- Email: [email protected]
Laboratory methods for detection of antineutrophil cytoplasm antibodies
Vol. 10, No. 11, 1990
ELISA using affinity-purifiedantigen. J Immtmol Methods 114:167-74, 1988. 16. LiidemannJ, Utecht B, Gross WL: Anticytoplasmic antibodies in Wegener's Granulomatosis are directed against proteinase 3. In: H6rl WH, Heidland A (eds): Proteasis III. New York: Plenum Press, 1990. 17. N~issbergerL, Jonsson H, Sj6hoim AG, et al.: Circulating anti-elastasein SystemicLupus Erythematosus. Lancet i:509, 1989. 18. N611eB, Specks U, LtidemarmJ, et al.: Anticytoplasmicautoantibodies:Their immunodiagnostic value in Wegener's granulomatosis. Ann Int Med 11:28-40, 1989.
Detection o f & n t l n e u t r o p h i l C ~ o p l m m &ntibodies
Continuedfrom page 159 antigens still remains a problem. The generally higher sensitivity of solid-phase assays compared to IIF is an advantage only if the tests are specific, and this requires the use of highly purified antigens. By design, all such assays measure autoantibodies by their interaction with the respective antigen, and the exact amount of antibody cannot be quantitated by precise physicochemical measurement.12 Because patients' sera generally contain mixtures of autoantibodies with different affinities, the autoantibody activity, rather than the concentration is measured with all the different assays. Despite this limitation, antigen-specific solid-phase assays offer the advantage of automation with more precise quantitation of autoantibody activity.
Indirect Immunofluorescence Autoantibodies directed against a cytoplasmic antigen of human neutrophils were detected by indirect immunofluorescence in patients with vasculitis and glomerulonephritis in the early 1980s. 5,jl These immunoglobulin G antibodies against neutrophils and monocytes, termed anti-cytoplasmic antibodies (ACPA) or anti-neutrophil cytoplasm antibodies (ANCA), were described to be a tool for diagnosis and a marker of disease activity in Wegener's granulomatosis (WG). 26,32 During the First International Workshop on ANCA in 1988, the presence of ACPA/ANCA was defined as the recognition of an uneven, granular staining of the neutrophil and monocyte cytoplasm iden-
C L I N I C A L I M M U N O L O G Y Newsletter 163
19. Pinching AJ, Rees AJ, Pussel BA, et at.: Relapses in Wegener'sgranulomatosis:The role of infection. Br Med J 281:836, 1980. 20. RosenboomS, RautmannA, Gross WL: Tumor necrosis factor in Wegener's granulomatosis. Abstract book of the 7th InternationalCongress of Immunology:Berlin, Stuttgart, New York, Gustav Fischer, 1989. 21. RosenboomS, RautmannA, Gross WL: AntizytoplasmatischeAntik6rper (ACPA) in der Immunfluoreszenz. Lab Med 12:339, 1988. 22. ThompsonRA, Lee SS: Antineutrophil Cytoplasmic Antibodies. Lancet i:Letter670-1, 1989.
23. UlmerM, RautmannA, LtidemannJ, Gross WL: ImmundiaguostischeRelevanzvon Antimyeloperoxidase-Antik6rpembei systemischen Vaskulitidien. 7th Int. Congressof Immunology, Berlin, July 30 to August 5, 1989. 24. van der Woude FJ, Daha MR, van Es LA: Review--The current status of neutrophil cytoplasmic antibodies. Clin Exp Immunol 78:143-8, 1989. 25. van der Woude FJ, RasmussenN, LobattoS, et ai.: Autoantibodiesagainst neutrophils and monocytes: tool for diagnosis and markerof disease activityin Wegener'sgranulomatosis. Lancet i:425-9, 1985.
tical to that of a standard reference serum, using a standardized technique with ethanol-fixed, smeared, or cytospun human leukocytes from normal donors, by indirect immunofluorescence.27 The ACPApositive standard reference serum is available from the Statens Seruminstitut, Copenhagen, Denmark, and the standard technique was published in the proceedings of the workshop. 33 However, the screening dilution that should be used is still under debate. Whereas we use undiluted serum without getting high background staining, others use threshold dilutions of 1:10, 1:16, or 1:20. t°,lT,23,2s Independent of these minor differences, it became clear, that some experience is required to obtain leukocyte preparations of high quality. This is important for differentiation between the various staining patterns. Therefore, it was recommended that the standard technique should be used and that it should not be applied for routine ACPA determination before at least 1000 sera have been examined. 27 Indeed, we obtained several sera from nonvasculitic patients, that were labeled as falsepositive by other laboratories, in which we could not detect ACPA either by IIF or by ELISA. Therefore, we suppose that at least some of the reports of ACPA in nonvasculitic patients might well be due to insufficient standardization of the immunofluorescence assay. Besides ACPA inducing the characteristic cytoplasmic staining, autoantibodies producing an artifactual perinuclear immunostaining of ethanol-fixed neutrophils were described. 7 This artifact is caused by the permeabilization of granule membranes while drying the cells onto slides.
Solubilized nucleophilic proteins diffuse and bind to nuclei when the dried cells are placed in an aqueous medium like ethanol. The redistribution can be prevented by fixing the cells with the cross-linking reagent formaldehyde. However, the use of formaldehyde fixation is not generally advisable, as several antigens lose their antigenicity if this fixative is used. 34 Moreover, just this artifact is desirable because it gives a hint to the autoantibody specificity. During the Second International Workshop on ANCA in 1989, it was recommended to adopt a terminology along the lines used for antinuclear antibody serology, where a descriptive term for the fluorescence pattern likewise gives a hint to the antibody specificity, for example, rim pattern for antibodies to DNA or speckled pattern for antibodies to extractable nuclear antigens. Clear cytoplasmic staining of ethanol-fixed neutrophils and monocytes should be termed C-ANCA and perinuclear staining P-ANCA. After IIF, immunochemical assays should be carried out to specify the autoantibody activity. 34 Although we could show that C-ANCA are directed in at least 95% of cases against proteinase 3 (PR-3), a neutral serine protease of human polymorphonuclear leukocytes and monocytes, ls,2°,2t P-ANCA are directed against a variety of different antigens. Originally, it was suspected that myeloperoxidase was the main target antigen of P-ANCA.7 In patients with pauci-immune necrotizing and crescentic glomerulonephritis, nearly 90% of P-ANCA sera were found to have antimyeloperoxidase reactivity. 6 In contrast, among patients with a wide variety of dif-
© 1990 Elsevier Science Publishing Co., Inc.
0197-1859/90/$0.00 + 2.20
164 C L I N I C A L I M M U N O L O G Y Newsletter
ferent diseases, anti-myeloperoxidase antibodies were only found in about 10% of sera inducing a perinuclear staining of ethanol-fixed neutrophils. 31 It has been shown that not only myeloperoxidase, but also elastase a,22 and lactoferrin3,3° are target antigens of autoantibodies that induce a perinuclear fluorescence pattern in neutrophils. Additionally, autoantibodies reacting with nuclear constituents can cause perinuclear staining. Thus, IIF with ethanol-fixed neutrophils is indeed an important screening procedure to detect antineutrophil antibodies, but the autoantibody specificity must be defined in antigen-specific assays. In order to optimize ANCA detection by IIF, several modifications of the standard technique have been investigated. To improve substrate standardization, and to avoid the use of human blood products, we evaluated ANCA reactivity with the promyelocytic cell line HL-60. Parallel IIF assays with sera from 72 patients demonstrated that the reactivity of HL-60 cells directly correlated with that of neutrophils in both their ability to detect ANCA-positive sera and to differentiate between C- and P-ANCA. 2 Antinuclear antibody binding that often mimics PANCA staining could selectively be impaired by using formalin-acetone-fixed chronic myelocytic leukemia cells for ANCA detection) Two staining patterns were identified using this substrate. Type I is characterized by diffuse granular staining preferentially of immature cells, whereas type II represents similar staining with additional granular staining of the basophils. Type I was suspected to be caused by anti-proteinase 3 antibodies and type II was mainly due to anti-myeloperoxidase antibodies. 3 Although these modifications offer promising perspectives, the standard IIF technique 27 is recommended.
Solid Phase Assays In the first attempts to develop solid-phase assays for detection of ANCA, crude extracts were used. The most crude preparations are an acid extract, ~6 or total nitrogen cavitated neutrophils. 7 These were used to develop a solid phase RIA or an ELISA, respectively. Both preparations not only contain proteinase 3, but the II 0197-1859/90/$0.00
+ 2.20
majority of soluble substances of neutrophils, probably also those that are known to be target antigens of autoantibodies, such as myeloperoxidase, elastase, and lactoferrin. Indeed, both assays lack specificity as sera of SLE patients also gave positive r e s u l t s . 7,29 Later, it became clear that at least the RIA with acid extract yields positive results with sera containing C-ANCA (e.g., anti-proteinase 3 antibodies), as well as with sera containing autoantibodies to myeloperoxidase. 24 Therefore, these extracts are not suitable for differentiation between the various autoantibodies. A more defined, but still crude, substrate that was used in an ELISA for detection of C-ANCA is a preparation of azurophil granules. 25 The azurophil granules were obtained by nitrogen bomb cavitation of neutrophils from healthy human donors and subsequent Percoll gradient centrifugation. After disruption of granules by detergent, the extract was coated to ELISA plates in a very alkaline buffer. This ELISA seems to be more specific but less sensitive for detecting CANCA than an ELISA based on the acid extract and several discrepancies between the results obtained by IIF and ELISA were observed.~4 Interestingly enough, although myeloperoxidase is a major component of the azurophil granules, antimyeloperoxidase antibodies were only detected with this ELISA if they were present in high concentrations. 24 It was not reported whether autoantibodies to other antigens of azurophil granules, like those directed against elastase, gave positive results with this ELISA. Another approach to solid-phase assays is the use of monoclonal antibodies with specificity for the respective antigen. Such antigen-catching ELISAs were developed with monoclonal antibodies against a 29 kDa protein of azurophil granules, against myeloperoxidase and against elastase. 8,9 The monoclonal antibodies were immobilized to the solid phase via an antiserum against mouse immunoglobulins and the respective antigens were caught by the monoclonal antibodies from a preparation of saline-extracted azurophil granules. The advantage of catching ELISAs is that the antigen is preI III © 1990 Elsevier Science Publishing Co., Inc.
Vol. 10, No. 1 1, 1990
sented in its native condition, but interactions between the anti-mouse antiserum or the monoclonal antibody and serum constituents, such as rheumatoid factors, might lead to false-positive results. However, using these catching ELISAs, reactions of ANCA-negative sera with high titers of rheumatoid factor of all three isotypes were not observed. 14 Comparison of titers of C-ANCA determined by IIF with (semi)-quantitative results from the 29 kDa antigen-catching ELISA revealed a reasonable, but not perfect, correlation between both assays. 14 Another limitation of catching ELISAs is due to the fact that binding of the antigen to the monoclonal antibody always occurs at the same epitope. If autoantibodies are directed against this epitope or adjacent regions, their binding will be prevented by steric hindrance. Almost all solid-phase assays for detection of a wide variety of different autoantibody specificities are based on purified antigens bound to the solid phase. Therefore, we used affinity chromatography with naturally occurring human autoantibodies to obtain a well-defined purified antigen as a substrate in an ELISA for detection and quantitation of C-ANCA. ~9 The target antigen of C-ANCA was released from human neutrophils during degranulation induced by phorbol myristate acetate and was further purified by affinity chromatography using a column with bound IgG from a C-ANCA-positive serum. The ELISA was sufficiently specific and sensitive. Autoantibodies directed against myeloperoxidase, elastase or lactoferrin did not lead to false-positive results and the arbitrarily defined units obtained with this ELISA correlated significantly with C-ANCA titers determined by IIF (Figure 1). 19 However, it has to be mentioned that some patients with Wegener's granulomatosis have high IIF titers and low ELISA values and vice versa. 23 In a recent study with a large population of clinically well-characterized patients, only three false-positive cases, all at the very low end of the positive range, were found when results from our ELISA test were evaluated against those of IIF. 23 As this ELISA seems to detect changes in C-ANCA levels in relation to changes in
C L I N I C A L I M M U N O L O G Y Newsletter 165
Vol. 10, No. 11, 1990
units/ml
~
•
SO00-
rs = 0.85 ¢ < 0.001
• •
WG(n:I05)
•
t I000.
•
5OO,
•
"
"
t
"
"
ie
•
•
: l 50
i •
•
: I
l
:•
"
• II
:!i 20J . . . . . . . . . . . . . . . . . . . . . . . . . . . I
IIF tttte (dilutions]
Figure I. Correlation between ACPA titre
determined by indirect immunofluorescence and ACPA concentration measured by ELISA. A highly significant positive correlation was observed between the values obtained by the two methods (Spearman's rank correlation coefficient = 0.85, p < 0.001). The limit for positive ELISA readings was set at 20 units/mL (from gel. 19).
disease activity more promptly than does IIF, 23 it represents a promising quantitative alternative to the semiquantitative IIF technique for serial C-ANCA determinations in patients with Wegener's granulomatosis. Recently, we were able to produce monoclonal antibodies against proteinase 3, 4 which may be of value in optimizing antigen purification. In addition, the chromatographic techniques originally employed for purification of proteinase 3 t5 can be used to establish a solid-phase assay, z° For the specific detection of autoantibodies against myeloperoxidase or elastase, basically two techniques have been used, either an ELISA with chromatographically purified enzymes or a sandwich ELISA using monoclonal antibodies to myeloperoxidase and elastase, s,9 The chromatographically purified enzymes were mainly purchased commercially, but myeloperoxidase was not 100% pure. 24 Impurities of antigen preparations may give rise to false-positive results, espeII
cially if contaminating proteins have a high affinity to the solid phase and are target antigens of different autoantibodies. Indeed, myeloperoxidase apparently binds poorly to the solid phase if present in a preparation of azurophil granules,25 which might be due to a rather low affinity to the polystyrene. In this context, it should be mentioned that factors determining the cut-off values of different solid-phase assays are not always clearly defined24 and confirmatory tests are carried out only occasionally. Immunoblotting, a method which is often used as a confirmatory test, is not very promising, because autoantigens like proteinase 3 and elastase are of very similar molecular weight and antigenic epitopes of myeloperoxidase and proteinase 3 are destroyed if electrophoresis is carried out under reducing conditions. 2° Future developments of laboratory methods for the detection of autoantibodies, in general, will involve tests based on synthetic antigens or peptides, including those produced by recombinant DNA technology.l Recently we have been successful in cloning proteinase 3, the main target antigen of C-ANCA t3 and, currently, we are developing a method to express the enzyme in its native form. This will enable us to generate large quantities of purified proteinase 3 that can be used to establish a sensitive and specific ELISA for detection and quantification of anti-proteinase 3 antibodies, c m
References 1. A Second IUIS/WHO Report: Laboratory investigations in clinical immunology: methods, pitfalls and clinical indications. Clin Exp lmmunol 74:494-503, 1988. 2. Charles LA, Falk RJ, Jennette JC: Reactivity of anti-neutrophil cytoplasmic autoantibodies with HL-60 cells. Ciin Immunol Immunopathol 53:243-53, 1989. 3. Chevailler A, Noel LH, Renier G, et al.: Unambiguous determination of antinentrophil cytoplasm antibodies (ANCA) specificity by immunofluorescence on chronic myelocytic leukemia ceils. J Immunol Methods (in press), 1990. 4. Csemok E, Ltidemarm J, Gross WL, Bainton DF: Ultra,structural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis. Am J Pathol (in press), 1990.
5. Davies DJ, Moran JE, Niall JF, Ryan GB: Segmental necrotising glomemlonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J 285:606, 1882. 6. Falk 1LI, Hogan SL, Wilkman AS, et al.: Myeloperoxidase specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA). Neth J Mud 36:121-5, 1990. 7. Falk PJ, Jennette JC; Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomemlonephritis. N Engl J Mud 318:1651-57, 1988. 8. Goldschmeding R, Cohen Tervaert JW, van der Schoot CE, et al.: ANCA, antimyeloperoxidase, and anti-elastase: three members of a novel class of autoantibodies against myeloid lysosomal enzymes. APMIS 97(Suppl. 6):48-9, 1989. 9. 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 84:1577-87, 1989. 10. Gross WL: Wegener's granulomatosis. New aspects of the disease course, immunodiagnostic procedures, and stage-adapted treatment. Sarcoidosis 6:15-29, 1989. 1I. Hall JB, Wadham BMcN, Wood CJ: Vasculitis and glomerulonepllfitis: a subgroup with antineutrophil cytoplasmic antibody. Aust NZ J Med 14:277-8, 1984. 12. Haynes DC, Homburger HA: International standards and reference reagents for measurement of serum proteins and autoantibodies. Clin Immunol Newsletter 10:39-41, 1990. 13. Jenne DE, Tschopp J, Liidemann J, et al.: Wegener's autoantigen decoded. Nature 346:520, 1990. 14. Kallenberg CGM, Rasmussen N: Solid phase assays for ANCA. Neth J Med 36:132-6, 1990. 15. Kao RC, Wehner NG, Skubitz KM, et al.: Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 82:1963-73, 1988. 16. Lockwood CM, Bakes D, Jones S, et al.: Association of alkaline phosphatase with an autoantigen recognized by circulating anti-neutrophil antibodies in systemic vasculitis. Lancet i:71620, 1987. 17. Liidemann G, N611e B, Rautmann A, et al.: Antizytoplasmatische Antik6rper als Seromarker und Aktivit~tsparameter der Wegener'schen Granulomatuse. Eine prospektive Studie. Dtsch med Wschr 113:413-7, 1988. 18. Liidemarm J, Csernok E, Ulmer M, et al.: Antineutrophil cytoplasm antibodies in Wegener's granulomatosis: immunodiagnostic value, monoclonal antibodies and characterization of the target antigen. Neth J Mud 36:157-62, 1990. 19. Liidemann J, Utecht B, Gross WL: Detection and quantification of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by
I
© 1990 Elsevier Science Publishing Co., Inc.
019%1859/90/$0.00 + 2.20
166 C L I N I C A L I M M U N O L O G Y Newsletter
ELISA using affinity-purified antigen. J lmmunol Methods 114:167-74, 1988. 20. Liidemann J, Utecht B, Gross WL: Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J Exp Med 171:357-62, 1990.
25.
21. Liidemann J, Utecht B, Gross WL: Anti-cytoplasmic antibodies in Wegener's granulomatosis are directed against proteinase 3. In: H6rl WH, Heidland A (eds): Proteases III. New York, Plenum Press (in press), 1990.
26.
22. Nhssberger L, Jonsson H, Sj6holm AG, et al.: Circulating anti-elastase in systemic lupus erythematosus. Lancet i:509, 1989. 23. N611e B, Specks U, Lt~demann J, et al.: Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 111:28-40, 1989. 24. Rasmussen N, Daha MR: Concluding remarks
27.
28.
on solid phase assays. Neth J Med 36:143-5, 1990. Rasmussen N, Sjrlin C, Isaksson B, et al.: An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA). J Immunol Methods 127:139-45, 1990. Rasmussen N, Wiik A: Autoimmunity in Wegener's granulomatosis. In: Veldman JE, McCabe BF, Huizing EH, Mygind N (eds): Immunobiology, Autoimmunity and Transplantation in Otorhinolaryngology. Amsterdam: Kugler Publications, pp. 231-6, 1985. Rasmussen N, Wiik A, H¢ier-Madsen M, et al.: Anti-neutrophil cytoplasm antibodies 1988. Lancet i:706-7, 1988. Rosenboom S, Rautmann A, Gross WL: Antizytoplasmatische Antikrrper (ACPA) in der indirekten lmmunfluoreszenz: Probleme beim Testaufbau und bei der Testbeurteilung. Lab Med 12:339-45, 1988.
Antlneutrolphil Cytoplasmic Autoantibody in Renal Disease Ronald J. Falk and J. Charles Jennette Departments of Medicine and Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
apidly progressive glomerulonephritis (RPGN) is a clinical syn,drome with aggressive glomerular injury that causes renal failure, which may be irreversible if not treated appropriately. 5 The pathologic lesion most frequently associated with RPGN is crescentic glomerulonephritis. RPGN may be observed in patients with disease limited to the kidney or as part of a systemic illness. To optimize patient management, practicing nephrologists must classify RPGN into one of several clinicopathologic syndromes. Some indication as to the classification of RPGN may be derived from three sources: (1) the clinical assessment as to which organ systems are involved in the disease process; (2) serologic studies; and (3) examination of the renal biopsy specimen by indirect immunofluorescence microscopy and electron microscopy for the presence and localization of immunoglobulin deposits. 21 Based on immunohistologic studies, three general categories of RPGN may be discerned. Immune-complex-mediated diseases are considered when granular de-
R
posits of immunoglobulin are observed in glomeruli, for example, in diseases such as Henoch-Schonlein purpura, systemic lupus erythematosus, postinfectious glomerulonephritis, and cryoglobulinemic glomerulonephritis. The diagnosis of these conditions is supported by the presence in the circulation of autoantibodies such as anti-DNA or antistreptococcal antibodies. The overall clinical picture aids in determining if the disease process is renal-limited or is a systemic process (e.g., IgA nephropathy versus HenochSchonlein purpura). Another category of disease to be considered in the diagnosis of RPGN is antiglomerular basement membrane antibody-mediated glomerulonephritis and Goodpasture's syndrome, in which aggressive glomerulonephritis is associated with linear staining for immunoglobulins along the glomerular basement membrane, and antiglomerular basement membrane autoantibodies in the circulation. A third category of disease is the pauci-immune form of glomerulonephritis; so called because of the few or no immune deposits seen by immunofluorescence mi-
Vol. 10, No. 1 1, 1990
29. Savage COS, Winearls CG, Jones S, et al.: Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet i:1389-93, 1987. 30. Thompson RA, Lee SS: Antineutrophil cytoplasmic antibodies. Lancet i:670-1, 1989. 31. Ulmer M, Liidemann J, Gross WL: Immunodiagnostic aspects of autoantibodies against myeloperoxidase. Kiln Nephrol (Submitted). 32. Van der Woude FJ, Rasmussen N, Lobatto S, et al.: Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet i:425-9, 1985. 33. Wiik A: Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 97(Suppl. 6):12-3, 1989. 34. Wiik A, Van der Woude FJ: The new ACPA/ ANCA nomenclature. Neth J Med 36:107-8, 1990.
croscopy. This renal lesion may be clinically manifest as a renal-limited process or as part of a systemic vasculitis such as polyarteritis nodosa or Wegener's granulomatosis. 6,2°,29 No serologic marker for this form of vascular injury was available until the recent discovery of antineutrophil cytoplasmic autoantibodies (ANCA).t8 In fact, ANCA-associated glomerulonephritis is the most common form of crescentic or rapidly progressive glomerulonephritis. 2°
Anca Nomenclature and ANCA Antigen Specificity The term anti-neutrophil cytoplasmic autoantibody was established as the official name for ANCA by the 2nd International ANCA Workshop held in Leiden, Netherlands in 1989. 33 This nomenclature includes two subgroups of ANCA recognized by indirect immunofluorescence microscopy: cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (PANCA). C-ANCA was first described by Davies, 7 and then by numerous investigators ]5,2t,3° to have a diffuse cytoplasmic staining pattern by indirect immunofluorescence microscopy. P-ANCA was first recognized by Falk and Jennette in 19888 and refers to a perinuclear/nuclear staining pattern produced when alcohol-fixed neutrophils are used as substrate. The perinuclear pattern is a consequence of permeabilization of the cell membranes, including granule membranes, allowing redistribution of soluble cationic molecules I
0197-1859/90/$0.00 + 2.20
© 1990 Elsevier Science Publishing Co., Inc.
ELISA using affinity-purifiedantigen. J Immtmol Methods 114:167-74, 1988. 16. LiidemannJ, Utecht B, Gross WL: Anticytoplasmic antibodies in Wegener's Granulomatosis are directed against proteinase 3. In: H6rl WH, Heidland A (eds): Proteasis III. New York: Plenum Press, 1990. 17. N~issbergerL, Jonsson H, Sj6hoim AG, et al.: Circulating anti-elastasein SystemicLupus Erythematosus. Lancet i:509, 1989. 18. N611eB, Specks U, LtidemarmJ, et al.: Anticytoplasmicautoantibodies:Their immunodiagnostic value in Wegener's granulomatosis. Ann Int Med 11:28-40, 1989.
Detection o f & n t l n e u t r o p h i l C ~ o p l m m &ntibodies
Continuedfrom page 159 antigens still remains a problem. The generally higher sensitivity of solid-phase assays compared to IIF is an advantage only if the tests are specific, and this requires the use of highly purified antigens. By design, all such assays measure autoantibodies by their interaction with the respective antigen, and the exact amount of antibody cannot be quantitated by precise physicochemical measurement.12 Because patients' sera generally contain mixtures of autoantibodies with different affinities, the autoantibody activity, rather than the concentration is measured with all the different assays. Despite this limitation, antigen-specific solid-phase assays offer the advantage of automation with more precise quantitation of autoantibody activity.
Indirect Immunofluorescence Autoantibodies directed against a cytoplasmic antigen of human neutrophils were detected by indirect immunofluorescence in patients with vasculitis and glomerulonephritis in the early 1980s. 5,jl These immunoglobulin G antibodies against neutrophils and monocytes, termed anti-cytoplasmic antibodies (ACPA) or anti-neutrophil cytoplasm antibodies (ANCA), were described to be a tool for diagnosis and a marker of disease activity in Wegener's granulomatosis (WG). 26,32 During the First International Workshop on ANCA in 1988, the presence of ACPA/ANCA was defined as the recognition of an uneven, granular staining of the neutrophil and monocyte cytoplasm iden-
C L I N I C A L I M M U N O L O G Y Newsletter 163
19. Pinching AJ, Rees AJ, Pussel BA, et at.: Relapses in Wegener'sgranulomatosis:The role of infection. Br Med J 281:836, 1980. 20. RosenboomS, RautmannA, Gross WL: Tumor necrosis factor in Wegener's granulomatosis. Abstract book of the 7th InternationalCongress of Immunology:Berlin, Stuttgart, New York, Gustav Fischer, 1989. 21. RosenboomS, RautmannA, Gross WL: AntizytoplasmatischeAntik6rper (ACPA) in der Immunfluoreszenz. Lab Med 12:339, 1988. 22. ThompsonRA, Lee SS: Antineutrophil Cytoplasmic Antibodies. Lancet i:Letter670-1, 1989.
23. UlmerM, RautmannA, LtidemannJ, Gross WL: ImmundiaguostischeRelevanzvon Antimyeloperoxidase-Antik6rpembei systemischen Vaskulitidien. 7th Int. Congressof Immunology, Berlin, July 30 to August 5, 1989. 24. van der Woude FJ, Daha MR, van Es LA: Review--The current status of neutrophil cytoplasmic antibodies. Clin Exp Immunol 78:143-8, 1989. 25. van der Woude FJ, RasmussenN, LobattoS, et ai.: Autoantibodiesagainst neutrophils and monocytes: tool for diagnosis and markerof disease activityin Wegener'sgranulomatosis. Lancet i:425-9, 1985.
tical to that of a standard reference serum, using a standardized technique with ethanol-fixed, smeared, or cytospun human leukocytes from normal donors, by indirect immunofluorescence.27 The ACPApositive standard reference serum is available from the Statens Seruminstitut, Copenhagen, Denmark, and the standard technique was published in the proceedings of the workshop. 33 However, the screening dilution that should be used is still under debate. Whereas we use undiluted serum without getting high background staining, others use threshold dilutions of 1:10, 1:16, or 1:20. t°,lT,23,2s Independent of these minor differences, it became clear, that some experience is required to obtain leukocyte preparations of high quality. This is important for differentiation between the various staining patterns. Therefore, it was recommended that the standard technique should be used and that it should not be applied for routine ACPA determination before at least 1000 sera have been examined. 27 Indeed, we obtained several sera from nonvasculitic patients, that were labeled as falsepositive by other laboratories, in which we could not detect ACPA either by IIF or by ELISA. Therefore, we suppose that at least some of the reports of ACPA in nonvasculitic patients might well be due to insufficient standardization of the immunofluorescence assay. Besides ACPA inducing the characteristic cytoplasmic staining, autoantibodies producing an artifactual perinuclear immunostaining of ethanol-fixed neutrophils were described. 7 This artifact is caused by the permeabilization of granule membranes while drying the cells onto slides.
Solubilized nucleophilic proteins diffuse and bind to nuclei when the dried cells are placed in an aqueous medium like ethanol. The redistribution can be prevented by fixing the cells with the cross-linking reagent formaldehyde. However, the use of formaldehyde fixation is not generally advisable, as several antigens lose their antigenicity if this fixative is used. 34 Moreover, just this artifact is desirable because it gives a hint to the autoantibody specificity. During the Second International Workshop on ANCA in 1989, it was recommended to adopt a terminology along the lines used for antinuclear antibody serology, where a descriptive term for the fluorescence pattern likewise gives a hint to the antibody specificity, for example, rim pattern for antibodies to DNA or speckled pattern for antibodies to extractable nuclear antigens. Clear cytoplasmic staining of ethanol-fixed neutrophils and monocytes should be termed C-ANCA and perinuclear staining P-ANCA. After IIF, immunochemical assays should be carried out to specify the autoantibody activity. 34 Although we could show that C-ANCA are directed in at least 95% of cases against proteinase 3 (PR-3), a neutral serine protease of human polymorphonuclear leukocytes and monocytes, ls,2°,2t P-ANCA are directed against a variety of different antigens. Originally, it was suspected that myeloperoxidase was the main target antigen of P-ANCA.7 In patients with pauci-immune necrotizing and crescentic glomerulonephritis, nearly 90% of P-ANCA sera were found to have antimyeloperoxidase reactivity. 6 In contrast, among patients with a wide variety of dif-
© 1990 Elsevier Science Publishing Co., Inc.
0197-1859/90/$0.00 + 2.20
164 C L I N I C A L I M M U N O L O G Y Newsletter
ferent diseases, anti-myeloperoxidase antibodies were only found in about 10% of sera inducing a perinuclear staining of ethanol-fixed neutrophils. 31 It has been shown that not only myeloperoxidase, but also elastase a,22 and lactoferrin3,3° are target antigens of autoantibodies that induce a perinuclear fluorescence pattern in neutrophils. Additionally, autoantibodies reacting with nuclear constituents can cause perinuclear staining. Thus, IIF with ethanol-fixed neutrophils is indeed an important screening procedure to detect antineutrophil antibodies, but the autoantibody specificity must be defined in antigen-specific assays. In order to optimize ANCA detection by IIF, several modifications of the standard technique have been investigated. To improve substrate standardization, and to avoid the use of human blood products, we evaluated ANCA reactivity with the promyelocytic cell line HL-60. Parallel IIF assays with sera from 72 patients demonstrated that the reactivity of HL-60 cells directly correlated with that of neutrophils in both their ability to detect ANCA-positive sera and to differentiate between C- and P-ANCA. 2 Antinuclear antibody binding that often mimics PANCA staining could selectively be impaired by using formalin-acetone-fixed chronic myelocytic leukemia cells for ANCA detection) Two staining patterns were identified using this substrate. Type I is characterized by diffuse granular staining preferentially of immature cells, whereas type II represents similar staining with additional granular staining of the basophils. Type I was suspected to be caused by anti-proteinase 3 antibodies and type II was mainly due to anti-myeloperoxidase antibodies. 3 Although these modifications offer promising perspectives, the standard IIF technique 27 is recommended.
Solid Phase Assays In the first attempts to develop solid-phase assays for detection of ANCA, crude extracts were used. The most crude preparations are an acid extract, ~6 or total nitrogen cavitated neutrophils. 7 These were used to develop a solid phase RIA or an ELISA, respectively. Both preparations not only contain proteinase 3, but the II 0197-1859/90/$0.00
+ 2.20
majority of soluble substances of neutrophils, probably also those that are known to be target antigens of autoantibodies, such as myeloperoxidase, elastase, and lactoferrin. Indeed, both assays lack specificity as sera of SLE patients also gave positive r e s u l t s . 7,29 Later, it became clear that at least the RIA with acid extract yields positive results with sera containing C-ANCA (e.g., anti-proteinase 3 antibodies), as well as with sera containing autoantibodies to myeloperoxidase. 24 Therefore, these extracts are not suitable for differentiation between the various autoantibodies. A more defined, but still crude, substrate that was used in an ELISA for detection of C-ANCA is a preparation of azurophil granules. 25 The azurophil granules were obtained by nitrogen bomb cavitation of neutrophils from healthy human donors and subsequent Percoll gradient centrifugation. After disruption of granules by detergent, the extract was coated to ELISA plates in a very alkaline buffer. This ELISA seems to be more specific but less sensitive for detecting CANCA than an ELISA based on the acid extract and several discrepancies between the results obtained by IIF and ELISA were observed.~4 Interestingly enough, although myeloperoxidase is a major component of the azurophil granules, antimyeloperoxidase antibodies were only detected with this ELISA if they were present in high concentrations. 24 It was not reported whether autoantibodies to other antigens of azurophil granules, like those directed against elastase, gave positive results with this ELISA. Another approach to solid-phase assays is the use of monoclonal antibodies with specificity for the respective antigen. Such antigen-catching ELISAs were developed with monoclonal antibodies against a 29 kDa protein of azurophil granules, against myeloperoxidase and against elastase. 8,9 The monoclonal antibodies were immobilized to the solid phase via an antiserum against mouse immunoglobulins and the respective antigens were caught by the monoclonal antibodies from a preparation of saline-extracted azurophil granules. The advantage of catching ELISAs is that the antigen is preI III © 1990 Elsevier Science Publishing Co., Inc.
Vol. 10, No. 1 1, 1990
sented in its native condition, but interactions between the anti-mouse antiserum or the monoclonal antibody and serum constituents, such as rheumatoid factors, might lead to false-positive results. However, using these catching ELISAs, reactions of ANCA-negative sera with high titers of rheumatoid factor of all three isotypes were not observed. 14 Comparison of titers of C-ANCA determined by IIF with (semi)-quantitative results from the 29 kDa antigen-catching ELISA revealed a reasonable, but not perfect, correlation between both assays. 14 Another limitation of catching ELISAs is due to the fact that binding of the antigen to the monoclonal antibody always occurs at the same epitope. If autoantibodies are directed against this epitope or adjacent regions, their binding will be prevented by steric hindrance. Almost all solid-phase assays for detection of a wide variety of different autoantibody specificities are based on purified antigens bound to the solid phase. Therefore, we used affinity chromatography with naturally occurring human autoantibodies to obtain a well-defined purified antigen as a substrate in an ELISA for detection and quantitation of C-ANCA. ~9 The target antigen of C-ANCA was released from human neutrophils during degranulation induced by phorbol myristate acetate and was further purified by affinity chromatography using a column with bound IgG from a C-ANCA-positive serum. The ELISA was sufficiently specific and sensitive. Autoantibodies directed against myeloperoxidase, elastase or lactoferrin did not lead to false-positive results and the arbitrarily defined units obtained with this ELISA correlated significantly with C-ANCA titers determined by IIF (Figure 1). 19 However, it has to be mentioned that some patients with Wegener's granulomatosis have high IIF titers and low ELISA values and vice versa. 23 In a recent study with a large population of clinically well-characterized patients, only three false-positive cases, all at the very low end of the positive range, were found when results from our ELISA test were evaluated against those of IIF. 23 As this ELISA seems to detect changes in C-ANCA levels in relation to changes in
C L I N I C A L I M M U N O L O G Y Newsletter 165
Vol. 10, No. 11, 1990
units/ml
~
•
SO00-
rs = 0.85 ¢ < 0.001
• •
WG(n:I05)
•
t I000.
•
5OO,
•
"
"
t
"
"
ie
•
•
: l 50
i •
•
: I
l
:•
"
• II
:!i 20J . . . . . . . . . . . . . . . . . . . . . . . . . . . I
IIF tttte (dilutions]
Figure I. Correlation between ACPA titre
determined by indirect immunofluorescence and ACPA concentration measured by ELISA. A highly significant positive correlation was observed between the values obtained by the two methods (Spearman's rank correlation coefficient = 0.85, p < 0.001). The limit for positive ELISA readings was set at 20 units/mL (from gel. 19).
disease activity more promptly than does IIF, 23 it represents a promising quantitative alternative to the semiquantitative IIF technique for serial C-ANCA determinations in patients with Wegener's granulomatosis. Recently, we were able to produce monoclonal antibodies against proteinase 3, 4 which may be of value in optimizing antigen purification. In addition, the chromatographic techniques originally employed for purification of proteinase 3 t5 can be used to establish a solid-phase assay, z° For the specific detection of autoantibodies against myeloperoxidase or elastase, basically two techniques have been used, either an ELISA with chromatographically purified enzymes or a sandwich ELISA using monoclonal antibodies to myeloperoxidase and elastase, s,9 The chromatographically purified enzymes were mainly purchased commercially, but myeloperoxidase was not 100% pure. 24 Impurities of antigen preparations may give rise to false-positive results, espeII
cially if contaminating proteins have a high affinity to the solid phase and are target antigens of different autoantibodies. Indeed, myeloperoxidase apparently binds poorly to the solid phase if present in a preparation of azurophil granules,25 which might be due to a rather low affinity to the polystyrene. In this context, it should be mentioned that factors determining the cut-off values of different solid-phase assays are not always clearly defined24 and confirmatory tests are carried out only occasionally. Immunoblotting, a method which is often used as a confirmatory test, is not very promising, because autoantigens like proteinase 3 and elastase are of very similar molecular weight and antigenic epitopes of myeloperoxidase and proteinase 3 are destroyed if electrophoresis is carried out under reducing conditions. 2° Future developments of laboratory methods for the detection of autoantibodies, in general, will involve tests based on synthetic antigens or peptides, including those produced by recombinant DNA technology.l Recently we have been successful in cloning proteinase 3, the main target antigen of C-ANCA t3 and, currently, we are developing a method to express the enzyme in its native form. This will enable us to generate large quantities of purified proteinase 3 that can be used to establish a sensitive and specific ELISA for detection and quantification of anti-proteinase 3 antibodies, c m
References 1. A Second IUIS/WHO Report: Laboratory investigations in clinical immunology: methods, pitfalls and clinical indications. Clin Exp lmmunol 74:494-503, 1988. 2. Charles LA, Falk RJ, Jennette JC: Reactivity of anti-neutrophil cytoplasmic autoantibodies with HL-60 cells. Ciin Immunol Immunopathol 53:243-53, 1989. 3. Chevailler A, Noel LH, Renier G, et al.: Unambiguous determination of antinentrophil cytoplasm antibodies (ANCA) specificity by immunofluorescence on chronic myelocytic leukemia ceils. J Immunol Methods (in press), 1990. 4. Csemok E, Ltidemarm J, Gross WL, Bainton DF: Ultra,structural localization of proteinase 3, the target antigen of anti-cytoplasmic antibodies circulating in Wegener's granulomatosis. Am J Pathol (in press), 1990.
5. Davies DJ, Moran JE, Niall JF, Ryan GB: Segmental necrotising glomemlonephritis with antineutrophil antibody: possible arbovirus aetiology? Br Med J 285:606, 1882. 6. Falk 1LI, Hogan SL, Wilkman AS, et al.: Myeloperoxidase specific anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA). Neth J Mud 36:121-5, 1990. 7. Falk PJ, Jennette JC; Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomemlonephritis. N Engl J Mud 318:1651-57, 1988. 8. Goldschmeding R, Cohen Tervaert JW, van der Schoot CE, et al.: ANCA, antimyeloperoxidase, and anti-elastase: three members of a novel class of autoantibodies against myeloid lysosomal enzymes. APMIS 97(Suppl. 6):48-9, 1989. 9. 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 84:1577-87, 1989. 10. Gross WL: Wegener's granulomatosis. New aspects of the disease course, immunodiagnostic procedures, and stage-adapted treatment. Sarcoidosis 6:15-29, 1989. 1I. Hall JB, Wadham BMcN, Wood CJ: Vasculitis and glomerulonepllfitis: a subgroup with antineutrophil cytoplasmic antibody. Aust NZ J Med 14:277-8, 1984. 12. Haynes DC, Homburger HA: International standards and reference reagents for measurement of serum proteins and autoantibodies. Clin Immunol Newsletter 10:39-41, 1990. 13. Jenne DE, Tschopp J, Liidemann J, et al.: Wegener's autoantigen decoded. Nature 346:520, 1990. 14. Kallenberg CGM, Rasmussen N: Solid phase assays for ANCA. Neth J Med 36:132-6, 1990. 15. Kao RC, Wehner NG, Skubitz KM, et al.: Proteinase 3: a distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters. J Clin Invest 82:1963-73, 1988. 16. Lockwood CM, Bakes D, Jones S, et al.: Association of alkaline phosphatase with an autoantigen recognized by circulating anti-neutrophil antibodies in systemic vasculitis. Lancet i:71620, 1987. 17. Liidemann G, N611e B, Rautmann A, et al.: Antizytoplasmatische Antik6rper als Seromarker und Aktivit~tsparameter der Wegener'schen Granulomatuse. Eine prospektive Studie. Dtsch med Wschr 113:413-7, 1988. 18. Liidemarm J, Csernok E, Ulmer M, et al.: Antineutrophil cytoplasm antibodies in Wegener's granulomatosis: immunodiagnostic value, monoclonal antibodies and characterization of the target antigen. Neth J Mud 36:157-62, 1990. 19. Liidemann J, Utecht B, Gross WL: Detection and quantification of anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis by
I
© 1990 Elsevier Science Publishing Co., Inc.
019%1859/90/$0.00 + 2.20
166 C L I N I C A L I M M U N O L O G Y Newsletter
ELISA using affinity-purified antigen. J lmmunol Methods 114:167-74, 1988. 20. Liidemann J, Utecht B, Gross WL: Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme. J Exp Med 171:357-62, 1990.
25.
21. Liidemann J, Utecht B, Gross WL: Anti-cytoplasmic antibodies in Wegener's granulomatosis are directed against proteinase 3. In: H6rl WH, Heidland A (eds): Proteases III. New York, Plenum Press (in press), 1990.
26.
22. Nhssberger L, Jonsson H, Sj6holm AG, et al.: Circulating anti-elastase in systemic lupus erythematosus. Lancet i:509, 1989. 23. N611e B, Specks U, Lt~demann J, et al.: Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 111:28-40, 1989. 24. Rasmussen N, Daha MR: Concluding remarks
27.
28.
on solid phase assays. Neth J Med 36:143-5, 1990. Rasmussen N, Sjrlin C, Isaksson B, et al.: An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA). J Immunol Methods 127:139-45, 1990. Rasmussen N, Wiik A: Autoimmunity in Wegener's granulomatosis. In: Veldman JE, McCabe BF, Huizing EH, Mygind N (eds): Immunobiology, Autoimmunity and Transplantation in Otorhinolaryngology. Amsterdam: Kugler Publications, pp. 231-6, 1985. Rasmussen N, Wiik A, H¢ier-Madsen M, et al.: Anti-neutrophil cytoplasm antibodies 1988. Lancet i:706-7, 1988. Rosenboom S, Rautmann A, Gross WL: Antizytoplasmatische Antikrrper (ACPA) in der indirekten lmmunfluoreszenz: Probleme beim Testaufbau und bei der Testbeurteilung. Lab Med 12:339-45, 1988.
Antlneutrolphil Cytoplasmic Autoantibody in Renal Disease Ronald J. Falk and J. Charles Jennette Departments of Medicine and Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
apidly progressive glomerulonephritis (RPGN) is a clinical syn,drome with aggressive glomerular injury that causes renal failure, which may be irreversible if not treated appropriately. 5 The pathologic lesion most frequently associated with RPGN is crescentic glomerulonephritis. RPGN may be observed in patients with disease limited to the kidney or as part of a systemic illness. To optimize patient management, practicing nephrologists must classify RPGN into one of several clinicopathologic syndromes. Some indication as to the classification of RPGN may be derived from three sources: (1) the clinical assessment as to which organ systems are involved in the disease process; (2) serologic studies; and (3) examination of the renal biopsy specimen by indirect immunofluorescence microscopy and electron microscopy for the presence and localization of immunoglobulin deposits. 21 Based on immunohistologic studies, three general categories of RPGN may be discerned. Immune-complex-mediated diseases are considered when granular de-
R
posits of immunoglobulin are observed in glomeruli, for example, in diseases such as Henoch-Schonlein purpura, systemic lupus erythematosus, postinfectious glomerulonephritis, and cryoglobulinemic glomerulonephritis. The diagnosis of these conditions is supported by the presence in the circulation of autoantibodies such as anti-DNA or antistreptococcal antibodies. The overall clinical picture aids in determining if the disease process is renal-limited or is a systemic process (e.g., IgA nephropathy versus HenochSchonlein purpura). Another category of disease to be considered in the diagnosis of RPGN is antiglomerular basement membrane antibody-mediated glomerulonephritis and Goodpasture's syndrome, in which aggressive glomerulonephritis is associated with linear staining for immunoglobulins along the glomerular basement membrane, and antiglomerular basement membrane autoantibodies in the circulation. A third category of disease is the pauci-immune form of glomerulonephritis; so called because of the few or no immune deposits seen by immunofluorescence mi-
Vol. 10, No. 1 1, 1990
29. Savage COS, Winearls CG, Jones S, et al.: Prospective study of radioimmunoassay for antibodies against neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet i:1389-93, 1987. 30. Thompson RA, Lee SS: Antineutrophil cytoplasmic antibodies. Lancet i:670-1, 1989. 31. Ulmer M, Liidemann J, Gross WL: Immunodiagnostic aspects of autoantibodies against myeloperoxidase. Kiln Nephrol (Submitted). 32. Van der Woude FJ, Rasmussen N, Lobatto S, et al.: Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet i:425-9, 1985. 33. Wiik A: Delineation of a standard procedure for indirect immunofluorescence detection of ANCA. APMIS 97(Suppl. 6):12-3, 1989. 34. Wiik A, Van der Woude FJ: The new ACPA/ ANCA nomenclature. Neth J Med 36:107-8, 1990.
croscopy. This renal lesion may be clinically manifest as a renal-limited process or as part of a systemic vasculitis such as polyarteritis nodosa or Wegener's granulomatosis. 6,2°,29 No serologic marker for this form of vascular injury was available until the recent discovery of antineutrophil cytoplasmic autoantibodies (ANCA).t8 In fact, ANCA-associated glomerulonephritis is the most common form of crescentic or rapidly progressive glomerulonephritis. 2°
Anca Nomenclature and ANCA Antigen Specificity The term anti-neutrophil cytoplasmic autoantibody was established as the official name for ANCA by the 2nd International ANCA Workshop held in Leiden, Netherlands in 1989. 33 This nomenclature includes two subgroups of ANCA recognized by indirect immunofluorescence microscopy: cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (PANCA). C-ANCA was first described by Davies, 7 and then by numerous investigators ]5,2t,3° to have a diffuse cytoplasmic staining pattern by indirect immunofluorescence microscopy. P-ANCA was first recognized by Falk and Jennette in 19888 and refers to a perinuclear/nuclear staining pattern produced when alcohol-fixed neutrophils are used as substrate. The perinuclear pattern is a consequence of permeabilization of the cell membranes, including granule membranes, allowing redistribution of soluble cationic molecules I
0197-1859/90/$0.00 + 2.20
© 1990 Elsevier Science Publishing Co., Inc.