anti - neutrophil p - anca antibodies are directed against antigens in the peripheral cytoplasm of both neutrophilic granulocytes and monocytes . we report a case of a 54-year - old female presenting with a pruritic rash , including purpura and diffuse erythema . a biopsy with hematoxylin and eosin ( h & e ) analysis , direct immunofluorescence ( dif ) , immunohistochemistry ( ihc ) and enzyme - linked immunosorbent assays for ancas the dif revealed evidence of vasculitis , the presence of p - ancas and neutrophil extracellular traps ( nets ) . by observing the deposition of neutrophil extracellular traps and myeloperoxidase in inflamed skin vessels , biopsy analysis may alert physicians for rapid therapeutic intervention in patients presenting with possible vasculitides . anti - neutrophil cytoplasmic antibodies ( ancas ) are a group of predominately igg antibodies , directed against antigens in the cytoplasm of both neutrophilic granulocytes and monocytes . they are detected in a number of autoimmune disorders , but are most often associated with forms of systemic vasculitis , the anca - associated vasculitides . ancas were first divided into two major classes , c - ancas ( cytoplasmic ) and p - ancas ( or perinuclear ) , based on their pattern of staining inside the affected cells . anca titers are classically measured using enzyme - linked immunosorbent assays ( elisas ) , and by indirect immunofluorescence ( iif ) . the perinuclear pattern occurs because the vast majority of the antigens targeted by p - ancas are strongly cationic ( positively charged ) at ph 7.00[13 ] . during fixation , the more cationic antigens migrate within the cell cytoplasm and congregate around the nucleus , attracted by its negatively charged deoxyribonucleic acid ( dna ) content[13 ] . immunofluorescence antibody staining then results in fluorescence of the region around the nucleus that reveals the classic p - anca perinuclear pattern . the most frequent p - anca target is myeloperoxidase ( mpo ) , a neutrophil granule protein whose primary role in classic metabolic processes is generation of oxygen radicals . ancas will , less commonly , form against alternative antigens , that may also result in the p - anca pattern . these antigens include lactoferrin , elastase , and cathepsin g. the presence of p - ancas is a sensitive , but not specific test for ulcerative colitis[13 ] . in contradistinction , c - ancas show a diffusely granular , cytoplasmic staining pattern . the c - anca pattern results from the binding of ancas to antigen targets throughout the neutrophil cytoplasm , and the most common protein targets at proteinase 3 ( pr3 ) . the pr3 c - anca pattern is often encountered in patients with wegener 's granulomatosis . other types of ancas that develop against antigens other than myeloperoxidase or proteinase 3 will sporadically result in patchy staining when visualized by immunofluorescence . the resultant pattern is commonly called the  snowdrift  prototype , and classically occurs in patients with non - vasculitic diseases that result in anca formation . it is not clear how these ancas are developed , although several hypotheses have been suggested . there is probably a genetic contribution ( predominantly in genes controlling the level of the immune response ) ; however , genetic susceptibility is likely to be linked to an environmental factor , as well as probable factors including vaccination or exposure to silicates . a 54-year - old female presented with a clinical history of a rapidly appearing , prutitic rash on her scalp , neck , hands , chest , abdomen , buttocks and back . a physical exam of the patient revealed an erythematous , macular rash in these areas , with lesions concentrated on the legs . no associated systemic symptoms were noted ; lung and kidney function were within normal limits . no known concurrent medical conditions were present , and the patient was also not under treatment with any medications or herbal supplements . a punch biopsy of the skin was performed for hematoxylin and eosin ( h & e ) analysis , as well as for multicolor direct immunofluorescence ( dif ) and for immunohistochemistry ( ihc ) . direct immunofluorescence ( dif ) : in brief , four um thickness skin cryosections were partially fixed in 3% paraformaldehyde , and then incubated with secondary antibodies , all conjugated with fluorescein isothiocyanate ( fitc ) . the following anti - human rabbit antibodies were utilized : anti - human igg (  chain ) , anti - human iga (  chain ) , anti - human igm ( -chain ) , anti - complement 3c , anti - fibrinogen , anti - albumin from ( all at either 1:20 to 1:40 dilutions ; all from dako , carpinteria , california , usa ) . in addition , we utilized goat anti - human ige - antiserum , conjugated with fitc from vector laboratories , bridgeport new jersey , usa , as well as goat - anti - human complement c1q antiserum from southern biotech , birmingham , alabama , usa . the slides were then counterstained with either 4 , 6-diamidino-2-phenylindole ( dapi ) ( pierce , rockford , illinois , usa ) , or with the carbocyanine monomer to - pro-3 dye ( molecular probes , invitrogen , carlsbad , california , usa ) , washed , and coverslipped . 1 . presence of neutrophil extracellular traps and antineutrophil cytoplasmic antibodies associated with vasculitides using dif and ihc . a through f , and i , dif . a. area of vasculitis , showing deposits of fitc - conjugated anti - human fibrinogen within dermal vessels ( red arrows ) . b. intra - cytoplasmic deposits of fitc - conjugated antihuman c1q within epidermal cells ( red arrow ) . c. positive p - ancas ( yellow arrow ) using fitc - conjugated anti - human igg . the nuclei are counterstained in red with to - pro-3 dye ( blue arrow ) ; vessel autoreactivity is displayed in green utilizing the fitc - conjugated anti - human igg ( white arrow ) . d. positive p - ancas ( yellow arrows ) in the dermis utilizing fitc - conjugated anti - human igg antibodies . e , same as d , but at higher magnification , and without nuclear counterstaining . f. shows a detail of the p - anca antigen(s ) in the epidermis and the nets ( yellow arrows ) . g ihc staining with collagen iv shows strong reactivity to the bmz , as well as around the superficial dermal vessels ( brown stain ) ( aqua arrows ) . h. h & e staining demonstrating leukocytoclastic vasculitis with fibrinoid necrosis ( blue arrow ) . .i , dif , and g , deposits of nuclear dust ( yellow ) on some superficial vessels utilizing fitc - conjugated anti - human iga antibodies ( yellow arrows ) . j an ihc stain using anti - cd34 on the vascular area in i , and showing strong , fragmented endothelial expression of this molecule ( aqua arrows ) . hematoxylin and eosin ( h&e ) stain : the h&e staining displayed histologic features of mild , diffuse epidermal spongiosis , and a mild , superficial dermal inflammatory process involving capillaries and small venules . a mild , superficial , perivascular dermal infiltrate of lymphocytes , histiocytes and neutrophils was present , with perivascular leukocytoclastic debris and focal vascular fibrinoid necrosis also appreciated ( fig . 1 ) . the h & e displayed other findings , including focal , perivascular extravasation of erythrocytes into the dermal interstitial tissue ; occasional neutrophils were also noted to be present within the epidermal stratum spinosum ( fig . 1 ) . direct immunofluorescence ( dif ) displayed the following results : anti - human igg , iga , and albumin ( all++ ) , and anti - human fibrinogen ( + + + ) deposits were noted , all in a linear pattern along the bmz , and surrounding the superficial dermal blood vessels . significant p - ancas were positive within selected neutrophils when utilizing anti - human igg , iga and igm . in addition , by dif we detected the presence of autoreactivity to intracytoplasmic keratinocytes with fitc - conjugated anti human c1q ( fig . 1 ) . immunohistochemistry ( ihc ) : we performed ihc to confirm the dif data , and to be able to distinguish between real , pathologic autoreactivity detected by direct immunofluorescence , versus non - pathologic , incidental autofluorescence of the dermal blood vessels . we performed ihc by using a dual endogenous peroxidase blockage , according to the dako instructions , with the addition of an envision dual link . we applied 3 , 3 diaminobenzidine ( dab ) as a chromogen marker , and then counterstained with hematoxylin . we stained for rabbit anti - human iga , igm , igd , ige , c1q , fibrinogen , albumin , and the blood vessel vascular endothelial antigens cd31 ( pecam ) and cd 34 ; in addition , we utilized d2 - 40 to highlight dermal lymphatic vessel endothelia . anti - myeloperoxidase ( mpo ) antibody from dako was also utilized in the ihc studies . our tests confirmed the autoreactivity detected by dif to the basement membrane zone ( bmz ) area of the skin , as well as to the superficial and intermediate dermal vessels ( fig . 2 ) . in addition , the ihc analysis revealed positive staining with myeloperoxidase ( mpo ) antibodies in the same areas where the p - ancas were observed in the epidermis and dermis by dif . the patient was treated immediately with topical corticosteroids , as well as with one injection of intramuscular steroids ( 60 mg i m ) . one and a half weeks later , the patient was tested for 1 ) blood complement c3 , and 2 ) an anca panel for vasculitis including myeloperoxidase ( mpo ) , proteinase 3 antibodies and immunoglobulin e antibodies . each of these test parameters were reported within normal limits , and the clinical lesions exhibited clearance at this time . real immunoreactivity versus intrinsic , non - pathologic background immunoreactivity in the dermal blood vessels using ihc . our results confirmed our previous dif findings . in panel a , note fragmented positivity for cd 34 ( red arrow ) . in b , autoreactivity to intermediate vessels using c1q ( yellow arrows ) . c. again using c1q , 1 ) an intracytoplasmic epidermal stratum spinosum reactivity is noted [ ( also shown with the dif ] , as well as 2 ) a reactivity to the superficial plexus dermal vessels , and 3 ) to the intermediate dermal vessels ( red arrows ) , ( also ihc colocalized with both anti - cd31/pecam and cd34 antibodies ) . d. interestingly , some of these vessels also demonstrated positive findings utilizing anti - human igd ( yellow arrows ) e , ( red arrows ) . in f , dermal perivascular reactivity was also noted utilizing anti - ige ( yellow arrows ) and utilizing iga ( data not shown ) . g. autoreactivity to the vessels utilizing anti - igm ( yellow arrows ) , as well as in h , to igg ( red arrows ) . in ( i ) note positive autoreactivity to the basal bmz area , and the superficial vascular plexus ( yellow arrows ) , and , in j , to intermediate dermal vessels ( blue arrows ) . finally , in k and l , we note compartmentalization or  tight cuffing  of the observed in the inflammatory areas utilizing anti - human fibrinogen ( red arrows ) , and anti - human albumin ( blue arrows ) , respectively . small - vessel vasculitides are autoimmune disorders , often associated with antineutrophil cytoplasmic autoantibodies ( ancas ) . the c - ancas are detected serum testing , as well as by direct and indirect immunofluorescence in a number of autoimmune disorders . ancas were originally divided into two primary classes , c - ancas and p - ancas , based on their pattern of staining in neutrophils . anca associated vasculitides classically affect the small vessels , and can present diverse clinical manifestations[13 ] . the primary clinical treatment of these disorders is systemic corticosteroid therapy , with or without other immunosuppressants . attempts to explain and model the presence of ancas have included the theory of molecular mimicry . for instance , microbial superantigens are molecules expressed by bacteria and other microrganisms that have the power to stimulate a strong immune response by activation of t lymphocytes . these molecules generally have regions that resemble self - antigens ; thus , the origin of the theory of molecular mimicry . the classic example of such superantigen pathophysiology is found in group a streptococcal rheumatic heart disease , where there is similarity between the m proteins of streptococcus pyogenes and those of cardiac myosin and laminin . it has also been shown that up to 70% of patients with wegener 's granulomatosis are chronic nasal carriers of staphylococcus aureus , with carriers having an eight times increased risk of disease presentation . in addition , an alternative theory of defective apoptosis has been suggested as a possible explanation of anca pathophysiology . neutrophil apoptosis , or programmed cell death , is critical in controlling the duration of the early inflammatory response , thus reducing injury to normal tissues by the neutrophils . ancas may be developed secondary to either 1 ) ineffective apoptosis or 2 ) ineffective removal of apoptotic cell fragments , leading to the exposure of the immune system to molecules normally sequestered inside the cells . the apoptosis theory solves the seeming pathophysiologic contradiction of how it could be possible for ancas to be raised against intracellular antigenic targets . the role of ancas in disease has been linked to three primary diseases that are consistently associated with ancas . the most commonly associated disorders are wegener 's granulomatosis , microscopic polyangiitis and glomerulonephritis[48 ] . the antibodies are assumed to be involved in the generation and/or progression of the clinical lesions . traditionally , c - ancas are associated with wegener 's granulomatosis ; p - ancas are associated with both microscopic polyangiitis and focal necrotizing and crescentic glomerulonephritis[48 ] . however , in recent years , ancas targeted against other autoantigens have been identified , including those involved in churg - strauss syndrome . patients with a number of other diseases , such as ulcerative colitis and ankylosing spondylitis , will frequently display ancas as well[48 ] . however , in these cases , there is classically no associated vasculitis , and the ancas are thought to be incidental or epiphenomena rather than part of the primary disease itself . it is unclear what the role of ancas may be wegener 's granulomatosis , glomerulonephritis , and churg strauss syndrome ; they may be markers of the diseases , or , alternatively , may play some part in the pathogenic process . in wegener 's granulomatosis , there is positive correlation between anca titer and disease activity ; further , in vitro studies have shown that ancas cause activation of primed neutrophils and react with endothelial cells expressing pr3[48 ] . ancas may thus act by causing release of lytic enzymes from the white blood cells , causing vasculitis in the nearby blood vessel walls . in our case , , microscopic findings of acute vasculitis such as fibrinoid necrosis , chronic signs such as endarteritis obliterans , and distant chronic signs such as acellular scarring were identified . our findings did not favor the diagnosis of microscopic polyangiitis due to the following : 1 ) no additional indicators of granulomatous inflammation were present ( e.g. , evidence of lung cavities or infiltrates present for over one month duration ) ; and 2 ) no biopsy findings of either a ) glomerulonephritis , nor b ) involvement of more than one organ system existed ( as documented by a biopsy or surrogate marker ) . the patient was not taking any medications at the time of biopsy . by definition , anca related vasculitides are usually  pauci - immune  , that is , they present few and weak vascular immunoreactants by dif .however , for more than fifty years , dermatopathologists and immunodermatologists were utilizing only monochromatic fitc dif , and not multicolor immunofluorescence as we did in our case . thus , the sensitivity and specificity of our analysis is increased by utilizing these techniques . we also utilized ihc ; this technique was employed to rule out nonpathologic , intense autofluorescence of the vessels ( i.e. , to be able to distinguish between real pathologic reactivity versus intrinsic self fluorescence of the vessels ) . our case also displays acute findings , and highlights the difficulty of making a specific diagnosis of vasculitis ( especially due to the presence of iga , igd , vascular deposits and p - ancas ) . specifically , we believe our case may represent a brief , largely subclinical presentation of henoch schonlein purpura , or , alternatively , an anca - positive small vessel vasculitis , i.e. , polyarteritis nodosa[1012 ] . other diseases where the combined , simultaneous findings of vasculitis and ancas can be found include goodpasture 's syndrome / anti - gbm - positive renal disease , and eleven per cent of cases of giant - cell arteritis . other rare , less likely diseases that should be considered include felty 's syndrome , atypical pneumonia , post - streptococcal glomerulonephritis , systemic lupus erythematosus , mixed connective tissue disease , and wegener 's granulomatosis . finally , it should be noted that many non - mpo p - ancas exist , including 1 ) those directed at other polycytoplasmic enzymes ( elastase , lactoferrin , lactoperoxidase , lysozyme , azurocidin , or cathepsin g ) , as well as 2 ) anas , and 3 ) granulocyte - specific anas ( gs - anas)[1012 ] . based on the clinical , histologic and dif findings in our case , a complete anca panel was ordered for the patient . one of the primary clinical problems encountered when evaluating patients with vasculitis is the fact that patients often present during an acute flare of the disease process . it is thus difficult for us to compare our case to other studies regarding treatment of early small vessel vasculitis . specifically , we found reports where early treatment of mpo - p - anca - associated small vessel vasculitis was felt to be crucial for the patient 's positive prognosis ( patients were treated with oral corticosteroid and cyclophosphamide therapy , in conjunction with plasmapheresis)[1012 ] . notably , one of the major problems physicians have to face when treating patients with early or chronic phase vasculitis is that patients may not respond to standard therapy , or may require unacceptably high doses of steroids or immunosuppressants[1013 ] . in our case , prompt medical intervention resulted in the clearance of the clinical as well as normalization of the laboratory findings in our patient . finally , specific predictors of treatment resistance and relapse of anca - associated small - vessel vasculitis have been recently described by utilizing clinical , pathologic and serologic predictors of treatment resistance and relapse in a community - based cohort of patients with anca - associated vasculitides[1013 ] . finally , we were also were able to demonstrate evidence of the p - anca antigen(s ) in the epidermis . these likely represent chromatin fibers , or so - called neutrophil extracellular traps ( nets ) , released by anca - stimulated neutrophils ; these antigens may contain the targeted autoantigens pr3 and mpo . we conclude that in these cases , early h&e and multicolor dif diagnosis and rapid treatment ( with systemic corticosteroid therapy , with or without other immunosuppressants ) may contribute to both a favorable prognosis , and to a lack of lesional clinical recurrence .
context : anti - neutrophil p - anca antibodies are directed against antigens in the peripheral cytoplasm of both neutrophilic granulocytes and monocytes . they are detected in several autoimmune disorders and are particularly associated with systemic vasculitis.case report : we report a case of a 54-year - old female presenting with a pruritic rash , including purpura and diffuse erythema . 
 a biopsy with hematoxylin and eosin ( h & e ) analysis , direct immunofluorescence ( dif ) , immunohistochemistry ( ihc ) and enzyme - linked immunosorbent assays for ancas were performed . 
 the h & e staining demonstrated leukocytoclastic vasculitis , with focal vascular fibrinoid necrosis . 
 the dif revealed evidence of vasculitis , the presence of p - ancas and neutrophil extracellular traps ( nets ) . 
 the ihc displayed autoreactivity to myeloperoxidase within the vessels . 
 the ihc aided in ruling out any intrinsic autofluorescence of the vessels.conclusions:by observing the deposition of neutrophil extracellular traps and myeloperoxidase in inflamed skin vessels , biopsy analysis may alert physicians for rapid therapeutic intervention in patients presenting with possible vasculitides .