Intern Emerg Med DOI 10.1007/s11739-014-1141-0
IM - ORIGINAL
Emergency testing for antineutrophil cytoplasmic antibodies combined with a dialog-based policy between clinician and biologist: effectiveness for the diagnosis of ANCA-associated vasculitis Johnny Sayegh • Caroline Poli • Alain Chevailler • Jean-Franc¸ois Subra Franc¸ois Beloncle • Pierre Antoine Deguigne • Ce´line Beauvillain • Jean-Franc¸ois Augusto
•
Received: 9 May 2014 / Accepted: 15 October 2014 Ó SIMI 2014
Abstract A prompt immunosuppressive treatment initiation is crucial in ANCA-associated vasculitis (AAV) to minimize organ injury. The aim of the present work was to analyze the accuracy of emergency ANCA screening to identify rapidly patients with AAV. In our Institution, emergency ANCA screening is based on a telephone call between a Clinician and a Biologist. Indirect immunofluorescence (IIF) for ANCA detection was performed using a commercial kit (EuroimmunÒ Granulocyte Mosaic 12). Positive serums for c- or p-ANCA at IIF are subsequently screened for antigenic specificity (MPO or PR3) by an immunodot technique (immunodot, D-TekÒ.) Positive samples with atypical c- or p-ANCA pattern at IIF are subsequently screened for antigenic specificity by ELISA. Data were retrieved from patients’ medical records and confronted to emergency ANCA screening results. Between 2005 and 2012, 114 patients were screened. IIF was positive in 27.2 % of patients, but c-/p-ANCA antiMPO/-PR3 was detected in 13.2 % of patients. The sensibility and specificity of IIF combined with immunodot for newly diagnosed AAV were 83.3 and 100 %, respectively. Ten patients were newly diagnosed with AAV. In these patients, a specific AAV treatment was initiated less than 24 h following ANCA screening. Emergency ANCA
J. Sayegh C. Poli A. Chevailler J.-F. Subra F. Beloncle P. A. Deguigne C. Beauvillain J.-F. Augusto LUNAM Universite´, Angers, France J. Sayegh J.-F. Subra F. Beloncle J.-F. Augusto (&) Service de Ne´phrologie-Dialyse-Transplantation, CHU Angers, 4 rue Larrey, 49933 Angers Cedex 9, France e-mail: [email protected] C. Poli A. Chevailler P. A. Deguigne C. Beauvillain Laboratoire d’Immunologie, CHU Angers, Angers, France
screening based on a clinical gating policy was relevant to identify patients with AAV diagnosis, and was associated with a rapid treatment initiation. Keywords vasculitis
ANCA Emergency ANCA-associated
Introduction Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against proteins of neutrophil cytoplasmic granules and monocytes lysosomes that are strongly associated with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) [1, 2]. The two main targets of ANCA are proteinase 3 (PR3), and myeloperoxidase (MPO). Anti-PR3 ANCA are usually associated with a cytoplasmic neutrophil fluorescence pattern (cANCA) using indirect immunofluorescence (IIF) on fixed neutrophils, and are mainly detected in GPA. On the other hand, anti-MPO ANCAs reproduce a perinuclear fluorescence pattern (p-ANCA), and are associated with MPA and CSS. When analyzed in ANCA-associated vasculitis (AAV) patients, the sensitivity and specificity of ANCAs have been reported to be as high as 95 % [3]. However, ANCAs have also been detected in numerous other conditions or diseases (i.e., rheumatoid arthritis and endocarditis) [4, 5]. Thus, in routine clinical practice, and especially when the clinician is looking for a diagnosis, the accuracy of ANCA testing to identify patients with AAV might be lower. In acute conditions, emergency ANCA testing may be of valuable information to help clinicians identify AAV. Given its non-specific clinical presentation, a fast
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identification of AAV is crucial to start treatment promptly. However, given the lower sensitivity/specificity of ANCA in unselected populations, such strategy may also be deleterious due to possible false positives. In the University Hospital of Angers, the emergency screening of ANCA has been available since January 2005. To the best of our knowledge, strategies evaluating the usefulness of ANCA in emergency conditions have been poorly reported to date [6]. We describe here the conditions that led clinicians to request emergency ANCA testing, and its usefulness for diagnosis and patient management.
Methods Study design and patients Emergency ANCA testing has been available since January 2005, 6 days a week (Mondays to Saturdays.) The request procedure is based on a telephone call between a senior clinician and a senior biologist. Acceptance of the request is left to the discretion of the senior biologist. Results are usually available within 4 to 8 h following the request, depending on the results of the IIF. When an emergency request is declined, ANCA screening is done without emergency, and results are given with a mean delay of 1 week. Even if no strict gating policy is applied, both clinical presentation and its confrontation to the international consensus statement outlining the clinical criteria for ANCA testing are determinant in accepting or declining the request [7, 8]. Only in-hospital patient requests were analyzed in the present study. A clinician (JFA) and a biologist (PAD) reviewed patients’ medical records, and were blinded to ANCA testing result. The final diagnosis was reviewed by two clinicians (JFA and JS) based on individual patient case notes, laboratory and biopsy results. The study protocol was approved by the Ethic Committee of the Angers University Hospital (CE 2013-65). Emergency ANCA detection Emergency ANCA detection was done using indirect immunofluorescence (IIF) with a commercial kit (EuroimmunÒ Granulocyte Mosaic 12.) Briefly, serum samples were assayed by IIF on slides with human ethanol, methanol or formaldehyde-fixed neutrophils and classified as c-, p or atypical-ANCA (a-ANCA). Positive serums for c- or p- or a-ANCA at IIF were subsequently screened for antigenic specificity (MPO or PR3) by an immunodot technique (immunodot, D-TekÒ). Sera with atypical c-, p- or a-ANCA were screened for antigenic specificity (BPI, azurocidin, elastase, cathepsin
123
Table 1 Clinical and biological characteristics of the study population All patients (n = 114)
Non-AAV patients (n = 96)
AAV patients (n = 18)
Age (years)
56.9 ± 25
55.6 ± 26
63.9 ± 14
Sex (men/women)
72/42
58/38
14/4
Clinical signs, n (%) Fever
39 (34.2)
33 (34.4)
6 (33.4)
Renal involvement
84 (73.7)
69 (71.9)
15 (83.3)
Hypertension
39 (34.2)
32 (33.3)
7 (38.9)
Acute renal failure
61 (53.5)
53 (55.2)
8 (44.4)
Proteinuria
75 (65.8)
62 (64.6)
13 (72.2)
Haematuria
57 (50)
47 (49)
10 (55.6)
Respiratory involvement
49 (43)
38 (39.6)
11 (61.1)
Dyspnoea
41 (36)
33 (34.4)
8 (44.4)
Hemoptysis
14 (12.3)
11 (11.5)
3 (16.7)
Arthritis
15 (13.2)
10 (10.4)
5 (27.8)
Purpura
20 (17.5)
17 (17.7)
3 (16.7)
Sinusitis
6 (5.3)
2 (2.1)
4 (22.2)**
Pericarditis/ myocarditis
5 (4.4)
4 (4.2)
1 (5.6)
Biological abnormalities, n (%) Inflammatory syndrome
93 (81.6)
75 (78.1)
18 (100)
Anemia
88 (77.2)
73 (76)
15 (83.3)
Hypereosinophilia
9 (7.9)
7 (7.3)
2 (11.1)
Antinuclear antibodies*
28 (24.6)
22 (22.9)
6 (33.3)
* Titre [1/200, **p \ 0.005
G, and lactoferrin) EuroimmunÒ).
by
ELISA
(ANCA
profil,
Statistical analysis Values were expressed as mean ± SD or percentage. Qualitative variables were compared using Chi squared test or Fisher exact test and quantitative variables using Mann– Whitney test. For all statistical analysis, p \ 0.05 was considered significant. Analysis was performed with the SPSS 15.0 software (SPSS, Inc., Chicago, IL).
Results A total of 114 emergency ANCA testings were performed in the study period. Patients had a mean age of 56.9 ± 25 [1.1–89.8], and were men in 63 % of cases. Routine ANCA
Intern Emerg Med Fig. 1 Flow chart of emergency ANCA screening and associated clinical diagnosis
Emergency ANCA screening (n=114)
Negave ANCA screening 83/114 (72.8%)
Posive ANCA screening 31/114 (27.2%)
Atypical ANCA 16/114 (14%)
Typical ANCA 15/114 (13.2%)
Vasculis: EGPA, n=2; IgA vasculis, n=1; GPA, n=1 Auto-immune diseases (2 Goodpasture, 1 myosis, 1 Crohn disease, 1 adult Sll disease, 1 thrombopenic pupura, 1 systemic sclerosis, 1 TINU syndrme, 1 Guillain Barre syndrome), n=9 Kidney diseases: acute renal faillure, n=7; glomerulonephris, n=12; renal sarcoidosis, n=1; undetermined nephropathies, n=6; others, n=4 Lymphoma, n=3 Infecous diseases: bacterial pneumonia, n=9; viral pneumonia, n=1; mycosis, n=1 Thromboc microangiopathy, n=5 Undetermined hypoxemic pneumonia, n=8 Others causes*, n=7 Undetermined, n=6
Atypical c-or p-ANCA 12/114 (10.5%) c-ANCA 6/114 (5.3%) Newly diagnosed GPA, n=4 Previously diagnosed GPA, n=2
p-ANCA 9/114 (7.9%) Newly diagnosed MPA, n=5 Newly diagnosed GPA, n=1 Previously diagnosed MPA, n=2 Previously diagnosed GPA, n=1
Undertermined GN, n=2 Acute renal failure, n=1 Bacterial pneumonia, n=2 IgA vasculis, n=1 Cholesterol embolizaon syndrome, n=1 Leukocytoclasc vasculis, n=1 Lymphoma, n=1 undetermined, n=3
a-ANCA 4/114 (3.5%) Vascular nephropathy, n=1 IgA vasculis, n=1 Brain sepc stroke embolism, n=1 Undetermined, n=1
*Stroke, n=1, cardiogenic shock, n=1; pancreas, n=1; lung cancer, n=1; status epilepcus, n=1; pulmonary hemosiderosis, n=1
testing was done in 11,147 patients during the analysis period, thus emergency ANCA testing represented a minor percentage of overall routine ANCA testing (around 1 %) There was no significant variation in the distribution of emergency ANCA requests between days in the week. Nephrology and Critical Care Medicine Departments represented 57.4 % of the emergency requests. The screening was performed at a median of 1.5 days from admission, and within the first 2 days from admission in 64 % of patients. The population was highly selected based on the clinical presentation neighboring criteria suggested by the 1999 International Consensus on ANCA testing [7]. As detailed in Table 1, most patients had fever, inflammatory syndrome, and renal or respiratory involvement. Renal involvement was present in 73.7 % of patients, and was mainly acute kidney injury associated with proteinuria or haematuria. Half of the patients had respiratory involvement with dyspnea, cough or hemoptysis. Digestive, rheumatologic, skin and respiratory tract involvement were less frequently observed. Among the 114 patients, 31 (27.2 %) had positive c- or p-ANCA (n = 6 and n = 9, respectively,) atypical positive c- or p-ANCA (n = 12), or positive a-ANCA (n = 4) at IIF. Figure 1 reports the results of emergency ANCA screening, and the patient’s final diagnosis. After reviewing the medical records, 12 patients (10.5 %) were newly diagnosed with AAV (5 MPA, 5 GPA and 2 EGPA.) The five MPA patients had positive p-ANCA anti-MPO,
whereas the patients with GPA had positive c-ANCA antiPR3 (n = 4) and positive p-ANCA anti-MPO (n = 1.) The two patients with EGPA were ANCA negative. The sensitivity and specificity of emergency IIF-ANCA testing for the diagnosis of newly identified AAV, after exclusion of the screened patients with previously known AAV, were 83.3 and 83.5 %, respectively. The positive predictive value of emergency ANCA testing for newly diagnosed AAV was 38.5 %, and the negative predictive value was 97.6 %. When the analysis was restricted to patients with typical c-ANCA anti-PR3 or p-ANCA anti-MPO, the sensitivity and specificity for the diagnosis of AAV were 82.3 and 100 %. The positive and negative predictive values were 100 and 98 %. Among clinical signs, only sinusitis was significantly more frequent in AAV patients (Table 1) as compared to other patients. We next analyzed the relationship between emergency ANCA testing results, histological results and treatment management. Among the 83 patients with negative ANCA screening, only 2 patients with EGPA received an AAV treatment. Among the 10 patients with typical positive ANCA screening and newly diagnosed with AAV, specific AAV treatment was initiated within the first 24 h of ANCA results in 7/10 patients. However, AAV histological confirmation was also available before or less than 24 h after ANCA results in 6/10 patients. In the other four patients, no histological confirmation was obtained due to noncontributive biopsies.
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Intern Emerg Med Table 2 Review of previous studies analyzing ANCA testing for the diagnosis of AAV
Sensitivity, specificity, positive predictive value, and negative predictive value for AAV diagnoses are given considering only IIF or IIF ? MPO- and PR3-ANCA positive samples N number, IIF Immunofluorescence, PPV positive predictive value, NPV negative predictive value, na non available
Reference
Gating policy
n
Positivity at IIF n (%)
MPO-/ PR3ANCA n (%)
Sensitivity (%) IIF/ ELISA
Specificity (%) IIF/ ELISA
PPV (%) IIF/ ELISA
NPV (%) IIF/ ELISA
Sinclair et al.
yes
287
57 (26.9)
na
na/na
na/na
73.7/na
na/na
Arnold et al.
yes
na
na (30.3)
na
na/na
na/na
na/na
na/na
Russell et al.
no
615
na
na
64/59.3
91.5/96
55/70.8
94/93.3
Mandl et al.
no
497
na
na (4.8)
na/81
na/98
na/54
na/99
Robinson et al.
no
1,127
110 (9.8)
21 (1.9)
na/100
na/99.9
na/75
na/100
Tsiveriotis et al.
no
10,803
644 (6)
129 (1.2)
na/na
na/na
na/84.5
na/na
Present study
yes
114
31 (27.2)
15 (13.2)
83.3/83.3
83.5/100
38.5/ 100
97.6/98
Among 16 patients with atypical c-/p- ANCA or a-ANCA, none had AAV as final diagnosis. The 83 patients without ANCA at IIF were diagnosed with various conditions or diseases. Only four were diagnosed with vasculitis: EGPA (n = 2), IgA vasculitis (n = 1) and giant cell arteritis (n = 1). Confrontation between ANCA detection and final clinical diagnosis is detailed in Fig. 1.
Discussion In the present study, we evaluated the performance of ANCA testing in the context of emergency screening. To the best of our knowledge, only one previous study has described such a procedure [6]. Our gating policy based on a dialog between the clinician and the biologist might be less selective than the guidelines on ANCA testing [7, 8]. However, given the clinical presentation of patients at ANCA emergency screening request (Table 1), we can presume that our gating policy was appropriate to select patients with clinical signs suggestive of AAV. Among clinical signs, only the presence of sinusitis was significantly associated with AAV, reflecting the relative specificity of sinusitis for GPA. We detected the presence of ANCA at IIF in 27.2 % of patients and of c-/p-ANCA anti-PR3/-MPO in 13.2 % of patients. We observed a good sensitivity/specificity and positive/negative predictive values for newly diagnosed AAV with IIF/Immunodot combination. Only two patients with EGPA were ANCA negative, a disease that is frequently seronegative [1]. Applying to ANCA request a gating policy based on the International conference recommendations enhances the rate of positivity of ANCA
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testing at IIF without causing missed diagnoses of AAV [9–11]. We observed in our study a rate of ANCA positivity at IIF that was in the same range of the other studies that have analyzed such gating policy (Table 2). This demonstrates that in an emergency context, our dialogbased policy was as effective as clinical guidelines [9–14]. Prompt detection and treatment initiation are critical to ensure recovery of organ AAV injury, especially when the kidney is involved [15, 16]. The impact of the result of emergency ANCA screening on patient management is difficult to analyze in our study given its retrospective design. However, specific AAV treatment was started within 24 h following the ANCA emergency result in seven of the ten patients with newly diagnosed AAV suggesting that emergency ANCA screen accelerated treatment initiation in these patients. In four patients with newly diagnosed AAV and with kidney involvement, a kidney biopsy showing a necrotizing crescentic glomerulonephritis was also available less than 24 h after ANCA results. Thus, adding emergency ANCA positivity may have been limited in these patients. Conversely, the other four patients without immediate or with inconclusive histology probably took the advantage of the emergency screening with respect to treatment initiation. Whether emergency ANCA detection is associated with a better outcome remains speculative. Moreover, the impact of having a negative ANCA screening is outside of what we can infer from this study. The main limitation of our study is that we were not able to analyze patients for whom emergency ANCA requests were declined by the biologist. It would have been interesting to verify that our gating policy was not responsible for missing or delayed AAV diagnoses. However, this
Intern Emerg Med
question has already been addressed in the study of Arnold et al. that applied a gating policy based on clinical guidelines for ANCA testing [9]. The authors demonstrate that such strategy is effective, as none of the excluded patients from ANCA testing subsequently developed AAV. Finally, we limited our analysis to in-hospital ANCA requests. However, emergency ANCA testing has been available for nearby hospitals of our region.
6.
7.
8.
Conclusion Emergency ANCA testing associated with a gating policy based on a senior clinician/biologist dialog was effective in identifying patients with AAV. The impact of such strategy on patients care is outside of what we can infer in our study.
9. Conflict of interest
None declared.
References 1. Beauvillain C, Delneste Y, Renier G, Jeannin P, Subra JF, Chevailler A (2008) Antineutrophil cytoplasmic autoantibodies: how should the biologist manage them? Clin Rev Allergy Immunol 35(1–2):47–58. doi:10.1007/s12016-007-8071-9 2. Jennette JC, Falk RJ, Hu P, Xiao H (2013) Pathogenesis of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Annu Rev Pathol 8:139–160. doi:10.1146/annurevpathol-011811-132453 3. Tervaert JW, van der Woude FJ, Fauci AS, Ambrus JL, Velosa J, Keane WF, Meijer S, van der Giessen M, van der Hem GK, The TH et al (1989) Association between active Wegener’s granulomatosis and anticytoplasmic antibodies. Arch Intern Med 149(11):2461–2465 4. Subra JF, Michelet C, Laporte J, Carrere F, Reboul P, Cartier F, Saint-Andre JP, Chevailler A (1998) The presence of cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA) in the course of subacute bacterial endocarditis with glomerular involvement, coincidence or association? Clin Nephrol 49(1):15–18 5. Tur BS, Suldur N, Ataman S, Tutkak H, Atay MB, Duzgun N (2004) Anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis: clinical, biological, and radiological
10.
11.
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13.
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16.
correlations. Jt Bone Spine : revue du rhumatisme 71(3):198–202. doi:10.1016/j.jbspin.2003.04.002 Aslam A, Newman TL, Misbah SA (2003) Audit of the clinical usefulness of a rapid qualitative ELISA screen for antimyeloperoxidase and antiproteinase 3 antibodies in the assessment of patients with suspected vasculitis. J Clin Pathol 56(10):775–777 Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, Hagen EC, Jayne D, Jennette JC, Paspaliaris B, Pollock W, Pusey C, Savage CO, Silvestrini R, van der Woude F, Wieslander J, Wiik A (1999) International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am J Clin Pathol 111(4):507–513 Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, McEvoy R, Pusey C, Pollock W, Trevisin M, Wiik A, Wong R, International Group for Consensus Statement on T, Reporting of Antineutrophil Cytoplasmic A (2003) Addendum to the international consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am J Clin Pathol 120(3):312–318. doi:10.1309/WAEPADW0-K4LP-UHFN Arnold DF, Timms A, Luqmani R, Misbah SA (2010) Does a gating policy for ANCA overlook patients with ANCA associated vasculitis? An audit of 263 patients. J Clin Pathol 63(8):678–680. doi:10.1136/jcp.2009.072504 Mandl LA, Solomon DH, Smith EL, Lew RA, Katz JN, Shmerling RH (2002) Using antineutrophil cytoplasmic antibody testing to diagnose vasculitis: can test-ordering guidelines improve diagnostic accuracy? Arch Intern Med 162(13):1509–1514 Sinclair D, Saas M, Stevens JM (2004) The effect of a symptom related ‘‘gating policy’’ on ANCA requests in routine clinical practice. J Clin Pathol 57(2):131–134 Robinson PC, Steele RH (2009) Appropriateness of antineutrophil cytoplasmic antibody testing in a tertiary hospital. J Clin Pathol 62(8):743–745. doi:10.1136/jcp.2009.064485 Russell KA, Wiegert E, Schroeder DR, Homburger HA, Specks U (2002) Detection of anti-neutrophil cytoplasmic antibodies under actual clinical testing conditions. Clin Immunol 103(2):196–203. doi:10.1006/clim.2001.5200 Tsiveriotis K, Tsirogianni A, Pipi E, Soufleros K, Papasteriades C (2011) Antineutrophil cytoplasmic antibodies testing in a large cohort of unselected greek patients. Autoimmun Dis 2011:626495. doi:10.4061/2011/626495 Moroni G, Ponticelli C (2014) Rapidly progressive crescentic glomerulonephritis: early treatment is a must. Autoimmun Rev 13(7):723–729. doi:10.1016/j.autrev.2014.02.007 Catanoso M, Pipitone N, Magnani L, Boiardi L, Salvarani C (2011) New indications for biological therapies. Intern Emerg Med 6(Suppl 1):1–9. doi:10.1007/s11739-011-0667-7
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IM - ORIGINAL
Emergency testing for antineutrophil cytoplasmic antibodies combined with a dialog-based policy between clinician and biologist: effectiveness for the diagnosis of ANCA-associated vasculitis Johnny Sayegh • Caroline Poli • Alain Chevailler • Jean-Franc¸ois Subra Franc¸ois Beloncle • Pierre Antoine Deguigne • Ce´line Beauvillain • Jean-Franc¸ois Augusto
•
Received: 9 May 2014 / Accepted: 15 October 2014 Ó SIMI 2014
Abstract A prompt immunosuppressive treatment initiation is crucial in ANCA-associated vasculitis (AAV) to minimize organ injury. The aim of the present work was to analyze the accuracy of emergency ANCA screening to identify rapidly patients with AAV. In our Institution, emergency ANCA screening is based on a telephone call between a Clinician and a Biologist. Indirect immunofluorescence (IIF) for ANCA detection was performed using a commercial kit (EuroimmunÒ Granulocyte Mosaic 12). Positive serums for c- or p-ANCA at IIF are subsequently screened for antigenic specificity (MPO or PR3) by an immunodot technique (immunodot, D-TekÒ.) Positive samples with atypical c- or p-ANCA pattern at IIF are subsequently screened for antigenic specificity by ELISA. Data were retrieved from patients’ medical records and confronted to emergency ANCA screening results. Between 2005 and 2012, 114 patients were screened. IIF was positive in 27.2 % of patients, but c-/p-ANCA antiMPO/-PR3 was detected in 13.2 % of patients. The sensibility and specificity of IIF combined with immunodot for newly diagnosed AAV were 83.3 and 100 %, respectively. Ten patients were newly diagnosed with AAV. In these patients, a specific AAV treatment was initiated less than 24 h following ANCA screening. Emergency ANCA
J. Sayegh C. Poli A. Chevailler J.-F. Subra F. Beloncle P. A. Deguigne C. Beauvillain J.-F. Augusto LUNAM Universite´, Angers, France J. Sayegh J.-F. Subra F. Beloncle J.-F. Augusto (&) Service de Ne´phrologie-Dialyse-Transplantation, CHU Angers, 4 rue Larrey, 49933 Angers Cedex 9, France e-mail: [email protected] C. Poli A. Chevailler P. A. Deguigne C. Beauvillain Laboratoire d’Immunologie, CHU Angers, Angers, France
screening based on a clinical gating policy was relevant to identify patients with AAV diagnosis, and was associated with a rapid treatment initiation. Keywords vasculitis
ANCA Emergency ANCA-associated
Introduction Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against proteins of neutrophil cytoplasmic granules and monocytes lysosomes that are strongly associated with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) [1, 2]. The two main targets of ANCA are proteinase 3 (PR3), and myeloperoxidase (MPO). Anti-PR3 ANCA are usually associated with a cytoplasmic neutrophil fluorescence pattern (cANCA) using indirect immunofluorescence (IIF) on fixed neutrophils, and are mainly detected in GPA. On the other hand, anti-MPO ANCAs reproduce a perinuclear fluorescence pattern (p-ANCA), and are associated with MPA and CSS. When analyzed in ANCA-associated vasculitis (AAV) patients, the sensitivity and specificity of ANCAs have been reported to be as high as 95 % [3]. However, ANCAs have also been detected in numerous other conditions or diseases (i.e., rheumatoid arthritis and endocarditis) [4, 5]. Thus, in routine clinical practice, and especially when the clinician is looking for a diagnosis, the accuracy of ANCA testing to identify patients with AAV might be lower. In acute conditions, emergency ANCA testing may be of valuable information to help clinicians identify AAV. Given its non-specific clinical presentation, a fast
123
Intern Emerg Med
identification of AAV is crucial to start treatment promptly. However, given the lower sensitivity/specificity of ANCA in unselected populations, such strategy may also be deleterious due to possible false positives. In the University Hospital of Angers, the emergency screening of ANCA has been available since January 2005. To the best of our knowledge, strategies evaluating the usefulness of ANCA in emergency conditions have been poorly reported to date [6]. We describe here the conditions that led clinicians to request emergency ANCA testing, and its usefulness for diagnosis and patient management.
Methods Study design and patients Emergency ANCA testing has been available since January 2005, 6 days a week (Mondays to Saturdays.) The request procedure is based on a telephone call between a senior clinician and a senior biologist. Acceptance of the request is left to the discretion of the senior biologist. Results are usually available within 4 to 8 h following the request, depending on the results of the IIF. When an emergency request is declined, ANCA screening is done without emergency, and results are given with a mean delay of 1 week. Even if no strict gating policy is applied, both clinical presentation and its confrontation to the international consensus statement outlining the clinical criteria for ANCA testing are determinant in accepting or declining the request [7, 8]. Only in-hospital patient requests were analyzed in the present study. A clinician (JFA) and a biologist (PAD) reviewed patients’ medical records, and were blinded to ANCA testing result. The final diagnosis was reviewed by two clinicians (JFA and JS) based on individual patient case notes, laboratory and biopsy results. The study protocol was approved by the Ethic Committee of the Angers University Hospital (CE 2013-65). Emergency ANCA detection Emergency ANCA detection was done using indirect immunofluorescence (IIF) with a commercial kit (EuroimmunÒ Granulocyte Mosaic 12.) Briefly, serum samples were assayed by IIF on slides with human ethanol, methanol or formaldehyde-fixed neutrophils and classified as c-, p or atypical-ANCA (a-ANCA). Positive serums for c- or p- or a-ANCA at IIF were subsequently screened for antigenic specificity (MPO or PR3) by an immunodot technique (immunodot, D-TekÒ). Sera with atypical c-, p- or a-ANCA were screened for antigenic specificity (BPI, azurocidin, elastase, cathepsin
123
Table 1 Clinical and biological characteristics of the study population All patients (n = 114)
Non-AAV patients (n = 96)
AAV patients (n = 18)
Age (years)
56.9 ± 25
55.6 ± 26
63.9 ± 14
Sex (men/women)
72/42
58/38
14/4
Clinical signs, n (%) Fever
39 (34.2)
33 (34.4)
6 (33.4)
Renal involvement
84 (73.7)
69 (71.9)
15 (83.3)
Hypertension
39 (34.2)
32 (33.3)
7 (38.9)
Acute renal failure
61 (53.5)
53 (55.2)
8 (44.4)
Proteinuria
75 (65.8)
62 (64.6)
13 (72.2)
Haematuria
57 (50)
47 (49)
10 (55.6)
Respiratory involvement
49 (43)
38 (39.6)
11 (61.1)
Dyspnoea
41 (36)
33 (34.4)
8 (44.4)
Hemoptysis
14 (12.3)
11 (11.5)
3 (16.7)
Arthritis
15 (13.2)
10 (10.4)
5 (27.8)
Purpura
20 (17.5)
17 (17.7)
3 (16.7)
Sinusitis
6 (5.3)
2 (2.1)
4 (22.2)**
Pericarditis/ myocarditis
5 (4.4)
4 (4.2)
1 (5.6)
Biological abnormalities, n (%) Inflammatory syndrome
93 (81.6)
75 (78.1)
18 (100)
Anemia
88 (77.2)
73 (76)
15 (83.3)
Hypereosinophilia
9 (7.9)
7 (7.3)
2 (11.1)
Antinuclear antibodies*
28 (24.6)
22 (22.9)
6 (33.3)
* Titre [1/200, **p \ 0.005
G, and lactoferrin) EuroimmunÒ).
by
ELISA
(ANCA
profil,
Statistical analysis Values were expressed as mean ± SD or percentage. Qualitative variables were compared using Chi squared test or Fisher exact test and quantitative variables using Mann– Whitney test. For all statistical analysis, p \ 0.05 was considered significant. Analysis was performed with the SPSS 15.0 software (SPSS, Inc., Chicago, IL).
Results A total of 114 emergency ANCA testings were performed in the study period. Patients had a mean age of 56.9 ± 25 [1.1–89.8], and were men in 63 % of cases. Routine ANCA
Intern Emerg Med Fig. 1 Flow chart of emergency ANCA screening and associated clinical diagnosis
Emergency ANCA screening (n=114)
Negave ANCA screening 83/114 (72.8%)
Posive ANCA screening 31/114 (27.2%)
Atypical ANCA 16/114 (14%)
Typical ANCA 15/114 (13.2%)
Vasculis: EGPA, n=2; IgA vasculis, n=1; GPA, n=1 Auto-immune diseases (2 Goodpasture, 1 myosis, 1 Crohn disease, 1 adult Sll disease, 1 thrombopenic pupura, 1 systemic sclerosis, 1 TINU syndrme, 1 Guillain Barre syndrome), n=9 Kidney diseases: acute renal faillure, n=7; glomerulonephris, n=12; renal sarcoidosis, n=1; undetermined nephropathies, n=6; others, n=4 Lymphoma, n=3 Infecous diseases: bacterial pneumonia, n=9; viral pneumonia, n=1; mycosis, n=1 Thromboc microangiopathy, n=5 Undetermined hypoxemic pneumonia, n=8 Others causes*, n=7 Undetermined, n=6
Atypical c-or p-ANCA 12/114 (10.5%) c-ANCA 6/114 (5.3%) Newly diagnosed GPA, n=4 Previously diagnosed GPA, n=2
p-ANCA 9/114 (7.9%) Newly diagnosed MPA, n=5 Newly diagnosed GPA, n=1 Previously diagnosed MPA, n=2 Previously diagnosed GPA, n=1
Undertermined GN, n=2 Acute renal failure, n=1 Bacterial pneumonia, n=2 IgA vasculis, n=1 Cholesterol embolizaon syndrome, n=1 Leukocytoclasc vasculis, n=1 Lymphoma, n=1 undetermined, n=3
a-ANCA 4/114 (3.5%) Vascular nephropathy, n=1 IgA vasculis, n=1 Brain sepc stroke embolism, n=1 Undetermined, n=1
*Stroke, n=1, cardiogenic shock, n=1; pancreas, n=1; lung cancer, n=1; status epilepcus, n=1; pulmonary hemosiderosis, n=1
testing was done in 11,147 patients during the analysis period, thus emergency ANCA testing represented a minor percentage of overall routine ANCA testing (around 1 %) There was no significant variation in the distribution of emergency ANCA requests between days in the week. Nephrology and Critical Care Medicine Departments represented 57.4 % of the emergency requests. The screening was performed at a median of 1.5 days from admission, and within the first 2 days from admission in 64 % of patients. The population was highly selected based on the clinical presentation neighboring criteria suggested by the 1999 International Consensus on ANCA testing [7]. As detailed in Table 1, most patients had fever, inflammatory syndrome, and renal or respiratory involvement. Renal involvement was present in 73.7 % of patients, and was mainly acute kidney injury associated with proteinuria or haematuria. Half of the patients had respiratory involvement with dyspnea, cough or hemoptysis. Digestive, rheumatologic, skin and respiratory tract involvement were less frequently observed. Among the 114 patients, 31 (27.2 %) had positive c- or p-ANCA (n = 6 and n = 9, respectively,) atypical positive c- or p-ANCA (n = 12), or positive a-ANCA (n = 4) at IIF. Figure 1 reports the results of emergency ANCA screening, and the patient’s final diagnosis. After reviewing the medical records, 12 patients (10.5 %) were newly diagnosed with AAV (5 MPA, 5 GPA and 2 EGPA.) The five MPA patients had positive p-ANCA anti-MPO,
whereas the patients with GPA had positive c-ANCA antiPR3 (n = 4) and positive p-ANCA anti-MPO (n = 1.) The two patients with EGPA were ANCA negative. The sensitivity and specificity of emergency IIF-ANCA testing for the diagnosis of newly identified AAV, after exclusion of the screened patients with previously known AAV, were 83.3 and 83.5 %, respectively. The positive predictive value of emergency ANCA testing for newly diagnosed AAV was 38.5 %, and the negative predictive value was 97.6 %. When the analysis was restricted to patients with typical c-ANCA anti-PR3 or p-ANCA anti-MPO, the sensitivity and specificity for the diagnosis of AAV were 82.3 and 100 %. The positive and negative predictive values were 100 and 98 %. Among clinical signs, only sinusitis was significantly more frequent in AAV patients (Table 1) as compared to other patients. We next analyzed the relationship between emergency ANCA testing results, histological results and treatment management. Among the 83 patients with negative ANCA screening, only 2 patients with EGPA received an AAV treatment. Among the 10 patients with typical positive ANCA screening and newly diagnosed with AAV, specific AAV treatment was initiated within the first 24 h of ANCA results in 7/10 patients. However, AAV histological confirmation was also available before or less than 24 h after ANCA results in 6/10 patients. In the other four patients, no histological confirmation was obtained due to noncontributive biopsies.
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Intern Emerg Med Table 2 Review of previous studies analyzing ANCA testing for the diagnosis of AAV
Sensitivity, specificity, positive predictive value, and negative predictive value for AAV diagnoses are given considering only IIF or IIF ? MPO- and PR3-ANCA positive samples N number, IIF Immunofluorescence, PPV positive predictive value, NPV negative predictive value, na non available
Reference
Gating policy
n
Positivity at IIF n (%)
MPO-/ PR3ANCA n (%)
Sensitivity (%) IIF/ ELISA
Specificity (%) IIF/ ELISA
PPV (%) IIF/ ELISA
NPV (%) IIF/ ELISA
Sinclair et al.
yes
287
57 (26.9)
na
na/na
na/na
73.7/na
na/na
Arnold et al.
yes
na
na (30.3)
na
na/na
na/na
na/na
na/na
Russell et al.
no
615
na
na
64/59.3
91.5/96
55/70.8
94/93.3
Mandl et al.
no
497
na
na (4.8)
na/81
na/98
na/54
na/99
Robinson et al.
no
1,127
110 (9.8)
21 (1.9)
na/100
na/99.9
na/75
na/100
Tsiveriotis et al.
no
10,803
644 (6)
129 (1.2)
na/na
na/na
na/84.5
na/na
Present study
yes
114
31 (27.2)
15 (13.2)
83.3/83.3
83.5/100
38.5/ 100
97.6/98
Among 16 patients with atypical c-/p- ANCA or a-ANCA, none had AAV as final diagnosis. The 83 patients without ANCA at IIF were diagnosed with various conditions or diseases. Only four were diagnosed with vasculitis: EGPA (n = 2), IgA vasculitis (n = 1) and giant cell arteritis (n = 1). Confrontation between ANCA detection and final clinical diagnosis is detailed in Fig. 1.
Discussion In the present study, we evaluated the performance of ANCA testing in the context of emergency screening. To the best of our knowledge, only one previous study has described such a procedure [6]. Our gating policy based on a dialog between the clinician and the biologist might be less selective than the guidelines on ANCA testing [7, 8]. However, given the clinical presentation of patients at ANCA emergency screening request (Table 1), we can presume that our gating policy was appropriate to select patients with clinical signs suggestive of AAV. Among clinical signs, only the presence of sinusitis was significantly associated with AAV, reflecting the relative specificity of sinusitis for GPA. We detected the presence of ANCA at IIF in 27.2 % of patients and of c-/p-ANCA anti-PR3/-MPO in 13.2 % of patients. We observed a good sensitivity/specificity and positive/negative predictive values for newly diagnosed AAV with IIF/Immunodot combination. Only two patients with EGPA were ANCA negative, a disease that is frequently seronegative [1]. Applying to ANCA request a gating policy based on the International conference recommendations enhances the rate of positivity of ANCA
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testing at IIF without causing missed diagnoses of AAV [9–11]. We observed in our study a rate of ANCA positivity at IIF that was in the same range of the other studies that have analyzed such gating policy (Table 2). This demonstrates that in an emergency context, our dialogbased policy was as effective as clinical guidelines [9–14]. Prompt detection and treatment initiation are critical to ensure recovery of organ AAV injury, especially when the kidney is involved [15, 16]. The impact of the result of emergency ANCA screening on patient management is difficult to analyze in our study given its retrospective design. However, specific AAV treatment was started within 24 h following the ANCA emergency result in seven of the ten patients with newly diagnosed AAV suggesting that emergency ANCA screen accelerated treatment initiation in these patients. In four patients with newly diagnosed AAV and with kidney involvement, a kidney biopsy showing a necrotizing crescentic glomerulonephritis was also available less than 24 h after ANCA results. Thus, adding emergency ANCA positivity may have been limited in these patients. Conversely, the other four patients without immediate or with inconclusive histology probably took the advantage of the emergency screening with respect to treatment initiation. Whether emergency ANCA detection is associated with a better outcome remains speculative. Moreover, the impact of having a negative ANCA screening is outside of what we can infer from this study. The main limitation of our study is that we were not able to analyze patients for whom emergency ANCA requests were declined by the biologist. It would have been interesting to verify that our gating policy was not responsible for missing or delayed AAV diagnoses. However, this
Intern Emerg Med
question has already been addressed in the study of Arnold et al. that applied a gating policy based on clinical guidelines for ANCA testing [9]. The authors demonstrate that such strategy is effective, as none of the excluded patients from ANCA testing subsequently developed AAV. Finally, we limited our analysis to in-hospital ANCA requests. However, emergency ANCA testing has been available for nearby hospitals of our region.
6.
7.
8.
Conclusion Emergency ANCA testing associated with a gating policy based on a senior clinician/biologist dialog was effective in identifying patients with AAV. The impact of such strategy on patients care is outside of what we can infer in our study.
9. Conflict of interest
None declared.
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