moderate agreement. The reverse pattern was observed for aPT with Cohen’s κ 0.29 for IgG (fair agreement) vs. 0.10 for IgM (poor agreement). Cohen’s κ from -0.10 to 0.00 for aPC, aPE, and aAnnV IgG and IgM pointed to poor agreements. A very good agreement could not be demonstrated for any non-criterion aPL  and highest for aPA (0.47) with the LIA. Sensitivities for the AES varied less and ranged from 0.15 for aPC and aPT IgM to 0.19 for aPE, aPI, and aPS IgM. The LIA was significantly more sensitive than AES for aPA IgM (0.47 vs. 0.17) and significantly less sensitive for aPC and aPE IgM . No further significant differences between the two investigated systems in the sensitivities for non-criteria aPL IgM were observed.Specificities for the non-criteria aPL were ≥ 0.88 and ≥ 0.91 with the LIA and ≥ 0.91 and ≥ 0.93 with AES for the IgG and IgM isotype, respectively. Differences of specificities were not significant for any non-criteria aPL.Eleven (20.8%) out of the 53 APS-patients were positive for LA, but negative for aCL or aβ2-GPI IgG or IgM as concluded from LCA. When using the LIA, 9 (81.8%) of these patients exhibited positivity for any of the non-criteria aPL. All 9 patients were positive for aPA IgG. In addition, aPS IgG was found in 6, aPG IgG in 5, aPI IgG in 4, and aPT IgM in 2 samples and aAnnV IgG, aPT IgG, and aPA IgM in one sample each. With the AES assays, 6 (54.5%) of the 11 patients were positive for non-criteria aPL: in 4 samples solely aPT IgG or IgM was detected, in one sample aPT IgG and aPE IgM, and in one sample a combination of aPG IgG, aPI IgG, aPS IgG, and aAnnV IgG. All 6 samples positive for non-criteria aPL with the AES system were also found positive for non-criteria aPL in the LIA.The diagnostic performance of a novel LIA for the detection of aPL was investigated. Sensitivities and specificities of the LIA for criteria aPL are well comparable to established immunoassays. With respect to non-criteria aPL, the LIA offers advantages in the sensitive detection of aPL against anionic PLs whereas the classical ELISA exhibits strengths in the sensitive detection of aPL against neutral PLs.Sensitivity of the LIA for aCL and aβ2-GPI IgG was significantly better than the worst and comparable to the three other established assays. Considering specificity for aCL and aβ2-GPI IgG, the LIA performed worse than the best and two best, but equal to three and two of the comparative assays, respectively. Moreover, with respect to aCL and aβ2-GPI IgM, no significant differences in sensitivity and specificity between the LIA and the established immunoassays were observed. As such, the diagnostic performance of the LIA for criteria aPL can be considered equal to the investigated established immunoassays.In contrast, considerable differences in the results for non-criteria aPL obtained by LIA and an established ELISA system were demonstrated. This is illustrated by Cohen’s κ indicating very good agreement for none and good agreement for only 5, but poor agreement for 7 out of the 16 analytes. Differences are based on varying sensitivities for non-criteria aPL. Sensitivity of the LIA was significantly better for aPA, aPI and aPS IgG, and aPA IgM and approached significance for aPS IgM. The ELISA, however, demonstrated a significantly higher sensitivity for aPC and aPE IgG and IgM. Phosphatidic acid, phosphatidyl-serine, -glycerol, and -inositol carry a negative net charge at physiological pH. Phosphatidyl-choline and–ethanolamine in contrast constitute neutral molecules at pH 7.4. Thus, the LIA exhibits higher sensitivities for antibodies against anionic and the ELISA for antibodies against neutral PL. Since the ELISAs contained β2-GPI as cofactor apart from the respective PL as autoantigenic target, positive serum reactivity in these assays could be due to aβ2-GPI, too. With the AES system, aPA, aPC, aPE, aPG, aPI, and aPS indeed exhibited a positive correlation with aβ2-GPI in our cohort. However, it remains to be shown, whether the aPL reactivity in the respective ELISAs is based on β2-GPI binding alone. Thus, further studies are warranted to investigate whether this phenomenon can be explained by actual analytical interference or if the respective aPL arise concurrently for biological reasons.The diagnostic performance of LIAs for aPL detection has been evaluated in two major studies: Egerer et al.  applied To the best of our knowledge, our work is the first study to compare non-criteria aPL detection of the LIA with established ELISAs thus revealing different strengths of the various assay surfaces. A LCA-based approached would clearly have been desirable here as well. A comprehensive investigation of all non-criteria aPL detected in the LIA, however, was only possible with one of the four assay systems that had been applied for the evaluation of the criteria aPL. We, therefore, choose to use the clinical diagnosis as the gold standard. One may argue that this approach is less critical for non-criteria aPL as they are not required for the diagnosis of APS.Characteristics of patient and control group entail some limitations of our study. First of all, the limited sample size may impair generalizability of our data. Furthermore, a high percentage of double and triple positive subjects in our patient cohort may have increased the sensitivities for criteria aPL. And finally, the lack of disease controls may have yielded rather high specificities. As such, selection of patient and control groups may explain the mostly higher sensitivities and specificities in our study as compared to previous ones. From an analytical point of view, the time interval between the “old” ELISA measurements of the criteria aPL and the “new” measurements with the LIA and of non-criteria aPL via ELISA may introduce some bias into our work. If at all, this bias would be to the disadvantage of the LIA and should not result in a false-good evaluation of this assay.Nevertheless, the observed high sensitivity of the LIA for aPL against anionic PLs sheds light on the optimal solid phases for testing of non-criteria aPL. The hydrophobic membrane of the LIA seems to be more suited for the detection of aPL against anionic PLs, whereas the polystyrene plastics of the ELISA plate detects neutral PLs with higher sensitivity. Of note, β2-GPI reactivity in these ELISAs cannot be excluded. Keeping in mind, that the most relevant autoantigenic epitopes for aPL are located on β2-GPI, our data support the hypothesis previously proposed , 30: theThe LIA proved to be a suitable tool for detection of criteria and presumably also of non-criteria aPL. As compared to classical ELISAs, the LIA offers several interesting features: in one single run a comprehensive aPL profile can be generated considerably quicker and with less reagent costs and effort as compared to ELISAs as one LIA test strip replaces 10 single ELISAs; it is suited for low as well as for high sample throughput and requires only a basic laboratory equipment. Our data demonstrate that the majority of APS patients with LA but without criteria aPL exhibits positivity for non-criteria aPL–thereby pointing to possible diagnostic benefits of the determination of non-criteria aPL. Of note, strength of positivity for non-criteria aPL as well as the time course of titers were not considered in this study. As such, better availability and convenient analysis of aPL profiles may foster research in this area and help to better elucidate the clinical significance of non-criteria aPL.In summary, our work demonstrates that the LIA exhibits a diagnostic performance comparable to established immunoassays for criteria aPL and offers advantages with respect to the detection of non-criteria aPL against anionic PL. It constitutes a valuable tool for aPL profiling in a clinical as well as in a research setting. Beyond that, our data will serve as a guide for assay developers when designing the solid-phase for detection of non-criteria aPL.S1 FigFor criteria aPL, the diagnostic performance was calculated against a “gold standard” derived from the results of the ALE, ACU, UNI, and AES systems via LCA. In contrast, the clinical diagnosis served as the gold standard to determine sensitivities and specificities for non-criteria aPL.(PPTX)Click here for additional data file.S2 FigSensitivities of the investigated assays for aCL IgG (A), aCL IgM (B), aβ2-GPI IgG (C), and aβ2-GPI IgM (D). Error bars denote a 95% confidence interval. Significant differences are marked with horizontal square brackets .(TIF)Click here for additional data file.S3 FigSpecificities of the investigated assays for aCL IgG (A), aCL IgM (B), aβ2-GPI IgG (C), and aβ2-GPI IgM (D). Error bars denote a 95% confidence interval. Significant differences are marked with horizontal square brackets .(TIF)Click here for additional data file.S4 FigLevels of aPA, aPC, aPE, aPG, aPI, and aPS as a function of aβ2-GPI as measured with the AES system for IgG (A) and IgM (B) for all samples included in the study. AES aPE IgG: samples > 300 U/ml were not further diluted; AES aPG IgG and AES aPI IgG: samples > 315 U/ml were not further diluted.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FileData underlying the findings described in the manuscript.(CSV)Click here for additional data file.