Infliximab Therapeutic monitoring by tryptic peptide LC-MS/MS method improvements lead to improved accuracy with decreased imprecision and turnaround time.

Introduction: Therapeutic drug monitoring of infliximab has become the standard of care for inflammatory bowel disease in the setting of loss of response to therapy, and occasionally in proactive therapy personalization. Measurement of infliximab by tryptic peptide HPLC-MS/MS has been available since 2015, mostly in reference laboratories. Objectives: Here, we present method improvements to our original published method leading to a more efficient, robust, and high throughput tryptic peptide HPLC-MS/MS assay for infliximab quantitation. Methods: Deidentified residual serum samples submitted for clinical testing were used for method comparison and infliximab was spiked into normal human serum for performance studies. Improvements included the addition of a stable isotope labeled full length infliximab internal standard (IS) replacing a surrogate IS, and immunoenrichment using Melon Gel for immunoglobulins replacing the saturated ammonium sulfate precipitation. Digestion and chromatography were optimized, and automation was added. The method improvements were validated to include precision, accuracy, reportable range, linearity, and analytical sensitivity. Results: The digestion time was reduced from overnight to 1 h. The assay analytical measuring range (AMR) remained the same throughout improvements, 1-100 µg/mL, with linearity of 0.98x + 0.50, R2 = 1.00. Intra- and inter-assay imprecision were less than 5 % CV at four different concentrations. Accuracy was assessed with 106 patients within the AMR; Passing-Bablok Regression yielded a slope of 1.00 and a y-intercept of 0.25. Turnaround time was reduced by 1 day, and imprecision of three levels of quality control trended down after new method implementation. Conclusions: Method improvements including automation have allowed for assay completion in half a day, improving robustness and turnaround time.

Diagnostic Performance of a Fecal Calprotectin Assay as a Biomarker for Mayo Endoscopic Subscore in Ulcerative Colitis: Result From a Tertiary Referral Center.

BACKGROUND: Fecal calprotectin (FC) is a promising biomarker for assessing ulcerative colitis (UC) endoscopic activity. However, the optimal FC cutoff to identify each Mayo endoscopic subscore (MES) remains inconclusive. METHODS: The electronic medical records of 177 adult UC patients evaluated at Mayo Clinic Rochester from January 2017 to March 2023 were retrospectively reviewed, obtaining clinical data and US-based Werfen Diagnostics FC levels collected within 30 days before colonoscopy or flexible sigmoidoscopy. Three independent inflammatory bowel disease specialist endoscopists blindly reviewed the most severe endoscopic images for grading MES. RESULTS: The median interval between FC collection and endoscopy was 2 days. Fecal calprotectin showed strong positive correlations with MES (Spearman's r = 0.709; P < .01) and other clinical parameters. Fecal calprotectin cutoff of 60 mcg/g effectively distinguished MES 0 from MES 1-3 (sensitivity, 0.78; specificity, 0.97; area under the receiver operating characteristic curve [AUC], 0.901) and predicted clinical remission (Total Mayo Score ≤2 and no subscore >1; sensitivity, 0.83; specificity, 0.98; AUC, 0.921). Fecal calprotectin cutoff of 110 mcg/g effectively differentiated MES 0-1 from MES 2-3 (sensitivity, 0.86; specificity, 0.87; AUC, 0.915), while a cutoff of 310 mcg/g distinguished MES 0-2 from MES 3 (sensitivity, 0.80; specificity, 0.76; AUC, 0.820). CONCLUSIONS: This study supports the reliability and applicability of FC as a valuable marker of endoscopic inflammation, particularly in distinguishing MES 0 from MES 1-3 using the FC cutoff of 60 mcg/g. Sensitivity analysis demonstrated robust results.

This study from a tertiary referral center evaluated 177 patients with ulcerative colitis and found that a fecal calprotectin cutoff of 60 mcg/g effectively distinguished between a Mayo endoscopic subscore (MES) 0 and MES 1-3, as well as clinical remission, with high specificity, supporting its reliability as a valuable biomarker.

Alpha-1-Antitrypsin (A1AT) Proteotyping by LC-MS/MS.

The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of specific allelic variants by phenotyping (isoelectric focusing (IEF) gel electrophoresis) and/or allele-specific genotyping. Various phenotyping and genotyping methodologies are available, and each has their own advantages and disadvantages. As an alternative, mass spectrometry is emerging as a powerful tool in the identification and quantitation of proteins and peptides. The method described here, referred to as proteotyping, is a proteomic method using trypsin digestion and tandem mass spectrometry that detects the most common deficiency alleles, S and Z, associated with A1AT deficiency.This qualitative mass spectrometry method is based on the principle that the S and Z mutations lead to amino acid changes which result in a change in the mass of the A1AT protein. When the A1AT protein is proteolytically digested, multiple peptides are generated, two of which include the sites of the S and Z mutations, respectively. Peptides generated from wild-type A1AT (M alleles) differ in sequence and mass from peptides generated from the S and Z alleles at these two specific locations. The mass difference allows for differentiation of S and Z peptides, representing the deficiency alleles, from non-S and non-Z peptides, representing the wild-type alleles (M). Interpretation of the peptide patterns in conjunction with A1AT quantitation by immunoassay allows for an accurate assessment for the presence of deficiency alleles in the majority of patients.

Lack of observed interference by therapeutic monoclonal antibodies in select commonly utilized immunoassays.

BACKGROUND: Therapeutic monoclonal antibodies (tmabs) have been hypothesized to interfere with immunoassay measurements, although studies investigating this potential new class of interference are lacking. This study evaluated the effects of tmabs used in cancers ipilimumab (Bristol Myers Squibb), nivolumab (Bristol Myers Squibb), pembrolizumab (Merck) and autoimmune disorders adalimumab (AbbVie), infliximab (Janssen) and vedolizumab (Takeda) in common immunoassays used in the clinical laboratory. METHODS: Residual sera from 10 randomly chosen patients were split into two tubes and spiked with same volume (approximately 5 % final volume) of either saline (control) or 6 tmabs (final concentration of 100 µg/mL each). Concentrations from sixteen analytes in 19 different assays were assessed: TSH (Roche and Beckman), free thyroxine (Roche and Siemens), cortisol (Beckman), Cancer Antigens (CA): CA19-9 (Beckman), CA15-3 (Roche), CA125 (Roche), and CA27.29 (Siemens), carcinoembryonic antigen (Beckman), alpha-fetoprotein (Beckman), thyroglobulin (Beckman) and thyroglobulin antibodies (Beckman), thyroid peroxidase antibody (Beckman), beta-human chorionic gonadotropin (Roche and Beckman), total prostate-specific antigen (Roche), parathyroid hormone (Roche) and antinuclear antibodies IgG (Werfen). The tmab spiked residual sera were compared with matched saline spiked sera and percent error was assessed against allowable total error defined from biological variation or CLIA limits. RESULTS: None of the tested immunoassays were affected by the presence of the tmabs, in samples within or outside assay reference intervals. The median % error among all immunoassays ranged between -2.0% (for TSH) to 2.7% (for TPO Ab assay). CONCLUSION: These findings demonstrate no detectable tmab interference for the assessed immunoassays using spiked preparations of the tmabs in residual human sera. The findings are limited to the tmabs and immunoassays studied here.

Outcomes of Infliximab-Treated inflammatory bowel disease patients undergoing therapeutic drug monitoring with two different assays.

OBJECTIVES: There are multiple assays for infliximab (IFX) drug level (IFX-DL) and antibody to infliximab (ATI) measurement. The aims of this study are to examine the correlation and outcomes of IFX-DL and ATI in inflammatory bowel disease (IBD) patients, simultaneously measured with different methods in different institutions. DESIGN AND METHODS: Residual samples of IFX-treated IBD patients undergoing drug monitoring for IFX-DL and ATI, both measured by ECLIA (Esoterix Laboratories) were used to simultaneously quantify IFX-DL via LC-MS/MS and ATI via an in-house ECLIA (ih-ECLIA) (Mayo Clinic Laboratories). Comparisons of IFX-DL and ATI detection between the assays from different institutions were performed, along with a comparison between the assays by association of IFX-DL and ATI obtained by each method with clinical remission, endoscopic healing (EH) and normal serum C-reactive protein (CRP ≤ 8 mg/L). RESULTS: A total of 151 patients were included (median age, 32 years (range, 12-84); 45.7% female). The median IFX-DL was 7 mcg/mL (IQR: 1.3, 19.4) and 6 mcg/mL (IQR: 0.9, 20) via LC-MS/MS and ECLIA, respectively (Spearman correlation coefficient r = 0.97). ATI was detected in 13/142 (9.2%) via ih-ECLIA of whom 100% had IFX-DL < 5 mcg/mL by LC-MS/MS. ATI was positive in 39/151 (25.8%) via ECLIA, and 84.6% of positives had IFX-DL < 5 mcg/mL by ECLIA. Compared to ECLIA, the frequency of ATI detection via ih-ECLIA was lower in patients in clinical remission (7.3% vs 36.6%; p = 0.0005), those with normal CRP (5.9% vs. 20.0%; p = 0.0005), and in patients with EH (5.3% vs 18.4%; p = 0.03). CONCLUSIONS: IFX-DL was comparable between LC-MS/MS and ECLIA assays. Rate of ATI detection via ih-ECLIA was lower than ECLIA, which was more aligned with favorable clinical outcomes.

Clinical Significance of Anti-U1 Ribonucleoprotein Antibody Is Analyte Dependent: Implications for Laboratory Reporting, Interpretation, and Interassay Correlations.

CONTEXT.­: Antibodies to U1 ribonucleoprotein (U1RNP) were first described more than 50 years ago, and although clinically relevant for antinuclear antibody-associated connective tissue disease (ANA-CTD), test results are challenging to interpret. OBJECTIVE.­: To evaluate the impact of anti-U1RNP analyte diversity in the assessment of patients at risk for ANA-CTD. DESIGN.­: Two multiplex assays for U1RNP (Smith [Sm]/RNP and RNP68/A) were used to test serum specimens from consecutive patients (n = 498) under evaluation for CTD in a single academic center. Discrepant specimens were further tested for Sm/RNP antibody by enzyme-linked immunosorbent assay and the BioPlex multiplex assay. Data were evaluated for antibody positivity per analyte and their method of detection, correlations between analytes, and impact on clinical diagnoses through retrospective chart review. RESULTS.­: Of the 498 patients tested, 47 (9.4%) were positive in the RNP68/A (BioPlex) and 15 (3.0%) were positive in the Sm/RNP (Theradiag) immunoassays. U1RNP-CTD, other ANA-CTD, and no ANA-CTD were diagnosed in 34% (16 of 47), 12.8% (6 of 47), and 53.2% (25 of 47) of the cases, respectively. The prevalence of antibody by method in patients with U1RNP-CTD was 100.0% (16 of 16), 85.7% (12 of 14), 81.5% (13 of 16), and 87.5% (14 of 16) for RNP68/A, Sm/RNP BioPlex, Sm/RNP Theradiag, and Sm/RNP Inova, respectively. For other ANA-CTD and no ANA-CTD, the highest prevalence was observed with RNP68/A; all others had comparable performance. CONCLUSIONS.­: In this study, the overall performance characteristics of Sm/RNP antibody assays were comparable; however, the RNP68/A immunoassay was very sensitive but less specific. In the absence of harmonization, reporting the type of U1RNP analyte in clinical testing may be useful in guiding interpretation and interassay correlations.

Pediatric Celiac Disease: A Review of Diagnostic Testing and Guideline Recommendations.

BACKGROUND: The history of how our knowledge of celiac disease (CD) evolved points to its importance in children. Although it is now appreciated that CD can present at any age, it was originally thought to occur only in children and, if untreated, led to serious consequences. CONTENT: This review includes a brief discussion of small bowel physiology and the pathogenesis of CD. Next, the varied clinical presentations of CD in children are reviewed, including both gastrointestinal and nongastrointestinal manifestations and how these contribute to the difficulty in diagnosis. In addition, information on specific conditions that are associated with CD is presented, particularly as it applies to diagnostic testing of apparently asymptomatic children. The review will also focus on diagnostic testing available for CD and their general performance characteristics. The review will end with a comparison between published guidelines from the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition for diagnosis of pediatric CD. In particular, this review will focus on differences in the incorporation of serologic and genetic testing, and the role of biopsies in the pediatric population. SUMMARY: It is important for laboratorians to understand the evolution of diagnostic guidelines for pediatric CD and how serologic and genetic testing are being applied to and interpreted in this particular patient group.

Correlation of ANA Characteristics with pANCA IFA Interference.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA) testing by the indirect immunofluorescence assay (IFA) is important for the diagnosis of autoimmune vasculitis. A common analytical interference for ANCA-IFA is the presence of an antinuclear antibody (ANA), which can cause an apparent perinuclear ANCA (pANCA) result on ethanol-fixed neutrophils. Here, the association of ANA patterns, titers, and concentrations with pANCA interference is investigated. METHODS: Samples positive for ANA by IFA with homogeneous, speckled, dense fine speckled (DFS), and centromere patterns were tested for ANA by enzyme immunoassay (EIA)] and for ANCA by IFA on ethanol-fixed neutrophils. Titers and concentrations were determined for the ANA-IFA and EIA, respectively, and correlated with the frequency of pANCA interpretations. RESULTS: For ANA-EIA positive samples (≥1.1U), 20.0% led to a pANCA interpretation compared to 5.1% for negative samples (≤1.0U). For samples positive by ANA-IFA, 12.9% resulted in a pANCA interpretation. Interference on pANCA correlated with ANA-IFA titer, with ANA titers ≥1:1280 identified as pANCA positive in 20.9% of samples compared to 9.7% for titers <1:1280. There was also a correlation with ANA pattern, as homogeneous samples were most likely to be called positive for pANCA (31.7%), followed by speckled (8.8%), DFS (6.8%), and centromere (3.6%). CONCLUSIONS: Positivity for ANA by EIA is associated with increased prevalence of pANCA interpretation. Samples positive for ANA by IFA also demonstrated this association, particularly with higher-titer, homogeneous patterns. Laboratories can use this information to determine an optimal workflow for when investigating potential pANCA interferences.

Detection of Plasma Cell Disorders by Mass Spectrometry: A Comprehensive Review of 19,523 Cases.

OBJECTIVES: To verify the analytical performance of a new mass spectrometry-based method, termed MASS-FIX, when screening for plasma cell disorders in a routine clinical laboratory. PATIENTS AND METHODS: Results from 19,523 unique patients tested for an M-protein between July 24, 2018, and March 6, 2020, by a combination serum protein electrophoresis (SPEP) and MASS-FIX were examined for consistency with pretest implementation performance. MASS-FIX's ability to verify abnormal results from SPEP and free light chain measurements was then compared with that of immunofixation electrophoresis (IFE) using a separate cohort of 52,586 patients tested by SPEP/IFE during the same period. RESULTS: Overall, 62.4% of our cohort was negative for an M-protein. Importantly, 7.3% of all specimens had an M spike on SPEP (0.1 to 8.5 g/dL) and MASS-FIX detected an M-protein in all these samples. Of all samples, 30.3% had M-proteins that were detected by MASS-FIX but the SPEP finding was too small for quantification. Of the positive samples, 5.7% contained a therapeutic monoclonal antibody. Of the positive samples, 4.1% had an N-glycosylated light chain (biomarker of high-risk plasma cell disorders). MASS-FIX confirmed a higher percentage of SPEP abnormalities than IFE. MASS-FIX was slightly more sensitive than IFE when confirming an M-protein in samples with an abnormal free light chain ratio. MASS-FIX had a very low sample repeat rate (1.5%). MASS-FIX was highly automatable resulting in a higher number of samples/technologist/day than IFE (∼30% more). CONCLUSION: Overall, MASS-FIX was successful in maintaining validation characteristics. MASS-FIX was more sensitive in confirming SPEP abnormalities when compared with IFE. Ability to detect therapeutic monoclonal antibodies and glycosylated light chains was distinctly advantageous.

MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins.

BACKGROUND: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins. METHODS: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples. RESULTS: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated. CONCLUSIONS: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs.

Synovial fluid α defensin has comparable accuracy to synovial fluid white blood cell count and polymorphonuclear percentage for periprosthetic joint infection diagnosis.

AIMS: The aim of this study was to determine the diagnostic accuracy of α defensin (AD) lateral flow assay (LFA) and enzyme-linked immunosorbent assay (ELISA) tests for periprosthetic joint infection (PJI) in comparison to conventional synovial white blood cell (WBC) count and polymorphonuclear neutrophil percentage (PMN%) analysis. METHODS: Patients undergoing joint aspiration for evaluation of pain after total knee arthroplasty (TKA) or total hip arthroplasty (THA) were considered for inclusion. Synovial fluids from 99 patients (25 THA and 74 TKA) were analyzed by WBC count and PMN% analysis, AD LFA, and AD ELISA. WBC and PMN% cutoffs of ≥ 1,700 cells/mm3 and ≥ 65% for TKA and ≥ 3,000 cells/mm3 and ≥ 80% for THA were used, respectively. A panel of three physicians, all with expertise in orthopaedic infections and who were blinded to the results of AD tests, independently reviewed patient data to diagnose subjects as with or without PJI. Consensus PJI classification was used as the reference standard to evaluate test performances. Results were compared using McNemar's test and area under the receiver operating characteristic curve (AUC) analysis. RESULTS: Expert consensus classified 18 arthroplasies as having failed due to PJI and 81 due to aseptic failure. Using these classifications, the calculated sensitivity and specificity of AD LFA was 83.3% (95% confidence interval (CI) 58.6 to 96.4) and 93.8% (95% CI 86.2 to 98.0), respectively. Sensitivity and specificity of AD ELISA was 83.3% (95% CI 58.6 to 96.4) and 96.3% (95% CI 89.6 to 99.2), respectively. There was no statistically significant difference between sensitivity (p = 1.000) or specificity (p = 0.157) of the two AD assays. AUC for AD LFA was 0.891. In comparison, AUC for synovial WBC count, PMN%, and the combination of the two values was 0.821 (sensitivity p = 1.000, specificity p < 0.001), 0.886 (sensitivity p = 0.317, specificity p = 0.011), and 0.926 (sensitivity p = 0.317, specificity p = 0.317), respectively. CONCLUSION: The diagnostic accuracy of synovial AD for PJI diagnosis is comparable and not statistically superior to that of synovial WBC count plus PMN% combined. Cite this article: Bone Joint J 2021;103-B(6):1119-1126.

Pneumococcal serotype-specific cut-offs based on antibody responses to pneumococcal polysaccharide vaccination in healthy adults.

Antibody responses to pneumococcal polysaccharide vaccination are frequently used as a diagnostic tool for humoral immunodeficiencies, part of the larger collection of inborn errors of immunity. Currently, arbitrary criteria, such as a serotype specific titer of >/= 1.3 µg/mL is most often used as a cut-off for interpretation of pneumococcal antibody responses. The magnitude of the antibody response to each of the 23 serotypes in Pneumovax®, and serotype-specific cut-offs in healthy pneumococcal vaccine-naïve adults has not been previously characterized. IgG antibody concentrations were measured prospectively for 23 pneumococcal serotypes pre and 4-6 weeks post-Pneumovax® vaccination in 100 healthy adults, using a multiplex bead-based assay. Antibodies to 19 of 23 serotypes were informative for distinguishing subjects who responded to vaccination, and the serotype threshold was determined to be 9 of 19 serotypes, which characterized an antibody response to pneumococcal vaccination. While this study may facilitate classification of IgG serotype-specific antibody responses post-pneumococcal vaccination in adult patients undergoing diagnostic immunological evaluation for antibody immunodeficiencies or other relevant contexts, additional studies in healthy children and S. pneumoniae protein-conjugate-vaccinated healthy adults will need to be undertaken in the future.

Highlights of the 33rd annual scientific meeting of the Association of Medical Laboratory Immunologists (AMLI).

The annual meeting of the Association of Medical Laboratory Immunologists (AMLI) was convened virtually over the month of August. Prior to the emergence of the COVID-19 pandemic, AMLI's scientific committee had chosen the following topics as the focus of its 2020 meeting: Histocompatibility Testing and Transplant Immunology; Secondary Immunodeficiency and Immunotherapy Monitoring; ANA Update; and Emerging Infectious Diseases and New Algorithms for Testing. Given the central role of the discipline in the evaluation of the host response to infection, it was apt to add a separate session on antibody testing for SARS-CoV-2 infections to the original program. The current report provides an overview of the subjects discussed in the course of this meeting.

Liquid Chromatography-Tandem Mass Spectrometry-Based α1-Antitrypsin (AAT) Testing.

OBJECTIVES: Failure to produce sufficient quantities of functional α1-antitrypsin (AAT) can result in AAT deficiency (AATD) and significant comorbidities. Laboratory testing plays a vital role in AATD, with diagnosis requiring documentation of both a low AAT level and a mutated allele. This retrospective evaluation examines the efficacy of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (proteotyping)-based algorithm for AATD detection. METHODS: A 16-month retrospective data analysis was performed on two cohorts: 5,474 samples tested with the proteotype-based algorithm and 16,147 samples directly tested by isoelectric focusing (IEF) phenotyping. RESULTS: LC-MS/MS reduced the rate of IEF testing by 97%. The 3% of cases reflexed to IEF resulted in 12 (0.2%) additional phenotype findings. Retrospectively applying the proteotype-based algorithm to the IEF cohort demonstrated a 99.9% sensitivity for the detection of deficiency-associated phenotypes. Most deficiency phenotypes missed by the proteotyping algorithm would come from heterozygous patients with an F, I, or P paired to an S or Z. In all of these cases, patient AAT levels were greater than 70 mg/dL, above the threshold for AAT augmentation therapy. CONCLUSIONS: The proteotype algorithm is a sensitive and cost-effective approach for the diagnosis of clinical AAT deficiency.

Clinical Utility of Serial Measurements of Antineutrophil Cytoplasmic Antibodies Targeting Proteinase 3 in ANCA-Associated Vasculitis.

Background: The utility of ANCA testing as an indicator of disease activity in ANCA-associated vasculitis (AAV) remains controversial. This study aimed to determine the association of ANCA testing by various methods and subsequent remission and examine the utility of a widely used automated addressable laser-bead immunoassay (ALBIA) to predict disease relapses. Methods: Data from the Rituximab vs. Cyclophosphamide for ANCA-Associated Vasculitis (RAVE) trial were used. ANCA testing was performed by direct ELISA, capture ELISA, and ALBIA. Cox proportional hazards regression models were used to evaluate the association of PR3-ANCA level and subsequent remission or relapse. The ALBIA results are routinely reported as >8 when the value is high. For this study, samples were further titrated. A decrease and increase in PR3-ANCA were defined as a halving or doubling in value, respectively. Results: A decrease in ANCA by ALBIA at 2 months was associated with shorter time to sustained remission (HR 4.52, p = 0.035). A decrease in ANCA by direct ELISA at 4 months was associated with decreased time to sustained remission (HR 1.77, p = 0.050). There were no other associations between ANCA decreases or negativity and time to remission. An increase in PR3-ANCA by ALBIA was found in 78 of 93 subjects (84%). Eleven (14%) had a PR3-ANCA value which required titration for detection of an increase. An increase of ANCA by ALBIA was associated with severe relapse across various subgroups. Conclusions: A decrease in ANCA by ALBIA at 2 months and by direct ELISA at 4 months may be predictive of subsequent remission. These results should be confirmed in a separate cohort with similarly protocolized sample and clinical data collection. A routinely used automated ALBIA for PR3-ANCA measurement is comparable to direct ELISA in predicting relapse in PR3-AAV. Without titration, 14% of the increases detected by ALBIA would have been missed. Titration is recommended when this assay is used for disease monitoring. The association of an increase in PR3-ANCA with the risk of subsequent relapse remains complex and is affected by disease phenotype and remission induction agent.