Antinuclear Antibodies Testing Method Variability: A Survey of Participants in the College of American Pathologists' Proficiency Testing Program.

OBJECTIVE: This study was conducted to determine the spectrum of laboratory practices in antinuclear antibody (ANA) test target, performance, and result reporting. METHODS: A questionnaire on ANA testing was distributed by the Diagnostic Immunology and Flow Cytometry Committee of the College of American Pathologists (CAP) to laboratories participating in the 2016 CAP ANA proficiency survey. RESULTS: Of 5847 survey kits distributed, 1206 (21%) responded. ANA screening method varied: 55% indirect immunofluorescence assay, 21% ELISA, 12% multibead immunoassay, and 18% other methods. The name of the test indicated the method used in only 32% of laboratories; only 39% stated the method used on the report. Of 644 laboratories, 80% used HEp-2 cell substrate, 18% HEp-2000 (HEp-2 cell line engineered to overexpress SSA antigen, Ro60), and 2% other. Slides were prepared manually (67%) or on an automated platform (33%) and examined by direct microscopy (84%) or images captured by an automated platform (16%). Only 50% reported a positive result at the customary 1:40 dilution. Titer was reported to endpoint routinely by 43%, only upon request by 23%, or never by 35%. Of the laboratories, 8% did not report dual patterns. Of those reporting multiple patterns, 23% did not report a titer with each pattern. CONCLUSION: ANA methodology and practice, and test naming and reporting varies significantly between laboratories. Lack of uniformity in testing and reporting practice and lack of transparency in communicating the testing method may misdirect clinicians in their management of patients.

Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice: A Survey of Participants in the College of American Pathologists Syphilis Serology Proficiency Testing Program.

CONTEXT: -Syphilis serology screening in laboratory practice is evolving. Traditionally, the syphilis screening algorithm begins with a nontreponemal immunoassay, which is manually performed by a laboratory technologist. In contrast, the reverse algorithm begins with a treponemal immunoassay, which can be automated. The Centers for Disease Control and Prevention has recognized both approaches, but little is known about the current state of laboratory practice, which could impact test utilization and interpretation. OBJECTIVE: -To assess the current state of laboratory practice for syphilis serologic screening. DESIGN: -In August 2015, a voluntary questionnaire was sent to the 2360 laboratories that subscribe to the College of American Pathologists syphilis serology proficiency survey. RESULTS: -Of the laboratories surveyed, 98% (2316 of 2360) returned the questionnaire, and about 83% (1911 of 2316) responded to at least some questions. Twenty-eight percent (378 of 1364) reported revision of their syphilis screening algorithm within the past 2 years, and 9% (170 of 1905) of laboratories anticipated changing their screening algorithm in the coming year. Sixty-three percent (1205 of 1911) reported using the traditional algorithm, 16% (304 of 1911) reported using the reverse algorithm, and 2.5% (47 of 1911) reported using both algorithms, whereas 9% (169 of 1911) reported not performing a reflex confirmation test. Of those performing the reverse algorithm, 74% (282 of 380) implemented a new testing platform when introducing the new algorithm. CONCLUSION: -The majority of laboratories still perform the traditional algorithm, but a significant minority have implemented the reverse-screening algorithm. Although the nontreponemal immunologic response typically wanes after cure and becomes undetectable, treponemal immunoassays typically remain positive for life, and it is important for laboratorians and clinicians to consider these assay differences when implementing, using, and interpreting serologic syphilis screening algorithms.

Comparison of 4th-Generation HIV Antigen/Antibody Combination Assay With 3rd-Generation HIV Antibody Assays for the Occurrence of False-Positive and False-Negative Results.

OBJECTIVE: To assess the false-positive and false-negative rates of a 4th-generation human immunodeficiency virus (HIV) assay, the Abbott ARCHITECT, vs 2 HIV 3rd-generation assays, the Siemens Centaur and the Ortho-Clinical Diagnostics Vitros. METHODS: We examined 123 patient specimens. In the first phase of the study, we compared 99 specimens that had a positive screening result via the 3rd-generation Vitros assay (10 positive, 82 negative, and 7 indeterminate via confirmatory immunofluorescent assay [IFA]/Western blot [WB] testing). In the second phase, we assessed 24 HIV-1 RNA-positive (positive result via the nuclear acid amplification test [NAAT] and negative/indeterminate results via the WB test) specimens harboring acute HIV infection. RESULTS: The 4th-generation ARCHITECT assay yielded fewer false-positive results (n = 2) than the 3rd-generation Centaur (n = 9; P = .02) and Vitros (n = 82; P <.001) assays. One confirmed positive case had a false-negative result via the Centaur assay. When specimens from the 24 patients with acute HIV-1 infection were tested, the ARCHITECT assay yielded fewer false-negative results (n = 5) than the Centaur (n = 10) (P = .13) and the other 3rd-generation tests (n = 16) (P = .002). CONCLUSIONS: This study indicates that the 4th-generation ARCHITECT HIV assay yields fewer false-positive and false-negative results than the 3rd-generation HIV assays we tested.

Marked Variability in Reported Minimal Residual Disease Lower Level of Detection of 4 Hematolymphoid Neoplasms: A Survey of Participants in the College of American Pathologists Flow Cytometry Proficiency Testing Program.

CONTEXT: Flow cytometry is often applied to minimal residual disease (MRD) testing in hematolymphoid neoplasia. Because flow-based MRD tests are developed in the laboratory, testing methodologies and lower levels of detection (LODs) are laboratory dependent. OBJECTIVES: To broadly survey flow cytometry laboratories about MRD testing in laboratories, if performed, including indications and reported LODs. DESIGN: Voluntary supplemental questions were sent to the 549 laboratories participating in the College of American Pathologists (CAP) FL3-A Survey (Flow Cytometry-Immunophenotypic Characterization of Leukemia/Lymphoma) in the spring of 2014. RESULTS: A total of 500 laboratories (91%) responded to the supplemental questions as part of the FL3-A Survey by April 2014; of those 500 laboratories, 167 (33%) currently perform MRD for lymphoblastic leukemia, 118 (24%) for myeloid leukemia, 99 (20%) for chronic lymphocytic leukemia, and 91 (18%) for plasma cell myeloma. Other indications include non-Hodgkin lymphoma, hairy cell leukemia, neuroblastoma, and myelodysplastic syndrome. Most responding laboratories that perform MRD for lymphoblastic leukemia reported an LOD of 0.01%. For myeloid leukemia, chronic lymphocytic leukemia, and plasma cell myeloma, most laboratories indicated an LOD of 0.1%. Less than 3% (15 of 500) of laboratories reported LODs of 0.001% for one or more MRD assays performed. CONCLUSIONS: There is major heterogeneity in the reported LODs of MRD testing performed by laboratories subscribing to the CAP FL3-A Survey. To address that heterogeneity, changes to the Flow Cytometry Checklist for the CAP Laboratory Accreditation Program are suggested that will include new requirements that each laboratory (1) document how an MRD assay's LOD is measured, and (2) include the LOD or lower limit of enumeration for flow-based MRD assays in the final diagnostic report.

Multiple factors contribute to positive results for hepatitis A virus immunoglobulin M antibody.

CONTEXT: In the United States, a successful vaccination program for hepatitis A virus (HAV) infection has decreased both its incidence and the true positive rate for diagnostic immunoglobulin M (IgM) antibody to HAV in acute hepatitis. OBJECTIVE: To survey positive results of HAV IgM tests and determine the effect of changing ordering options. DESIGN: We reviewed all positive results for IgM antibody to HAV between January 2007 and December 2010. Patient demographics, clinical history, and laboratory data were recorded and the encounter, order, and reason for test reviewed. Each result was categorized as indicating acute, recent, resolved, or indeterminate HAV infection. RESULTS: A total of 10,735 tests were performed; 35 patients had 49 positive results. Most positive test results were associated with outpatient visits and were ordered in the assessment of patients with liver disease, but not clinical acute hepatitis. In the final analysis, 4 patients had acute hepatitis A and 20 individual patients had recent and/or resolved hepatitis. All but 1 of the remaining 11 patients had another established cause of liver disease with a positive IgM HAV antibody test result; data to determine causality were insufficient. The total number of tests requested annually decreased more than 35% with the introduction of computerized physician order entry. CONCLUSIONS: Current assays for IgM HAV antibodies are overused in the absence of clinical acute hepatitis; future clinical decision support may improve patterns of order entry. Most patients have findings consistent with HAV exposure but not acute hepatitis; dormant viral infection may be a continuing source of antigen.

Risk factors associated with false positive HIV test results in a low-risk urban obstetric population.

OBJECTIVE: To examine risk factors for false positive HIV enzyme immunoassay (EIA) testing at delivery. STUDY DESIGN: A review of pregnant women who delivered at Parkland Hospital between 2005 and 2008 was performed. Patients routinely received serum HIV EIA testing at delivery, with positive results confirmed through immunofluorescent testing. Demographics, HIV, hepatitis B surface antigen (HBsAg), and rapid plasma reagin (RPR) results were obtained. Statistical analyses included Pearson's chi-square and Student's t-test. RESULTS: Of 47,794 patients, 47,391 (99%) tested negative, 145 (0.3%) falsely positive, 172 (0.4%) positive, and 86 (0.2%) equivocal or missing HIV results. The positive predictive value of EIA was 54.3%. Patients with false positive results were more likely nulliparous (43% versus 31%, P < 0.001) and younger (23.9 ± 5.7 versus 26.2 ± 5.9 years, P < 0.001). HIV positive patients were older than false positive patients and more likely positive for HBsAg and RPR. CONCLUSION: False positive HIV testing at delivery using EIA is associated with young maternal age and nulliparity in this population.

Multiparameter flow cytometry in the diagnosis and management of acute leukemia.

CONTEXT: Timely and accurate diagnosis of hematologic malignancies is crucial to appropriate clinical management. Acute leukemias are a diverse group of malignancies with a range of clinical presentations, prognoses, and preferred treatment protocols. Historical classification systems relied predominantly on morphologic and cytochemical features, but currently, immunophenotypic, cytogenetic, and molecular data are incorporated to define clinically relevant diagnostic categories. Multiparameter flow cytometry provides rapid and detailed determination of antigen expression profiles in acute leukemias which, in conjunction with morphologic assessment, often suggests a definitive diagnosis or a narrow differential. Many recurrent molecular or cytogenetic aberrations are associated with distinct immunophenotypic features, and therefore flow cytometry is an important tool to direct further testing. In addition, detection of specific antigens may have prognostic or therapeutic implications even within a single acute leukemia subtype. After initial diagnosis, a leukemia's immunophenotypic fingerprint provides a useful reference to monitor response to therapy, minimal residual disease, and recurrence. OBJECTIVE: To provide an overview of the application of flow cytometric immunophenotyping to the diagnosis and management of acute leukemias, including salient features of those entities described in the 2008 World Health Organization classification. DATA SOURCES: Published articles pertaining to flow cytometry, acute leukemia classification, and experiences of a reference flow cytometry laboratory. CONCLUSION: Immunophenotypic evaluation is essential to accurate diagnosis and classification of acute leukemia. Multiparameter flow cytometry provides a rapid and effective means to collect this information, as well as providing prognostic information and a modality for minimal residual disease evaluation.