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CLINICAL LABORATORY IMMUNOLOGY

Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease

Maria Papp, Istvan Altorjay, Gabriella Lakos, Judit Tumpek, Sandor Sipka, Tamas Dinya, Karoly Palatka, Gabor Veres, Miklos Udvardy, Peter Laszlo Lakatos
Maria Papp
12nd Department of Medicine
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  • For correspondence: drpappm@yahoo.com
Istvan Altorjay
12nd Department of Medicine
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Gabriella Lakos
2Laboratory of Clinical Immunology
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Judit Tumpek
2Laboratory of Clinical Immunology
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Sandor Sipka
2Laboratory of Clinical Immunology
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Tamas Dinya
31st Department of Surgery, University of Debrecen, Debrecen
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Karoly Palatka
12nd Department of Medicine
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Gabor Veres
41st Department of Pediatrics
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Miklos Udvardy
12nd Department of Medicine
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Peter Laszlo Lakatos
51st Department of Medicine, Semmelweis University, Budapest, Hungary
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DOI: 10.1128/CVI.00002-09
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    FIG. 1.

    Immunofluorescent staining patterns produced by four different ANCA assays, both on ethanol- and on formalin-fixed human neutrophils, for two different patients. Atypical P-ANCA present as broad, nonhomogeneous rim-like staining of the nuclear periphery on an ethanol slide and show little or no perinuclear or cytoplasmic staining on a formalin slide. P-ANCA (perinuclear staining) show a fine, homogeneous fluorescent staining of the perinuclear cytoplasm on an ethanol slide and diffusely label the cytoplasm (i.e., convert to C-ANCA) on a formalin slide. C-ANCA (cytoplasmic staining) yield a coarse, speckled cytoplasmic fluorescent pattern with accentuated fluorescence between the nuclear lobes on an ethanol slide, and this pattern becomes more pronounced on a formalin slide. (A) All four ANCA assays show atypical P-ANCA patterns for the serum sample of UC patient 104. (B) The four ANCA assays show distinct ANCA patterns for the serum sample of UC patient 95. Magnification, ×40.

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  • TABLE 1.

    Clinical data for UC and CD patients and non-IBD gastrointestinal-disease controls

    Group and characteristicValue for group
    UC patients (n = 96)
        Males/females46/50
        Age (yr) (mean ± SD)39.8 ± 14.9
        Disease location (%)
            Left-sided colitis67.4
            Extensive23.1
            Proctitis9.5
        Surgery (%)5.2
        Extraintestinal manifestations (%)28.4
    CD patients (n = 108)
        Males/females43/65
        Age (yr) (mean ± SD)34.0 ± 12.2
        Disease behavior (%)
            Nonstricturing, nonpenetrating (B1)39.8
            Stricturing (B2)26.7
            Penetrating (B3)33.5
        Disease location (%)
            Terminal ileum (L1)22.8
            Colon (L2)25.1
            Ileocolon (L3)51.3
            Upper gastrointestinal tract (L4)0.9
        Perianal disease (%)29.2
        Surgery (%)40.9
        Extraintestinal manifestations (%)41.7
    Non-IBD gastrointestinal-disease controls
        Irritable bowel syndrome/diverticulosis patients (n = 64)
            Males/females30/34
            Age (yr) (mean ± SD)38.2 ± 15.1
        Celiac disease patients (n = 96)
            Males/females23/73
            Age (yr) (mean ± SD)35.7 ± 17.4
  • TABLE 2.

    Results of the interassay study with four ANCA assays

    Patient group and ANCA patternNo. (%) of patients with ANCA patterns observed by the following assay:
    InovaImmcoEuroimmunImmuno Concepts
    ANA-negative UC patients (n = 79)
        Total ANCA43 (54.4)40 (50.6)31 (39.2)30 (38.0)
        Atypical P-ANCA34 (43.0)5 (6.3)23 (29.1)23 (29.1)
        P-ANCA6 (7.6)30 (38.0)8 (10.1)4 (5.1)
        C-ANCA3 (3.8)5 (6.3)0 (0.0)3 (3.8)
    ANA-negative CD patients (n = 99)
        Total ANCA18 (18.2)23 (23.2)11 (11.1)11 (11.1)
        Atypical P-ANCA16 (16.2)9 (9.1)8 (8.1)9 (9.1)
        P-ANCA2 (2.0)10 (10.1)3 (3.0)1 (1.0)
        C-ANCA0 (0.0)4 (4.0)0 (0.0)1 (1.0)
    Non-IBD gastrointestinal-disease control groups
        Irritable bowel syndrome/ diverticulosis patients without inflammation (n = 64)
            Total ANCA2 (3.1)3 (4.7)
            Atypical P-ANCA1 (1.6)1 (1.6)
            P-ANCA1 (1.6)2 (3.1)
            C-ANCA0 (0.0)0 (0.0)
        Celiac disease patients (n = 96)
            Total ANCA5 (5.2)4 (4.2)
            Atypical P-ANCA2 (2.1)2 (2.1)
            P-ANCA3 (3.1)2 (2.1)
            C-ANCA0 (0.0)0 (0.0)
  • TABLE 3.

    Concordance between four different immunofluorescent ANCA tests with regard to atypical P-ANCA

    TestConcordancea with the following assay:
    InovaImmcoEuroimmunImmuno Concepts
    Inova1
    Immco0.111
    Euroimmun0.390.031
    Immuno Concepts0.350.120.291
    • ↵ a Agreement was characterized by the κ coefficient.

  • TABLE 4.

    Distribution of the immunofluorescent patterns detected by different assays in IBD patients with antibodies against lactoferrin, BPI, or lysozyme

    TestNo. of casesa
    Atypical P-ANCATypical P-ANCAC-ANCANegative
    Immco71464
    Inova164011
    Euroimmun94018
    Immuno Concepts91120
    • ↵ a A total of 31 patients were tested. Four patients had antibodies against both lactoferrin and BPI, and six patients had antibodies against MPO or PR3 as well.

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Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease
Maria Papp, Istvan Altorjay, Gabriella Lakos, Judit Tumpek, Sandor Sipka, Tamas Dinya, Karoly Palatka, Gabor Veres, Miklos Udvardy, Peter Laszlo Lakatos
Clinical and Vaccine Immunology Apr 2009, 16 (4) 464-470; DOI: 10.1128/CVI.00002-09

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Evaluation of the Combined Application of Ethanol-Fixed and Formaldehyde-Fixed Neutrophil Substrates for Identifying Atypical Perinuclear Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease
Maria Papp, Istvan Altorjay, Gabriella Lakos, Judit Tumpek, Sandor Sipka, Tamas Dinya, Karoly Palatka, Gabor Veres, Miklos Udvardy, Peter Laszlo Lakatos
Clinical and Vaccine Immunology Apr 2009, 16 (4) 464-470; DOI: 10.1128/CVI.00002-09
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KEYWORDS

Antibodies, Antineutrophil Cytoplasmic
Fixatives
Inflammatory Bowel Diseases
Neutrophils
Specimen Handling
Tissue Fixation

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