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Variability in Testing for Antineutrophil Cytoplasmic Antibodies: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

Laboratory evaluation for the presence of antineutrophil cytoplasmic antibodies (ANCAs) is used diagnostically in the evaluation of patients suspected of having systemic vasculitis. (1-4) The main utility in ANCA testing is for small-vessel vasculitides, including microscopic polyangiitis; eosinophilic granulomatosis with polyangiitis (previously known as Churg-Strauss syndrome); and small and medium vasculitides, such as granulomatosis with polyangiitis (previously known as Wegener granulomatosis). Together, these diseases are known as ANCA-associated vasculitides. Some drugs are associated with positive ANCA results with or without clinical vasculitis, such as propylthiouracil, methimazole, and carbimazole. (5-7)

Antineutrophil cytoplasmic antibodies are not specific for defined syndromes nosologically classed as vasculitis.

Positive results are associated with autoimmune diseases, such as rheumatoid arthritis, scleroderma, systemic lupus erythematosus, and others. (8-10) Positive results are seen in many patients with ulcerative colitis (but less commonly in Crohn disease) and primary sclerosing cholangitis. (11,12) Additionally, cystic fibrosis patients commonly have a positive ANCA test result resulting from antibodies to bactericidal/permeability-increasing protein. (13) Chronic bacterial infections and chronic liver disease have also been associated with positive ANCA test results. (14,15)

The classic method for detection of ANCAs uses an indirect immunofluorescence assay (IFA) technique where patient serum is incubated on a human neutrophil substrate followed by application of anti-human antibodies conjugated to fluorescein. The cells are reviewed with a fluorescence microscope. In positive cases, 2 main fluorescent patterns will be detected in ethanol-fixed neutrophils: one that reacts with the azurophilic granules distributed throughout the cytoplasm (cytoplasmic pattern, called cANCA) and one that reacts only with antigens located in the cytoplasm surrounding the nucleus (perinuclear pattern, called pANCA). The pANCA pattern results from ethanol-induced leaching of cationic proteins from the azurophilic granules that then collect around the negatively charged nucleus. In formalin-fixed neutrophils, cationic proteins are cross-linked and remain in the azurophilic granules; the immunofluorescent patterns of both cANCA and pANCA reactive sera seen on formalin-fixed cells, respectively, are cytoplas mic granule staining. In most cases, cANCA results from proteinase 3 antibodies (PR3). Perinuclear pattern ANCA results from myeloperoxidase antibodies (MPOs) and occasionally leukocyte elastase or azurocidin. (16,17) Enzyme-linked immunoassays (EIAs) for detection of PR3 and MPO antibodies are also available.

An international consensus statement on ANCA in 1999 recommended that screening be performed with IFA using both ethanol-fixed neutrophils and formalin-fixed neutrophils, and that EIA be performed on positive IFA samples. (18) However, the consensus report recommended that EIA optimally be performed on all samples because the 2 methodologies are discordant in some cases. Current general practice is to screen with IFA and confirm positive results with EIA. A newer multiplex bead assay is also available which uses a combination of flow cytometry and antibody sandwich technique to detect antibodies to PR3 and MPO simultaneously in samples. (19) Thus, the multiplex bead assay could be considered a variant of the EIA technique.

A third ANCA pattern, often noted as an atypical pattern, is rare in vasculitides and can show features of either cANCA (often with a diffuse or dull cytoplasmic pattern) or pANCA (often with intense nuclear membrane staining) on ethanol-fixed neutrophils. The specificities for these atypical ANCAs include lactoferrin, cathepsin G, elastase, lysozyme, bactericidal/permeability-increasing protein, catalase, alpha-enolase, actin, histone H1, high-mobility group protein-1, high-mobility group protein-2, and lamin B1, and the 50-kDa laminlike neutrophil nuclear envelope protein. (20) In addition, the presence of antinuclear antibodies (ANAs) may interfere with evaluation of IFA patterns. Use of both ethanol-fixed neutrophils and formalin-fixed neutrophils improves interpretation of ANCA patterns. (21) In many cases, the atypical patterns are absent or decreased in formalin-fixed neutrophils. However, some difficulties arise in testing formalin-fixed neutrophils, including autofluorescence of cells. (22) Indirect immunofluorescence testing for ANA using HEp-2 cell substrate can be used as a supplemental test to determine whether there is ANA interference.

Detection of ANCAs is not always straightforward and use of these tests clinically is occasionally controversial. Issues regarding the methods of detection, laboratory testing algorithms, and clinical utility of serial testing are common. Additionally, the reported prevalence of ANCAs in different disease states varies widely depending on disease stage, disease activity, prior therapy, and testing methods used. (23) Laboratory testing practices vary because of the complexity of testing and possible interferences.

The Diagnostic Immunology Resource Committee acts as the expert scientific and educational resource for the College of American Pathologists (CAP) in diagnostic immunology and flow cytometry. Volunteer members from a variety of academic institutions and private laboratories oversee the proficiency testing (PT) for hundreds of analytes in our surveys. The primary purpose of this study was to examine the testing practices used by laboratories for detection of ANCAs among the participants in the PT survey.

MATERIALS AND METHODS

A supplemental questionnaire was sent to laboratories participating in the ANCA PT survey (CAP Special Immunology S2 Survey). A total of 189 laboratories reported results for IFA on ethanol-fixed neutrophils, and 263 laboratories reported results for EIA testing (including laboratories that performed both). The brief questionnaire (Figure 1) was developed by members of the Diagnostic Immunology Resource Committee, and the questions were included as part of the S2-A mailing in the first part of 2014.

RESULTS

A total of 333 unique laboratories reported ANCA results, and 315 laboratories responded to the questionnaire, a response rate of 95%. The first question asked laboratories about the algorithm used for ANCA testing. As seen in Figure 2, laboratories reported a variety of algorithms, including IFA only (84 of 315; 27%), EIA only (92 of 315; 29%), or a combination of both (88 of 315; 28%). A small percentage of laboratories (29 of 315; 9%) reported "Other" for their algorithm, which included laboratories running a combination of IFA and multiplex bead assay.

Several follow-up questions asked laboratories about the specifics of their testing algorithm. Of the total respondents, 190 laboratories reported using IFA in some capacity (shown in the Table), and of these, the vast majority (170 of 190; 89.5%) used both ethanol- and formalin-fixed neutrophil substrates. A total of 304 laboratories responded to the question about ANA being run on all samples (shown in the Table). A minority of these laboratories reported running ANA on all samples (43 of 304; 14%), and a similar minority (39 of 304; 13%) reported running ANA on pANCA cases only. The final question asked if laboratories reported results for atypical ANCA, to which 245 labs responded, and 135 laboratories (55%) responded affirmatively.

COMMENT

In this study, we sought to determine the current state of laboratory testing for ANCA. We found great variety in the testing algorithms used by laboratories in the detection of ANCA. More than a third reported using EIA or multiplex bead assays alone (114 of 315, or 36% of responses for either). Although these laboratories will detect the most common forms of ANCA associated with vasculitides, they will not be able to detect an atypical ANCA of any form. It is most likely that laboratories choose to perform this type of testing alone because EIA and multiplex bead assays can be run on automated platforms, and the IFA technique requires more specialized training of personnel to recognize the fluorescent patterns microscopically. One additional benefit of the EIA-only methods is that interference by the presence of an ANA will be very unlikely.

Laboratories reporting IFA testing only accounted for 84 of 315 total respondents (27%). Although the IFA remains the gold standard for ANCA testing, the EIA (and presumably the multiplex bead assay) are considered somewhat more sensitive for the presence of low-level anti-PR3 or anti-MPO antibodies. Laboratories performing IFA only will likely miss these low-level antibodies. It is unclear whether low-level antibody can be responsible for disease, but some studies have shown that testing with a combination of both IFA and EIA is more sensitive for ANCA-associated vasculitis than either alone. Clinicians ordering from these laboratories would either have to order the EIA separately, or wait for a negative result on the IFA and reflexively order the EIA if medically necessary.

Only 62 of 315 laboratories (20%) reported running IFA followed by EIA for positive cases, and an additional 16 of 315 laboratories (5%) reported running both IFA and EIA on all cases. Either of these approaches fully complies with the International Consensus Statement on ANCA testing. Since this statement was published in 1999, current recommendations for testing continue to advocate for running both IFA and EIA. Interestingly, 10 of 315 laboratories (3%) reported running the EIA on all cases, and then running IFA on the negative cases (a reverse algorithm). Technically, this approach would also result in a similar sensitivity for disease. This reverse algorithm would provide labs with the ability to automate testing in a first-pass screening, and subsequently reduce the number of IFAs being run, but it would miss EIA-negative, ANCA-positive cases.

Given the possibility of interference by ANA, it is interesting to note that only 82 labs (of the 190 labs that reported using IFA, shown in the Table) reported running an ANA test either on all cases (43 labs) or on pANCA-positive cases (39 labs). Presumably many clinicians are likely to order ANA in addition to ANCA, because some vasculitis cases can be associated with ANA-positive disorders (such as lupus). Additionally, some ANA cases will look negative or only weakly positive on formalin-fixed neutrophils; thus, laboratories that run testing on formalin-fixed neutrophils may not feel the need to run this additional testing. Some labs may feel that running the EIA as a "confirmatory" assay after the IFA would provide evidence of interference. Well-trained observers can usually distinguish nuclear staining from perinuclear cytoplasmic staining, but this can be difficult in some cases when antinuclear staining is rim or homogeneous in pattern.

The final question asked participants whether an atypical ANCA pattern was reported, and 135 labs reported yes. If only ethanol-fixed neutrophils were used, it is unlikely for the laboratory to detect an atypical pattern. Atypical patterns are most often associated with diseases other than vasculitis, including chronic infections and ulcerative colitis. Recent reports have also shown an atypical pattern in patients exposed to levamisole. (24,25)

CONCLUSIONS

We believe optimal testing includes IFA on ethanol- and formalin-fixed neutrophil substrates and PR3 and MPO EIAs. At the least, laboratories should clearly communicate to ordering physicians the tests performed and the methodologies used in their ANCA evaluation. Some recent advances in laboratory testing technology may help improve ANCA testing in the near future. Instruments are available that help automate the processing of IFA slides. Automated image analysis may streamline the now subjective, operator-dependent nature of evaluation of these slides. (26,27) Antineutrophil cytoplasmic antibody continues to be a valuable tool for evaluation of vasculitides, and newer studies suggest ANCA can be a useful diagnostic test in other diseases as well. As new research begins to show genetic linkages to many of these diseases, ANCA will continue to be useful in the future as well. The College of American Pathologists hopes that laboratory personnel and clinical staff will continue dialogue about testing algorithms and strategies that will maximize benefit to patients.

Please Note: Illustration(s) are not available due to copyright restrictions.

References

(1.) Van der Woude FJ, Rasmussen N, Lobatto S, et al. Autoantibodies against neutrophils and monocytes: tools for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet. 1985; 325(8426): 425-429.

(2.) Savage COS, Winearls CG, Jones S, et al. Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet. 1987; 329(8547):1389-1393.

(3.) Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides: proposal of an International Consensus Conference. Arthritis Rheum. 1994; 37(2):187-192.

(4.) Jennette JC, Falk RJ, Bacon A, et al. 2012 Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum. 2013; 65(1):1-11.

(5.) Slot MC, Links TP, Stegeman CA, et al. Occurrence of antineutrophil cytoplasmic antibodies and associated vasculitis in patient with hyperthyroidism treated with antithyroid drugs: a long-term followup study. Arthritis Rheum. 2005; 53(1):108-113.

(6.) Guma M, Salinas I, Reverter JL, et al. Frequency of antineutrophil cytoplasmic antibody in Graves' disease patients treated with methimazole. J Clin Endocrinol Metab. 2003; 88(5):2141-2146.

(7.) Miller RM, Savige J, Nassis I, et al. Antineutrophil cytoplasmic antibody (ANCA)-positive cutaneous leucocytoclastic vasculitis associated with antithyroid therapy in Graves' disease. Australas J Dermatol. 1998; 39(2):96-99.

(8.) Wiik A. Granulocyte-specific antinuclear antibodies, possible significance for the pathogenesis, clinical features and diagnosis of rheumatoid arthritis. Allergy. 1980; 35(4):263-289.

(9.) Nassberger L, Sjoholm AG, Jonsson H, et al. Autoantibodies against neutrophil cytoplasm components in systemic lupus erythematosus and in hydralazine-induced lupus. Clin Exp Immunol. 1990; 81(3): 380-383.

(10.) Targan SR, Landers C, Vidrich A, et al. High-titer antineutrophil cytoplasmic antibodies in type-1 autoimmune hepatitis. Gastroenterology. 1995; 108(4):1159-1166.

(11.) Oudkerk Pool M, Ellerbroek PM, Ridwan BU, et al. Serum antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease are mainly associated with ulcerative colitis: a correlation study between perinuclear antineutrophil cytoplasmic autoantibodies and clinical parameters, medical, and surgical treatment. Gut. 1993; 34(1):46-50.

(12.) Terjung B, Spengler U, Sauerbruch T, et al. Atypical p-ANCA in IBD and hepatobiliary disorders react with a 50-kilodalton nuclear envelope protein of neutrophils and myeloid cell lines. Gastroenterology. 2000; 119(2):310-322.

(13.) Zhao MH, Jayne DRW, Ardiles LG, et al. Autoantibodies against bactericidal/permeability-increasing protein in patient with cystic fibrosis. QJM. 1996; 89(4):259-265.

(14.) Bonaci-Nikolic B, Andrejevic S, Pavlovic M, et al. Prolonged infections associated with antineutrophil cytoplasmic antibodies specific to proteinase 3 and myeloperoxidase: diagnostic and therapeutic challenge. Clin Rheumatol. 2010; 29(8):893-904.

(15.) Csernok E, Lamprecht P, Gross WL. Clinical and immunological features of drug-induced and infection-induced proteinase 3-antineutrophil cytoplasmic antibodies and myeloperoxidase-antineutrophil cytoplasmic antibodies and vasculitis. Curr Opin Rheumatol. 2010; 22(1):43-48.

(16.) Goldschmeding R, van der Schoot CE, ten Bokkel Huinink D, et al. Wegener's granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils. J Clin Invest. 1989; 84(5):1577-1587.

(17.) Zhao MH, Lockwood CM. Azurocidin is a novel antigen for antineutrophil cytoplasmic autoantibodies (ANCA) in systemic vasculitis. Clin Exp Immunol. 1996; 103(3):397-402.

(18.) Savige J, Gillis D, Benson E, et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol. 1999; 111(4):507-513.

(19.) Artemissia-Phoebe N, Notas G, Mamoulaki M, et al. Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum. J Immunol Methods. 2006; 311(1-2):189-197.

(20.) Lesavre P, Noel LH, Nusbaum P, et al. Atypical autoantigen targets of perinuclear antineutrophil cytoplasm antibodies (P-ANCA): specificity and clinical associations. J Autoimmun. 1993; 6(2):185-195.

(21.) Lin MW, Silvestrini RA, Culican S, et al. A dual-fixed neutrophil substrate improves interpretation of antineutrophil cytoplasmic anti-bodies by indirect immunofluorescence. Am J Clin Pathol. 2014; 142(3): 325-330.

(22.) Chowdhury SMZ, Broomhead V, Spickett GP, et al. Pitfalls of formalin fixation for determination of antineutrophil cytoplasmic antibodies. J Clin Pathol. 1999; 52(6):475-477.

(23.) Chen M, Yu F, Wang SX, et al. Antineutrophil cytoplasmic autoantibody-negative pauci-immune crescentic glomerulonephritis. J Am Soc Nephrol. 2007; 18(2):599-605.

(24.) Rongioletti F, Ghio L, Ginevri F, et al. Purpura of the ears: a distinctive vasculopathy with circulating autoantibodies complicating long-term treatment with levanmisole in children. Br J Dermatol. 1999; 140(5):948-951.

(25.) Walsh NMG, Green PJ, Burlingame RW, et al. Cocaine-related retiform purpura: evidence to incriminate the adulterant, levamisole. J Cutan Pathol. 2010; 37(12):1212-1219.

(26.) Sowa M, Grossmann K, Knutter I, et al. Simultaneous automated screening and confirmatory testing for vasculitis-specific ANCA. PLOS One. 2014; 9(9):e107743.

(27.) Csernok E, Moosig F. Current and emerging techniques for ANCA detection in vasculitis. Nat Rev Rheumatol. 2014; 10(8):494-501.

William J. Karlon, MD, PhD; Stanley J. Naides, MD; John T. Crosson, MD; Mohammad Qasim Ansari, MD

Accepted for publication September 30, 2015.

From the Department of Laboratory Medicine, University of California, San Francisco (Dr Karlon); Quest Diagnostics Nichols Institute, San Juan Capistrano, California (Dr Naides); the Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis (Dr Crosson); and the Department of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio (Dr Ansari).

No financial support was provided by any entity except the College of American Pathologists, which provided the questionnaire sent to the subscribing laboratories and helped analyze the data.

Reprints: M. Qasim Ansari, MD, Department of Clinical Pathology, Cleveland Clinic, 9500 Euclid Ave, Main Campus, Cleveland, OH 44195 (email: ansarim@ccf.org).
Figure 1. Copy of supplemental questions and answer choices
provided to participants
in the S2 survey (Special Immunology). Abbreviations:
ANCA, antineutrophil cytoplasmic antibodies; P-ANCA, perinuclear
antineutrophil cytoplasmic antibodies.

ANCA Supplemental Questions

1. What is your laboratory's testing algorithm for ANCA testing?

** 2638 Enzyme immunoassay (EIA) followed by
    immunofluorescent assay (IFA)

** 2639 IFA followed by EIA

** 2640 EIA testing only

** 2641 IFA testing only

** 0010 Other, specify: __

** 1871 ANCA testing not performed

If your laboratory does not perform ANCA testing, please stop here.

2. If your laboratory is using IFA for ANCA testing, does you
  laboratory use:

** 2642 Ethanol-fixed slides only

** 2643 Formalin-fixed slides only

** 2644 Both ethanol- and formalin-fixed slides

** 2645 We do not do IFA testing

3. Do you report titers on all positive IF As?

** 1341 Yes
** 1342 No

4. Is your laboratory running a routine antinuclear antibody (ANA)
  test on all ANCA requests?

** 1341 Yes
** 1342 No
** 2646 Only on P-ANCA positive patterns

5. Do you report the atypical P-ANCA pattern on IFA?

** 1341 Yes
** 1342 No

Figure 2. Summary of testing algorithms
used by laboratories responding to Supplemental
Question 1. Abbreviations: ANCA,
antineutrophil cytoplasmic antibodies; EIA,
enzyme-linked immunoassay; IFA, indirect
immunofluorescence assay.

Algorithm Used for ANCA Testing

                 Number of
               Participants
EIA followed        10
by IFA
IFA followed        62
by EIA
IFA and EIA         16
on all
EIA only            92
IFA only            84
Multiplex           22
Bead only
Other               29

Note: Table made from bar graph.

Responses to Supplemental Questions 2 and 4

Question                                       No. (%)
                                            of Respondents

Question 2: If your laboratory is using       190 (100)
IFA for ANCA testing, does your
laboratory use:
  Ethanol- and formalin-fixed slides          170 (89.5)
  Ethanol-fixed only                           20 (10.5)
  Formalin-fixed only                            0
Question 4: Is your laboratory running        304 (100)
a routine antinuclear antibody (ANA)
test on all ANCA requests?
  No                                          222 (73)
  Yes                                          43 (14)
  Only on pANCA cases                          39 (13)

Abbreviations: ANCA, antineutrophil cytoplasmic antibodies; IFA,
indirect immunofluorescence assay; pANCA, perinuclear
antineutro-phil cytoplasmic antibodies.
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Author:Karlon, William J.; Naides, Stanley J.; Crosson, John T.; Ansari, Mohammad Qasim
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Survey
Date:Jun 1, 2016
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