Optimal cut-off value for detecting colorectal cancer with fecal immunochemical tests according to age and sex.

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.

Cut-off values of serum IgG4 among three reagents, including a novel IgG4 reagent: a multicenter study.

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.

Improving hexaminolevulinate enabled cancer cell detection in liquid biopsy immunosensors.

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.

Evaluation of microscopy, serology, circulating anodic antigen (CAA), and eosinophil counts for the follow-up of migrants with chronic schistosomiasis: a prospective cohort study.

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.

Factors associated with differential T cell responses to antigens ESAT-6 and CFP-10 in pulmonary tuberculosis patients.

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.

Effects of antithrombotic drugs on the results of fecal immunochemical test in colorectal neoplasms screening.

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.

Three years' experience of quality monitoring program on pre-analytical errors in china.

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.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

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.

Hepatitis C virus antigen detection is an appropriate test for screening and early diagnosis of hepatitis C virus infection in at-risk populations and immunocompromised hosts.

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.

Analytical performance of lateral flow immunoassay for SARS-CoV-2 exposure screening on venous and capillary blood samples.

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.

Well-Based Antibody Arrays.

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.

Comparison of double antigen sandwich and indirect enzyme-linked immunosorbent assay for the diagnosis of hepatitis C virus antibodies.

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.

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

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).

Improving accuracy of myasthenia gravis autoantibody testing by reflex algorithm.

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.

Analytical performance evaluation of thyroid-stimulating hormone receptor antibody (TRAb) immunoassays.

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.

Use of Humanized Fluorescent Reporter Cell Line RBL NFAT-DsRed for the Detection of Allergen-Specific IgE in Patient Sera Using Allergen Microarrays.

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).

Diagnostic performance of culture filtered protein 10-specific perforin in pediatric patients with active tuberculosis.

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.

Sample size and rejection limits for detecting reagent lot variability: analysis of the applicability of the Clinical and Laboratory Standards Institute (CLSI) EP26-A protocol to real-world clinical chemistry data.

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.

Williams-Beuren Syndrome and celiac disease: A real association?

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.

The new IVD Regulation 2017/746: a case study at a large university hospital laboratory in Belgium demonstrates the need for clarification on the degrees of freedom laboratories have to use lab-developed tests to improve patient care.

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.