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In the fecal immunological test, a suitable cut-off value may be selected to classify results as either positive or negative. Our aim is to estimate the optimal cut-off value for detecting colorectal cancer in different age and sex groups. This is a multicentric retrospective cohort study of participants in CRC screening programs with FIT between 2006 and 2012. A total of 545,505 participations were analyzed. Cancers diagnosed outside of the program were identified after a negative test result (IC_test) up until 2014. The Wilcoxon test was used to compare fecal hemoglobin levels. ROC curves were used to identify the optimal cut-off value for each age and sex group. Screening program results were estimated for different cut-off values. The results show that the Hb concentration was higher in colorectal cancer (average = 179.6μg/g) vs. false positives (average = 55.2μg/g), in IC_test (average = 3.1μg/g) vs. true negatives (average = 0μg/g), and in men (average = 166.2μg/g) vs. women (average = 140.2μg/g) with colorectal cancer. The optimal cut-off values for women were 18.3μg/g (50-59y) and 14.6μg/g (60-69y), and 16.8μg/g (50-59y) and 19.9μg/g (60-69y) for men. Using different cut-off values for each age and sex group lead to a decrease in the IC_test rate compared to the 20μg/g cut-off value (from 0.40‰ to 0.37‰) and an increase in the false positive rate (from 6.45% to 6.99%). Moreover, test sensitivity improved (90.7%), especially in men and women aged 50-59y (89.4%; 90%) and women aged 60-69y (90.2%). In conclusion, the optimal cut-off value varies for different sex and age groups and the use of an optimal cut-off value for each group improves sensitivity and leads to a small decrease in IC_tests, but also to a larger increase in false positives., Competing Interests: The authors have declared that no competing interests exist.

Early diagnosis remains key for effective prevention and treatment. Unfortunately, current screening with anti-hepatitis C virus antibody (anti-HCV Ab) test may have limited utility in the diagnosis of HCV infection and reinfection. This is of special concern to at-risk population, such as immunocompromised hosts and end-stage renal failure patients on hemodialysis. HCV antigen (Ag) could be useful in identifying the ongoing infection in such clinical scenarios. Hence, we aimed to study the utility of HCV Ag testing for the diagnosis of acute and chronic hepatitis C. Of 89 samples studied, 19 were from acute hepatitis C patients who were immunocompromised or were on hemodialysis, 43 were from active chronic hepatitis C patients and 27 were from patients treated for chronic hepatitis C. All samples were tested for HCV Ag using the Abbott ARCHITECT HCV Ag assay. HCV Ag was reactive in 19/19 samples from acute hepatitis C patients and 42/43 samples from active chronic hepatitis C patients. It was nonreactive in all samples from treated patients. The test showed a sensitivity and specificity of 98.4% and 100.0%, respectively. The positive and negative predictive values were 100.0% and 96.4%, respectively. The HCV antigen test has high clinical sensitivity and specificity and is useful for the diagnosis of acute and chronic hepatitis C infection in at-risk and immunocompromised patients. Its short turnaround time and relatively low cost are advantageous for use in patients on hemodialysis and other at-risk patients who require monitoring of HCV infection and reinfection., (© 2020 Wiley Periodicals LLC.)

Elevated serum IgG4 is a useful marker of IgG4-related disease (IgG4-RD) activity. However, there is no uniformity in the cut-off values of IgG4 among the various reagents. The aim of this study was to compare the measured and cut-off values of IgG4 assessed using three different reagents. This study enrolled 466 IgG4-RD and non-IgG4-RD patients who required measurement of serum IgG4 levels to diagnose or treat IgG4-RD. Serum IgG4 was measured using three reagents: N-assay LA IgG4 Nittobo (Nittobo), BS-NIA IgG4 (TBS), and N Latex IgG4 (Siemens). The values obtained using the three reagents were compared, and cut-off values were calculated for each. Although there was good correlation among the results with the three reagents, the measured and cut-off values were all different. The Nittobo values were 1.4 times the TBS values and the TBS values were almost half those of the Siemens values. ROC curve analysis showed cut-off values for the Nittobo, TBS, and Siemens reagents of 1.42, 1.31, and 2.38 g/L, respectively. The measured and cut-off values of serum IgG4 vary depending on the reagents used for the assay, although there is good correlation among the values measured by the three reagents.

Hexaminolevulinate (HAL) induced Protoporphyrin IX (PpIX) fluorescence is commonly used to differentiate cancer cells from normal cells in vivo, as for instance in blue light cystoscopy for bladder cancer diagnosis. A detailed approach is here provided to use this diagnostic principle ex vivo in an immunosensor device, towards enabling non-invasive cancer diagnostic from body fluids, such as urine. Several factors susceptible to affect the applicability of HAL-assisted diagnosis in body fluids were tested. These included the cell viability and its impact on PpIX fluorescence, the storage condition and shelf life of HAL premix reagent, light exposure (360-450 nm wavelengths) and its corresponding effect on both intensity and bleaching of the PpIX fluorescence as a function of the microscopy imaging conditions. There was no significant decrease in the viability of bladder cancer cells after 6 h at 4 °C (student's t-test: p > 0.05). The cellular PpIX fluorescence decreased in a time-dependent manner when cancer cells were kept at 4 °C for extended period of time, though this didn't significantly reduce the fluorescence intensity contrast between cancer and non-cancer cells kept in the same condition for 6 h. HAL premix reagent kept in long term storage at 4 °C induced stronger PpIX fluorescence than reagent kept in the - 20 °C freezer. The PpIX fluorescence was negatively affected by repeated light exposure but increased with illumination intensity and exposure time. Though this applied to both healthy and cancer cell lines, and therefore did not statistically improved the differentiation between cell types. This study revealed important experimental settings that need to be carefully considered to benefit from the analytical potential of HAL induced fluorescence when used in technologies for the diagnosis of cancer from body fluids.

Background: An accurate test for the diagnosis and post-treatment follow-up of patients with schistosomiasis is needed. We assessed the performance of different laboratory parameters, including the up-converting reporter particle technology lateral flow assay to detect circulating anodic antigen (UCP-LF CAA), for the post-treatment follow-up of schistosomiasis in migrants attending a dedicated outpatient clinic in a non-endemic country., Methods: Routine anti-Schistosoma serology results and eosinophil counts were obtained of patients with positive urine/stool microscopy and/or PCR (confirmed cases) or only positive serology (possible cases), and at least one follow-up visit at 6 (T6) or 12 (T12) months after praziquantel treatment. All sera samples were tested with the UCP-LF CAA assay., Results: Forty-eight patients were included, 23 confirmed and 25 possible cases. The percentage seropositivity and median antibody titers did not change significantly during follow-up. UCP-LF CAA was positive in 86.9% of confirmed and 20% of possible cases. The percentage positivity and median CAA levels decreased significantly post-treatment, with only two patients having positive CAA levels at T12., Conclusions: The UCP-LF CAA assay proved useful for the diagnosis of active infection with Schistosoma spp. and highly valuable for post-treatment monitoring in migrants, encouraging the development of a commercial test.

Background: Various errors in the procedure of specimen collection have been reported as the primary causes of pre-analytical errors. The aim of this study was to monitor and assess the reasons and frequencies of rejected samples in China., Methods: A pre-analytical external quality assessment (EQA) scheme involving six quality indicators (QIs) was conducted from 2017 to 2019. Rejection rate was calculated for each QI. The difference of the rejection rates over the time was checked by Chi-square test. Furthermore, the 25th, 50th, and 75th percentiles of the results from total laboratories each year were calculated as optimum, desirable, and minimum level of performance specifications., Results: In total, 423 laboratories submitted data continuously for six EQA rounds. The overall rejection rates were 0.2042%, 0.1709%, 0.1942%, 0.1689%, 0.1593%, and 0.1491%, respectively. The most common error was sample hemolysed (0.0514%-0.0635%), and the least one was sample not received (0.0008%-0.0014%). A significant reduction in percentages was observed for all QIs. For biochemistry and immunology, hemolysis accounted for more than half of the rejection causes, while for hematology, the primary cause shifted from incorrect fill level to sample clotted. The quality specifications had improved over time, except for the optimum level., Conclusion: The significant reduction in error rates on sample rejection we observed suggested that laboratories should pay more attention to the standardized specimen collection. We also provide a benchmark for QIs performance specification to help laboratories increase awareness about the critical aspects in the need of improvement actions., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Abstract: The T-SPOT.TB assay detects cellular immune responses to 2 core Mycobacterium tuberculosis antigens, early secreted antigenic target of 6-kDa protein (ESAT-6) and culture filtrate protein-10 (CFP-10). T-SPOT.TB has been recently used for auxiliary diagnosis of active pulmonary tuberculosis (PTB). However, testing can produce inconsistent results due to differential PTB patient immune responses to these antigens, prompting us to identify factors underlying inconsistent results.Data were retrospectively analyzed from 1225 confirmed PTB patients who underwent T-SPOT.TB testing at 5 specialized tuberculosis hospitals in China between December 2012 and November 2015. Numbers of spot-forming cells (SFCs) reflecting T cell responses to ESAT-6 and CFP-10 antigens were recorded then analyzed via multivariable logistic regression to reveal factors underlying discordant T cell responses to these antigens.The agreement rate of 84.98% (82.85%-86.94%) between PTB patient ESAT-6 and CFP-10 responses demonstrated high concordance. Additionally, positivity rates were higher for ESAT-6 than for CFP-10 (84.8% vs 80.7%, P < .001), with ESAT-6 and CFP-10 microwell SFC numbers for each single positive group not differing significantly (P > .99), while spot numbers of the single positive group were lower than numbers for the double positive group (P < .001). Elderly patients (aged ≥66 years) and patients receiving retreatment were most likely to have discordance results.ESAT-6 promoted significantly more positive T-SPOT.TB results than did CFP-10 in PTB patients. Advanced age and retreatment status were correlated with discordant ESAT-6 and CFP-10 results. Assessment of factors underlying discordance may lead to improved PTB diagnosis using T-SPOT.TB., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Fecal immunochemical test (FIT) is widely used as a colorectal cancer screening tool. Antithrombotic drugs may affect the screening performance of FIT for colorectal tumors. The aim of this study was to clarify the effect of antithrombotic agents on FIT accuracy in screening for colorectal neoplasms. This retrospective study enrolled a total of 758 patients who underwent both FIT and total colonoscopy. The effect of antithrombotic drugs on FIT accuracy in detecting colorectal neoplasms (CN), including colorectal cancer (CRC), advanced adenoma (AA), and non-advanced adenoma (NAA), was examined. Of the 758 patients, 144 (19%) received antithrombotic drugs (administration group). In administration group, 61/144 (42%) cases had CN [CRC:14, AA:15, NAA:32] and 217/614 (35%) cases had CN (CRC:43, AA:56, NAA:118) in non-administration group. The prevalence of CN was not significantly different between the two groups (p = 0.1157). There was no significant difference in sensitivity or specificity of the detection of all types of CN with or without taking antithrombotic drugs. Neither the positive predictive value nor negative predictive value of FIT was affected by antithrombotic drug administration. Taking antithrombotic drugs may not have a large impact on sensitivity, specificity, positive predictive value, or negative predictive value of FIT in screening for CN.

Objectives: We validate the use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample., Methods: Samples collected by venous blood draw and finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients. Samples were tested with Biolidics 2019-nCoV IgG/IgM Detection Kit lateral flow immunoassay, and antibody calls were compared with ELISA., Results: Biolidics LFI showed clinical sensitivity of 92% with venous blood at 7 days after PCR diagnosis of SARS-CoV-2. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p = 1.00), except for detection of IgM at D7 (p = 0.04). Capillary blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection., Conclusions: Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit is comparable to ELISA and was consistent across sample types. This provides an opportunity for decentralized rapid testing and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom ® FXIII Activity, a functional ammonia release assay, and the HemosIL ™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

Background: Thyroid-stimulating hormone receptor (TSHR)-activating autoantibodies stimulate thyroid growth and hormone synthesis/secretion, causing hyperthyroidism of Graves' disease (GD). TRAb measurement helps diagnose GD and is an important first test in evaluating hyperthyroidism according to the recent American Thyroid Association guidelines. We compared the performance of the BRAHMS TRAK Kryptor (Thermo Scientific) and Roche cobas TRAb immunoassays for use in GD., Method: Method comparison (n = 40) and clinical agreement were assessed between the Kryptor, cobas e411, and cobas e601. The analytical performance of Kryptor and cobas e411 were assessed for within- and between-day imprecision across 20 days, linearity, functional assay sensitivity (FAS), dilution recovery, and cut-off verification., Results: The Kryptor, e411, and e601 TRAb immunoassays correlated well (r > 0.95, overall percent agreement = 0.95, Cohen's kappa = 0.90). With a total allowable error of 20%, percent bias was within 13%, which was minimally negative at <20 IU/L, but highly positive (33%-34%) >20 IU/L. The Kryptor, but not e411, was linear across the claimed analytical measuring range (AMR). The claimed functional assay sensitivity (FAS), which was close to the clinical GD cut-off 1.8 IU/L, was verified for Kryptor and e411., Conclusion: Overall, our evaluation demonstrates acceptable comparability between TRAb immunoassays with in-house imprecision up to 13% and 10% on Kryptor and e411, respectively. While Roche has preferable calibration frequency and on-board reagent stability, both platforms demonstrate acceptable imprecision using patient samples at their claimed FAS, which is important for GD diagnosis. Diluted results (using a negative patient pool as diluent) exhibits proportional positive bias on the Kryptor relative to the Roche methods., (Copyright © 2020 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objective: To improve myasthenia gravis (MG) autoantibody testing., Methods: MG serologic tests with confirmatory or refuting clinical-electrodiagnostic (EDX) testing and cancer evaluations were reviewed over 4 years (2012-2015). All patients had acetylcholine receptor-binding (AChR-Bi), modulating (AChR-Mo), and striational (STR) autoantibody testing, and negatives reflexed to muscle-specific kinase (MuSK). Thymoma and cancer occurrences were correlated with STR and reflexed glutamic acid decarboxylase 65 (GAD65), ganglionic acetylcholine receptor (α3), collapsin response mediating protein-5, and voltage-gated potassium channel complex autoantibodies., Results: Of 433 samples tested, 133 (31%) met clinical-EDX criteria for MG. Best sensitivity (90%) occurred at AChR-Bi >0.02 nmol/L, leaving 14 negative (6 ocular MG, 7 generalized MG, 1 MuSK MG) with specificity 90% (31 false-positives). Using AChR-Mo antibodies (>20% loss), specificity was better (92%, 24 false-positives), but sensitivity dropped (85%). Specificity improved (95%) by testing AChR-Mo when AChR-Bi are positive, resulting in 45% reduction of false-positives (31-17), maintaining AChR-Bi 90% sensitivity. Cutoff values recommended by area under the curve analysis did not outperform this approach. AChR-Bi and AChR-Mo values were significantly higher in true-positives. CT evaluations in 121 MG samples revealed 16 thymomas. Historical or subsequent cancers occurred in 22. STR and reflexed autoantibodies were not more common in MG with thymoma or other cancers. Full-body CT (n = 34) was performed in those with STR and reflex autoantibody positivity, but without additional cancers found., Conclusion: Accuracy of MG serologic testing is improved by reflexing AChR-Bi-positive cases to AChR-Mo. STR and other reflexed cancer evaluation autoantibodies did not provide value beyond standard CT chest imaging at the time of MG diagnosis. Diagnostic certainty is informed by AChR-Bi and AChR-Mo with higher values increasing specificity., (© 2020 American Academy of Neurology.)

Background: The aim of this study is to compare double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA in the diagnosis of hepatitis C virus（HCV）infection., Methods and Materials: A total of 176 samples from the Tumor Hospital Affiliated to Xin Jiang Medical University were utilized to comparison. All serum samples were tested using double-antigen sandwich ELISA and indirect ELISA. Cohen's kappa statistics were used to assess the agreement between the two assays, and multivariate analysis was used to evaluate risk factors for the discordance between the double-antigen ELISA and indirect ELISA., Results: The positivities of indirect ELISA (Beijing Wantai), double-antigen sandwich ELISA (Beijing Wantai), and indirect ELISA (Beijing Jinhao) were 74.43%, 68.75%, and 73.30%, respectively. The agreement between the indirect ELISA (Beijing Wantai) and double-antigen sandwich ELISA (Beijing Wantai) was high (κ = 0.829;P < .001), and the agreement between the double-antigen sandwich ELISA (Beijing Wantai) and indirect ELISA (Beijing Jinhao) was high (κ = 0.847;P < .001). Variables associated with discordant results between the double-antigen sandwich and indirect ELISA in multivariate analysis were as follows: female (OR:1.462; P < .05), age (<35 years old; OR:3.667; P < .05), and cancer (suffer from malignant tumor; OR:3.621; P < .05)., Conclusion: In detection of HCV, high agreement was found between the double-antigen sandwich ELISA and indirect ELISA. Female, younger age, and suffer from malignant tumor were significant risk factors for the discordance. Based on double-antigen sandwich ELISA has distinct methodological advantages over indirect ELISA. It is recommended for the diagnosis of HCV infection., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

Background: Mycobacterium tuberculosis (Mtb)-specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb-specific perforin in pediatric patients with tuberculosis., Methods: Diagnostic performance of perforin levels induced by 6-kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty-six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF-γ of CD4 + , CD8 + T cells in response to CFP10 stimulation., Results: After ex vivo stimulation, levels of ESAT6/CFP10-specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non-tuberculosis infection (non-TB), and health control (HC) individuals. The diagnostic efficacy of CFP10-specific perforin for TB diagnosis was significantly higher than ESAT6-specific perforin and T-SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10-specific perforin in both CD4 + and CD8 + T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF-γ was mainly secreted by CD4 + T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10-specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively., Conclusions: CFP10-specific perforin may be used as a novel cellular immunity-based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Celiac disease (CD) screening in patients with Williams-Beuren Syndrome (WBS) is suggested, although data described in literature are discordant regarding CD prevalence in WBS. We retrospectively collected data from 101 WBS Italian patients [mean age: 13.5 years], to clarify the CD prevalence in a large cohort. All patients underwent a CD biochemical screening: IgA and anti-transglutaminase reflex antibodies (tTGA). CD-specific HLA typing was available for 42 patients. Small intestinal biopsy was performed in patients according to ESPGHAN guidelines. In 7 WBS patients an overt celiac disease was diagnosed. In 3 patients CD was confirmed by symptoms, HLA-DQ heterodimers and CD specific antibodies title, whereas in 4 patients, it was confirmed by a small intestinal biopsy. CD prevalence in our cohort is 6.9% (7/101). In 42/101 patients the CD-specific HLA typing was available, detecting 29/42 (69%) patients genetically predisposed to CD. The CD prevalence and CD-specific HLA prevalence are both higher than in the general population (p < 0.001; p < 0.001). Our cohort is the most numerous described confirming that the CD risk in WBS patients is significantly greater than in general population. Moreover, our HLA typing results, as well as scientific literature, suggest that the higher CD prevalence in WBS patients might not be intrinsically related to the genetic disease itself but with the higher HLA prevalence. However, HLA typing should be performed in bigger WBS cohorts to confirm this hypothesis. Our data confirms that HLA typing is mandatory in WBS patients and that CD screening should be performed only if genetically predisposed., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Objectives: The new European In Vitro Diagnostic (IVD) Regulation 2017/746 (IVDR) restricts the use of lab-developed tests (LDT) after 26th May 2022. There are no data on the impact of the IVDR on laboratories in the European Union., Methods: Laboratory tests performed in UZ Leuven were divided in four groups: core laboratory, immunology, special chemistry, and molecular microbiology testing. Each test was classified as Conformité Européenne (CE)-IVD, modified/off-label CE-IVD, commercial Research Use Only (RUO) or LDT. Each matrix was considered a separate test., Results: We found that 97.6% of the more than 11.5 million results/year were generated with a CE-IVD method. Of the 922 different laboratory tests, however, only 41.8% were CE-IVD, 10.8% modified/off-label CE-IVD, 0.3% RUO, and 47.1% LDT. Off-label CE-IVD was mainly used to test alternative matrices not covered by the claim of the manufacturer (e.g., pleural or peritoneal fluid). LDTs were mainly used for special chemistry, flow cytometry, and molecular testing. Excluding flow cytometry, the main reasons for the use of 377 LDTs were lack of a CE-IVD method (71.9%), analytical requirements (14.3%), and the fact the LDT was in use before CE-IVD available (11.9%)., Conclusions: While the large majority of results (97.6%) were generated with a CE-IVD method, only 41.8% of laboratory tests were CE-IVD. There is currently no alternative on the market for 71.5% of the 537 LDTs performed in our laboratory which do not fall within the scope of the current IVD directive (IVDD). Compliance with the IVDR will require a major investment of time and effort.

Objectives: To maintain the consistency of laboratory test results, between-reagent lot variation should be verified before using new reagent lots in clinical laboratory. Although the Clinical and Laboratory Standards Institute (CLSI) document EP26-A deals with this issue, evaluation of reagent lot-to-lot difference is challenging in reality. We aim to investigate a practical way for determining between-reagent lot variation using real-world data in clinical chemistry., Methods: The CLSI EP26-A protocol was applied to 83 chemistry tests in three clinical labs. Three criteria were used to define the critical difference (CD) of each test as follows: reference change value and total allowable error, which are based on biological variation, and acceptable limits by external quality assurance agencies. The sample size and rejection limits that could detect CD between-reagent lots were determined., Results: For more than half of chemistry tests, reagent lot-to-lot differences could be evaluated using only one patient sample per decision level. In many cases, the rejection limit that could detect reagent lot-to-lot difference with ≥90% probability was 0.6 times CD. However, the sample size and rejection limits vary depending on how the CD is defined. In some cases, impractical sample size or rejection limits were obtained. In some cases, information on sample size and rejection limit that met intended statistical power was not found in EP26-A., Conclusions: The CLSI EP26-A did not provide all necessary answers. Alternative practical approaches are suggested when CLSI EP26-A does not provide guidance.

Objective: To determine frequencies, interlaboratory reproducibility, clinical ratings, and prognostic implications of neural antibodies in a routine laboratory setting in patients with suspected neuropsychiatric autoimmune conditions., Methods: Earliest available samples from 10,919 patients were tested for a broad panel of neural antibodies. Sera that reacted with leucine-rich glioma-inactivated protein 1 (LGI1), contactin-associated protein-2 (CASPR2), or the voltage-gated potassium channel (VGKC) complex were retested for LGI1 and CASPR2 antibodies by another laboratory. Physicians in charge of patients with positive antibody results retrospectively reported on clinical, treatment, and outcome parameters., Results: Positive results were obtained for 576 patients (5.3%). Median disease duration was 6 months (interquartile range 0.6-46 months). In most patients, antibodies were detected both in CSF and serum. However, in 16 (28%) patients with N-methyl-D-aspartate receptor (NMDAR) antibodies, this diagnosis could be made only in cerebrospinal fluid (CSF). The two laboratories agreed largely on LGI1 and CASPR2 antibody diagnoses (κ = 0.95). The clinicians (413 responses, 71.7%) rated two-thirds of the antibody-positive patients as autoimmune. Antibodies against the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), NMDAR (CSF or high serum titer), γ-aminobutyric acid-B receptor (GABABR), and LGI1 had ≥ 90% positive ratings, whereas antibodies against the glycine receptor, VGKC complex, or otherwise unspecified neuropil had ≤ 40% positive ratings. Of the patients with surface antibodies, 64% improved after ≥ 3 months, mostly with ≥ 1 immunotherapy intervention., Conclusions: This novel approach starting from routine diagnostics in a dedicated laboratory provides reliable and useful results with therapeutic implications. Counseling should consider clinical presentation, demographic features, and antibody titers of the individual patient.

Background: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT)., Study Design and Methods: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages., Results: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients., Conclusion: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn., (© 2020 AABB.)

Identifying analytical interference is a challenge for the medical biologist in providing advice to the prescriber. Indeed, these analytical interferences often have deleterious consequences on the care of patients. Understanding their mechanisms and mastering corrective procedures is essential to limit these management errors. Faced with the many questions from clinicians in current practice, we propose an algorithm for managing a sample when interference is suspected.

Urinary detection of Mycobacterium tuberculosis lipoarabinomannan (LAM) for tuberculosis (TB) diagnosis is well characterized, but the utility of serum LAM detection remains unclear. We developed an assay for serum LAM detection using single-molecule array (Simoa), purified M. tuberculosis LAM, and anti-LAM monoclonal antibodies and evaluated performance on diluted/heat-treated serum samples from patients with and without active TB and/or HIV. The Simoa assay had a limit of detection of 0.35 pg/mL and lower limit of quantification of 0.942 pg/mL. Corrected serum LAM concentrations ranged from 0 to 132.0 pg/mL [median 1.71, interquartile range (IQR) 0.94-6.80] in 90 TB+ patients and from 0 to 2.29 pg/mL (median 1.03, IQR 0.47-1.69) in 55 TB- patients. Using a cutoff of 2.3 pg/mL for 100% specificity, assay sensitivity was 37% in all TB+ subjects (33/90; 95% CI 0.27-0.48), 47% in TB+/HIV+ subjects (26/55; 0.34-0.61), and 60% in TB+/HIV+/smear+ subjects (21/35; 0.42-0.76). Mycobacterial LAM is detectable in serum with high specificity and reasonable sensitivity using Simoa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: IgG4-related disease (IgG4-RD) is a new syndrome characterized by elevated serum IgG4 concentration and tissue infiltration of IgG4-positive plasma cells. Here, we evaluated the analytical performance of a new IgG4 assay reagent featuring a wide dynamic range, highly specific monoclonal antibody, and the reversed passive latex agglutination assay and determined the IgG4 reference interval (RI) for the Japanese population., Methods: Performance evaluations were conducted on precision, linearity, sensitivity, interference, and method comparison with The Binding Site (TBS) and Siemens reagents. The RI was derived by the parametric method from 619 apparently healthy Japanese 18 to 65 years of age., Results: Between-day precisions ranged from 1.99 to 5.52 CV%. Linearity was confirmed up to 5.0 g/l. The limit of quantitation was 0.085 g/l. Interfering substances did not significantly influence values. Method comparison among the 3 reagents yielded correlation coefficients between 0.973 and 0.988. Values for the new reagent matched those of TBS reagent except at a higher concentration range, where reactivity dissociated. The RI was 0.11-1.21 g/l without distinction by sex and age., Conclusion: The novel IgG4 assay reagent demonstrated satisfactory analytical performance for clinical use. Because of matched value with TBS reagent at low concentrations, it is possible to use the IgG4-RD cut-off value determined by TBS reagent., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose of the Study: Among various immunological tests available for diagnosis of neurocysticercosis (NCC), only EITB (Electroimmunotransfer blot for detection of anticysticercal antibodies) had gained widespread acceptance. However EITB is not available widely and is costly (Indian rupees 15,000/- approximately). We evaluated utility of Loop mediated isothermal amplification (LAMP) assay for detection of Taenia solium cox1 gene in blood of patients with NCC., Patients and Methods: Current study included 100 consecutive patients of NCC at a tertiary teaching hospital in Northern India. All the patients underwent detailed history and examinations as well as gadolinium enhanced magnetic resonance imaging of brain. LAMP assay was performed in all the patients. The results were compared with 50 controls., Results: LAMP detected Taenia Solium cox1 gene in 74% of all blood samples in patients of NCC.T he overall sensitivity of LAMP assay for detection of cox1 gene was 74% in all patients with NCC, 71.8% in patients with intraparenchymal brain cysts only and 86.7% of patients with extraparenchymal brain cysts with or without intraparenchymal brain cysts. The overall specificity of LAMP assay was 90% in all these three subgroups. The positive predictive value of real time LAMP assay was close to 93% for almost all forms of NCC- both solitary and multiple while negative predictive value ranged from 57 to 64%., Conclusion: Real time LAMP assay of blood for detection of Taenia solium cox1 gene appears to be a promising toll for diagnosis of NCC., (Copyright © 2019. Published by Elsevier B.V.)

Many biotechnological applications require the simultaneous binding of affinity reagents to nonoverlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ∼10 9 variants. We used RAPIDS to develop highly specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or nontargets, yielding diagnostic assays that are sensitive and specific by design.

After tier 1 and 2 cut points for anti-drug antibody (ADA) assays are derived during pre-study assay validation in a population, there is a need to verify the continued appropriateness of the previously derived cut points during sample analysis in the same or different populations, per FDA guidance (US HHS, FDA, CDER, CBER, 2019). Proper sample size-dependent criteria with statistical underpinning were derived and presented in this technical note to aid in assessing the appropriateness of tier 1 and tier 2 cut points, respectively.

The presence of allergen-specific IgE (sIgE) in human sera can be determined by measuring the binding of sIgE to solid phase-bound preparations containing the allergens to be tested. These can be complex extracts, purified or recombinant allergens, or peptides. Older methods, such as the IgE CAP test, only allow sIgE measurements to multiple allergens in individual measurements. Newer technologies such as the ImmunoCAP ® ISAC test allows semiquantitative testing of sIgE to over a hundred allergens on a protein array. Allergen arrays have higher numerical power, allowing testing to many allergens at the same time, using only a small amount of serum. We have previously demonstrated how allergen arrays can be used in combination with purified peripheral blood basophils, introducing a clinically relevant readout. Here, we describe a protocol and materials that allow the testing of sIgE with multiple allergens in array format, using a humanized fluorescent IgE reporter system (RBL NFAT-DsRed).

"With the increasing application of biotechnology to the realm of pharmacology and therapeutics, the types of biological treatments available have significantly expanded. Currently, recombinant proteins, humanized antibodies, or rationally engineered monoclonal antibodies are used on a regular basis in the clinical setting. Moreover, cell-based therapeutics with molecularly rewired antigenic specificities are becoming increasingly common in oncology and are actively being developed for a broad range of diseases. Nonetheless, there has been a significant lag between the development of these technologies and the emergence of assays that can monitor these novel interventions.", (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: κ and λ free light chains (FLCs) are monitored to aid in the diagnosis of plasma cell disorders. Our goal was to validate the Diazyme Human κ and λ assays on Beckman Coulter UniCel DxC 800 Synchron and compare to Freelite κ and λ assays on Roche Cobas Integra., Methods: Linearity verification, within- and between-run precision, method comparison, and reference range (RR) verification were conducted using CLSI guidelines. Statistical analysis was performed using EP Evaluator ® . Mean, SD, CV, and bias were determined., Results: Diazyme κ FLC assay was linear within 0.00-191.00 mg/L. Diazyme λ FLC assay was linear within 0.00-205.30 mg/L. Diazyme κ FLC QC1 had a mean of 16.70 mg/L, CV of 7.0%. QC2 had a mean of 33.37 mg/L, CV of 2.6%. Diazyme λ FLC QC1 had a mean of 21.73 mg/L, CV of 2.3%. QC2 had a mean of 42.05 mg/L, CV of 1.5%. Bias of DxC-Diazyme FLCs compared to Integra-Freelite FLCs was -2.55 mg/L (κ FLC), and 4.54 mg/L (λ FLC). Qualitative comparison of κ FLC assays indicated 100% agreement for both normal and abnormal values. For λ FLC assay, agreement was 95% for normal values and 75% for abnormal values. For κ/λ ratio there was 50% agreement for normal values, and 100% for abnormal values. For RR verification, 1 sample was outside the Diazyme κ RR. For λ, all samples were within the manufacturer's RR., Conclusions: Diazyme assays for FLCs have excellent precision and accuracy and are comparable to Freelite assays., (© 2019 American Association for Clinical Chemistry.)

Immunodiagnostic tests performed at the point of care (POC) today usually employ antibodies for biorecognition and are read out either visually or with specialized equipment. Availability of alternative biorecognition elements with promising features as well as smartphone-based approaches for signal readout, however, challenge the described established configuration in terms of analytical performance and practicability. Assessing these developments' clinical relevance and their impact on POC immunodiagnostics is demanding. The first part of this review will therefore give an overview on suitable diagnostic biosensors based on alternative recognition elements (such as nucleic acid-based aptamers or engineered binding proteins) and exemplify advantages and drawbacks of these biomolecules on the base of selected assays. The second part of the review then focuses on smartphone-connected diagnostics and discusses the indispensable considerations required for successful future clinical POCT implementation. Together, the joint depiction of two of the most innovative and exciting developments in the field will enable the reader to cast a glance into the distant future of POC immunodiagnostics.

Understanding of the determinants of immunogenicity, the testing paradigm, the impact of antibody attributes on clinical outcomes and regulatory guidance is leading to harmonized practices for immunogenicity assessment of biotherapeutics. However, generation of robust immunogenicity data for inclusion in product labels to support clinical practice continues to be a challenge. Assays, protocols and antibody positive controls/standards need to be developed in sufficient time to allow assessment of clinical immunogenicity using validated methods and optimized protocols. Standardization and harmonization play a significant role in achieving acceptable results. Harmonization in the postapproval setting is crucial for a valid interpretation of the product's immunogenicity and its clinical effects. Efforts are ongoing to standardize assays where possible for antibody measurement and for measuring product/drug levels by producing reference standards. Provision of such standards will help toward personalized treatment strategies with better patient outcomes.

Background: The Mindray SAL 8000 is an integrated serum analyzer for photometric, electrochemical, and im-munological assays. The technical, analytical, and workflow performance of the system were evaluated in this study., Methods: The technical evaluation was performed using protocols adopted from the guidelines of the China Food and Drug Administration (CFDA). The precision, linearity, interference, and method comparison were carried out according to the Clinical and Laboratory Standards Institute (CLSI) protocols. The verification of carryover and turnaround time were conducted using specimens containing different analytes., Results: The technical performance was acceptable for all evaluated aspects. The repeatability and within-labora-tory coefficients of variation (CVs) ranged between 0.22% and 4.23% for routine chemistry and between 1.05% and 6.89% for immunochemistry, respectively. All evaluated analytes exhibited linearity over the ranges claimed by the manufacturer. Significant interferences were observed during low concentration TBIL and P measure-ments due to the presence of lipemia. Method comparisons showed good agreement with the comparison systems and with the correlation coefficients ≥ 0.988 except for anti-HBs (r = 0.812). No significant intra-module and inter-module carryovers were detected. For all the 1,220 samples, 25%, 54%, 63%, 79%, 91%, and 100% samples com-pleted analysis in 16.3 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 320 minutes, respectively., Conclusions: The Mindray SAL 8000 integrated system achieved optimal technical performance and met most of the criteria regarding analytical performance. The workflow study of the system met the turnaround time (TAT) requirements of laboratories. Therefore, it is a good candidate to be used in medium and large-sized laboratories.

Techniques with high sensitivity and specificity are required for an accurate diagnosis in low-transmission settings, where the conventional parasitological methods are insensitive. We determined the accuracy of an up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF CAA) assay in urine and serum for Schistosoma mansoni diagnosis in low-prevalence settings in Ceará, Brazil, before and after praziquantel treatment. Clinical samples of a total of 258 individuals were investigated by UCP-LF CAA, point-of-care-circulating cathodic antigen (POC-CCA), soluble worm antigen preparation (SWAP)-ELISA and Kato-Katz (KK); a selection of 128 stools by real-time PCR technique. Three and 6-weeks after treatment, samples were collected and evaluated by detection Schistosoma circulating antigens (CAA and CCA). The UCP-LF CAA assays detected 80 positives (31%) with urine and 82 positives (31.8%) with serum. The urine POC-CCA and serum SWAP-ELISA assays detected 30 (11.6%) and 107 (40.7%) positives, respectively. The Kato-Katz technique revealed only 4 positive stool samples (1.6%). Among the 128 individuals with complete data records, 19 cases were identified by PCR (14.8%); Sensitivities and specificities of the UCP-LF CAA assays, determined versus a combined reference standard based on CCA/KK/PCR positivity, ranged from 60-68% to 68-77%, respectively. In addition only for comparative purposes, sensitivities of the different assays were determined vs. a comparative reference based on CAA/KK/PCR positivity, showing the highest sensitivity for the urine CAA assay (80%), followed by the serum CAA (70.9%), SWAP-ELISA (43.6%), PCR (34.5%), POC-CCA (29.1%), whilst triplicate Kato-Katz thick smears had a very low sensitivity (3.6%). CAA concentrations were higher in serum than in urine and were significantly correlated. There was a significant decrease in urine and serum CAA levels 3 and 6-weeks after treatment. The UCP-LF CAA assays revealed 33 and 28 S. mansoni -infected patients at the 3- and 6-week post-treatment follow-up, respectively. The UCP-LF CAA assays show high sensitivity for the diagnosis of S. mansoni in low-endemicity settings. It detects a considerably higher number of infections than microscopy, POC-CCA or PCR. Also it shows to be very useful for evaluating cure rates after treatment. Hence, the UCP-LF CAA assay is a robust and promising diagnostic approach in low-transmission settings.

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible., (© 2019 International Society on Thrombosis and Haemostasis.)

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting.Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards.This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe in vitro methods in detail.

Summary: Background and Objective. Drug-induced anaphylaxis (DIA) is the most common cause of fatal anaphylaxis. We aimed to characterize patients with DIA and their allergological workup. Methods. Systematic review of patients with history of DIA referred to our center over 7 years. Results. Included 125 patients (10% pediatric age), being 36 years the median age of first episode (from 1 to 74 years). The main culprits were nonsteroidal anti-inflammatory drugs (NSAIDs) (43%), antibiotics (42%) and anesthetic agents (6%). In 24% the reactions occurred in hospital setting and 14% perioperative. The etiology was confirmed in 75% through allergological workup. Conclusions. NSAIDs and antibiotics were responsible for most of DIA. The heterogeneity of mechanisms, the severity of the reactions and the lack of standardized in vivo and/or in vitro tests for some drugs do not allow to confirm the diagnosis in all cases. Patients with DIA should be evaluated in specialized centers to perform accurate diagnosis, to prevent recurrence and to find safe alternatives.

Purpose of Review: To present the most recent evidence on beta-lactam hypersensitivity reactions in children., Recent Findings: Drug provocation tests (DPTs) are the gold standard when investigating beta-lactam allergy in children and evidence is increasingly supporting DPTs without skin tests as a safe approach when evaluating children with nonimmediate mild reactions to beta-lactams. Of note, data are limited in the adolescent population, and this attitude may not apply to this age group. Standardization of DPT protocols is required in nonimmediate reactions, as many protocols ranging from 1 to 10 days have been described. The optimal duration of DPT is still unknown, with extended protocols providing slightly more sensitivity and possible higher long-term compliance, at the expense of potential side effects associated with prolonged antibiotic use. On the other hand, 1-day DPTs will identify the vast majority of patients, and the rest will only develop a mild rash during a subsequent full treatment. The natural history of beta-lactam allergy in children is not well studied with recent evidence pointing to the resolution of most confirmed beta-lactam allergies after 3 years., Summary: Further studies are needed for the standardization of DPT protocols and to confirm the favourable natural history of beta-lactam drug allergies in children. In addition, multicentric studies are required to confirm the increasingly accepted position of performing DPTs without skin tests in nonimmediate mild reactions to beta-lactams and to further evaluate the possibility of performing DPTs in benign immediate reactions to beta-lactams in children.

Autoantibodies against double-stranded DNA (dsDNA) were first described >60 years ago and although they are still one of the most clinically relevant autoantibodies, test results may be more challenging to interpret compared to other autoantibody tests. They are a serological hallmark of systemic lupus erythematosus (SLE) and are included in both the American College of Rheumatology (ACR) and the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria for SLE. Furthermore, anti-dsDNA antibodies (a-dsDNA) have been shown to associate with SLE disease activity and coincide with renal involvement. Given their importance and long history, one might assume that immunological tests for a-dsDNA are standardized and give comparable results. However, even though there has been an international reference standard serum (the WHO Wo/80), different methods for the detection of a-dsDNA and tests from different manufacturers give different results for the same samples. This disparity is due to the diversity of possible antibodies generated to this biochemically complex antigen, which may have different clinical associations. The goal of this review is to summarize the current knowledge regarding the clinical associations with a-dsDNA, highlight challenges in a-dsDNA testing, and elucidate the reasons for discrepant results between methods or manufacturers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose of Review: NSAIDs are the drugs most frequently involved in hypersensitivity reactions (HSR). These are frequently prescribed at all ages. HSR are of great concern and can affect people at any age. These drugs can induce reactions by stimulating the adaptive immune system (IgE or T cell), known as selective responders or more frequently by abnormalities in biochemical pathways related with prostaglandin metabolism. These are known as cross-intolerant. With some exceptions, skin testing and in-vitro studies are of little value in selective responders., Recent Findings: In the last years, several classifications have been provided based on clinical symptoms, time interval between drug intake and appearance of symptoms, response to other nonchemically related NSAIDs and the underlying disease. Based on this classification, several well differentiated categories within each group of entities cross-intolerant and selective responders are now recognized. The most complex groups for evaluation are cross-intolerant in which three major groups exist: NSAIDs exacerbated respiratory disease, NSAIDs exacerbated cutaneous disease and NSAIDs-induced urticaria/angioedema in the absence of chronic spontaneous urticaria. Within the selective responders, there are two mechanisms involved: drug-specific IgE or T-cell effector responses. New entities have been added to this classification like mixed reactions within the cross-intolerant category, that must manifest as anaphylaxis and multiple immediate selective reactions., Summary: The precise evaluation of patients with NSAIDs hypersensitivity following established guidelines will improve not only our understanding but also the management of these entities. As the number of patients affected with NSAIDs is important, further studies are warranted.

An estimated 10% to 30% of the global population has an allergic disease. Clinical presentations of allergic diseases, respiratory infections, and autoimmune conditions have similar features. Allergy and immunologic testing can help clarify the diagnosis and guide treatment. Immediate immunoglobulin E (IgE) and delayed T cell-mediated reactions are the main types of allergic responses. The allergens suspected in an immediate IgE-mediated response are identified through serum IgE-specific antibody or skin testing. For patients with an inhalant allergy, skin or IgE-specific antibody testing is preferred. In patients with food allergies, eliminating the suspected allergenic food from the diet is the initial treatment. If this is ineffective, IgE-specific antibody or skin testing can exclude allergens. An oral food challenge should be performed to confirm the diagnosis. Patients with an anaphylactic reaction to an insect sting should undergo IgE-specific antibody or skin testing. Skin testing for penicillin has a high negative predictive value and can help when penicillin administration is indicated and there are limited alternatives. Testing for other drug allergies has less well-determined sensitivity and specificity, but can guide the diagnosis. Patch testing can help identify the allergen responsible for contact dermatitis.

The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the occurrence of venous and arterial thromboses, often multiple, and obstetric-related adverse events in the presence of antiphospholipid antibodies (aPL). APS, first described in 1983, as thrombosis, abortion and cerebral disease, is nowadays recognised as a systemic disease with a wide constellation of clinical manifestations related to acute and chronic vascular lesions. The presence of aPL is the serological hallmark of APS representing a heterogeneous population of autoantibodies with many antigenic specificities directed to phospholipid-binding proteins, either alone or in combination with phospholipids. Many assays have been developed for aPL detection. Particularly, anticardiolipin antibodies, anti-β2-glycoprotein I antibodies and lupus anticoagulant are essential tools for APS diagnosis. The cumulative evidence indicates that aPL are pathogenic autoantibodies binding to target cells and promoting thrombosis and pregnancy complications through a wide range of pathological mechanisms not yet fully understood. Finally, the recognition of the important role of aPL to assess the individual risk of thrombosis or pregnancy complications has expanded the concept of aPL, and currently aPL profile is regarded as a major risk factor for clinical thrombotic events.

Opsonophagocytic assays are used to measure functional antibodies important in protection against pneumococcal capsular antigens. There have been efforts to standardize these methods, as the assays are commonly used to measure vaccine immunogenicity. We report here the results from three international laboratories using their own methods, based on the recommended WHO standard method. We tested 30 pediatric sera, before and after administration of a 13-valent conjugate pneumococcal vaccine, against all 13 serotypes. The three laboratories demonstrated good agreement using their own standardized multiplex opsonophagocytosis assay protocols, particularly postimmunization for those serotypes in the vaccine. While serotype-specific IgG methods have already been internationally standardized and are currently used as a measure of vaccine immunogenicity, this report demonstrates that despite minor differences in methods and a minor variation in response to nonvaccine serotypes, the results from opsonophagocytic assays across the three laboratories may be compared with confidence. IMPORTANCE When measuring a functional antibody response to pneumococcal immunization, it is imperative that a specific, reproducible, accurate, and standardized assay with acceptable inter- and intra-assay variation be advocated internationally to allow for meaningful comparison of results between laboratories. We report here the results of a collaboration between 3 international laboratories testing 30 pediatric samples against the 13 serotypes in Prevenar13., (Copyright © 2018 Balloch et al.)

Cut-off values for anti-dsDNA, anti-nucleosome and anti-C1q antibodies tests and for complementmediated hemolytic activity (CH50) were explored to identify patients with high risk of developing severe lupus nephritis (LN). Forty-one patients with confirmed systemic lupus erythematosus (SLE) were identified; their levels for the three antibodies and complement had been measured on a same serum sample. These patients were classified based on the presence of renal involvem ent; sixteen had active proliferative LN. With the cut-off values accepted in the laboratory for SLE diagnosis (anti-dsDNA > 100 UI/ml, anti-nucleosome > 50 U/ml or CH50 < 190 UCH50%) no significant differences were found between patients with and without LN. Anti-C1q > 40 U/ml showed a statistically significant association with LN and had 80% of specificity. Cut-off values for LN identified by Receiver Operating Characteristic curves (ROC) were higher for anti-dsDNA (> 455 IU/ml) and antinucleosome (>107 U/ml), lower for CH50 (< 150 UCH50%) and, for anti-C1q (> 41 U/ml) coincided with the cut-off values accepted for SLE. Anti-C1q > 134 U/ml had a 92% of specificity, 56% of sensibility and was associated with a fifteen-fold increased risk of LN. The simultaneous presence of anti-nucleosome > 107 U/ml and anti-C1q > 134 U/ml was associated with a 27-fold higher probability for LN. According to these results, the cut-off values used to detect SLE activity could be inadequate to identify patients at high risk of severe LN.

Background: Laboratory quantification of IgG subclasses (IgGSc) is a well-established second-line tool for differential diagnosis of immune deficiencies. However, so far there is still no internationally approved standard available for IgGSc, and different assays are prone to produce divergent results. In this study, we evaluated the comparability and equivalence of two commercially available IgGSc assays, one being the Siemens IgGSc assay on a BN ProSpec analyzer and the other being The Binding Site (TBS) IgGSc assay on a Roche cobas c502 analyzer., Methods: We analyzed a total of 50 patient plasma samples obtained over a 3-month period with both IgGSc assays and compared the resulting data based and the CLSI EP09-A3 method comparison guideline., Results: Depending on the analyzed IgGSc type, the average relative differences in IgGSc concentration (g/L) between the two assays were considerable, starting with -13.5% for IgG1 and 11.3% for IgG2, over -47.3% for IgG4, and up to 52.9% for IgG3. Applying the assay-specific reference intervals, the classification agreement (below, within, or above the reference range) ranged from 88% to 90% for the individual subclasses. However, only 68% of samples showed an overall classification agreement., Conclusion: The comparability of the two IgGSc assays proved to be limited and might be considered similar at best on the diagnostic level. Laboratory specialists as well as clinicians therefore should be cautious when using and interpreting IgGSc measurements obtained with different assays or analyzers., (© 2017 Wiley Periodicals, Inc.)

This study was designed to determine the sensitivity and reproducibility of recovering anti-streptolysin O titres (ASOT) from dried blood spot (DBS) samples, a methodologic subcomponent of the penicillin pharmacokinetic studies in children receiving secondary prophylaxis with intramuscular benzathine penicillin for acute rheumatic fever., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

The clinical utility of molecular diagnostic approaches in allergy investigation is being recognized increasingly to play a significant role in the management of allergic patients. Determining the sensitization pattern, which is best achieved through the use of component resolved diagnostics (CRD), allows effective risk stratification, appropriate treatment and patient selection for immunotherapy. In order to assess the diagnostic service provisions for in-vitro allergy testing across Europe, a survey was carried out via the total immunoglobulin (Ig)E and specific IgE external quality assurance schemes run by UK National External Quality Assessment Service (NEQAS) Immunology, Immunochemistry and Allergy. This survey assessed allergy testing, and in particular allergen components offered by the laboratories, and found a wide variability in service provision, particularly between the United Kingdom and other European Union (EU) countries. Furthermore, there was lack of standardization for acquisition of clinical information to aid allergen (and component) selection, gating strategy, testing algorithms and clinical interpretation. Interestingly, a significant proportion of laboratories (the majority from EU) stated that they 'used' the results for peanut components for risk stratification. However, the vast majority of participants were unaware of guidelines relating to the use of allergen component testing, and agreed that further education would assist in reaching a common platform. Hence, this survey has highlighted that although CRD has been adopted into routine diagnostics across Europe, it is potentially compromised by lack of standardized protocols and guidance sources. Consequently, there is a need for local or national standards and education through External Quality Assurance services on the performance and application of CRD into allergy investigation., (© 2017 Crown copyright. Clinical and Experimental Immunology, © 2017 British Society for Immunology.)