Prevalence of Antineutrophil cytoplasmic Antibodies in Systemic Lupus Erythematosus and its relevance with Clinical manifestations

Bitan Naik¹, Mahima Yadav², Vikas Kailashiya³, Anu Singh^4*, Sandip Kumar⁵

¹MD Pathology, Assistant Professor, Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh, India.

²MD Pathology, Assistant Professor, Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh. India.

³MD Pathology, Assistant Professor, Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh. India.

⁴MD Pathology, Assistant Professor, Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh. India.

Address for communication- Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh. India.

⁵MD Pathology, Professor & Head , Department of Pathology,

Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi, Uttar Pradesh. India.

*Corresponding Author E-mail: anusingh@bhu.ac.in

ABSTRACT:

Background: Role of anti-neutrophil cytoplasmic antibodies (ANCA) in pathogenesis of systemic lupus erythematous (SLE) and its association with clinical manifestations is not completely understood.  Prevalence data of ANCA in SLE patients of Indian population is limited. Aims and Objectives: The aim of the present study is to measure the prevalence of ANCAs in SLE patients and study its association with clinical manifestations of SLE. Material and Methods: Total 92 patients of SLE cases were included in this prospective observational study. Demographic, clinical and laboratory data was collected in all patients. Serum levels of myeloperoxidase (MPO)-ANCA, proteinase 3 (PR3)-ANCA, antinuclear antibody (ANA), anti-dsDNA antibody and extractable nuclear antigen (ENA) antibodies were measured by enzyme immune assay methods. Serum Complement C3 and C4 estimation was done by nephelometer. Unpaired t test was used to find the significance difference in mean value between ANCA positive and ANCA negative group. Chi-square test was used to compare categorical data of two groups. Results: Nineteen cases (20.65%) showed ANCA positivity. Ten cases were positive for PR3-ANCA and seven cases were positive for MPO-ANCA. Two cases were detected with dual MPO and PR3-ANCA. Nephritis was significantly more common in ANCA positive SLE patients. Rest all of clinical manifestations, anti-dsDNA antibody positivity, ENAs antibodies positivity and reduction in complement level did not show any significant correlation with presence of ANCA antibody.

Conclusion: In contrast to results of earlier studies, PR3-ANCA was more prevalent in our study population. Renal system involvement was significantly high in ANCA positive SLE patients as compared to ANCA negative patients.

KEYWORDS: Anti-neutrophil cytoplasmic antibody, Systemic lupus erythematosus, prevalence.

INTRODUCTION: 

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with formation of many different types of antinuclear antibodies (ANAs). Antibody like anti-double stranded DNA(Anti-dsDNA) and anti-Smith antibody (anti-Sm) have an important role in the pathogenesis and diagnosis of SLE. Anti-neutrophilic cytoplasmic antibodies (ANCAs) are formed against cytoplasmic antigen present in azurophilic granules of neutrophils and lysozyme of monocytes.¹ Proteinase- 3(PR3) and myeloperoxidase (MPO) are two important antigens against which ANCAs are produced in diseases of small vessel vasculitis. PR3-ANCA is elevated in Granulomatosis with polyangiitis. Microscopic polyangiitis and Eosinophilic granulomatosis with polyangiitis are associated with elevated serum level of MPO-ANCA.² Besides small vessel vasculitis, ANCAs are also detected in systemic autoimmune disease like SLE.³ The occurrence of ANCAs in SLE patients varies from 16% to 42%.^4,5 The exact role of ANCAs in pathogenesis of SLE is still not clear. Previous studies reveal conflicting results about association between ANCA positivity in SLE patients and different clinical manifestations of SLE.^4-9In recent past there are limited numbers of studies in Indian population that estimated the prevalence of ANCA in SLE patients. Very few earlier studies have excluded patients taking immunosuppressive treatment, thus potentially giving biased results. The aim of our study is to measure the prevalence of ANCA in SLE patients and analyse any possible association with clinical features, serologic evidence of disease activity and serum antinuclear antibodies.

MATERIAL AND METHODS:

The present study is a prospective observational study conducted between July 2017 to June 2019 in UGC Advanced Immunodiagnostic Training and Research Centre of the department of Pathology, Institute of Medical Sciences, Banaras Hindu University. Total 92 cases of SLE diagnosed by revised 1997 American College of Rheumatology (ACR) criteria and Systemic Lupus International Collaborating Clinics classification criteria (SLICC,2002) were included in the study. Patients taking immunosuppressive drugs and cases of drug induced SLE and  SLE/AAV overlap syndrome were excluded from the study. Ethical approval from Institute ethical committee and written consent from patients were taken prior to starting the study. Clinical and demographic details were collected in all patients. Blood samples were collected in vacutainers for serum ANCAs, ANAs, complements and haematological parameter estimation. Serum samples were stored at -80°C. Renal involvement of SLE was defined by presence of one of the following ACR criteria; 1- persistent proteinuria greater than 500 mg per 24 hours, 2- presence of cellular cast, 3- biopsy proven lupus nephritis. Haematological abnormities were defined as follows; leukopenia- total leukocyte count less than 4000/mm³, lymphopenia-total lymphocyte count less than 1500/mm³ and thrombocytopenia-platelet count less than 100000/mm³.

Estimation of MPO and PR-3 ANCA

Estimation of MPO and PR-3 ANCA was done by indirect non-competitive enzyme immunoassay Kit of MPO and PR3 ANCA respectively. (AESKU, Germany)

Principle of the ANCA assay: Microwell of the enzyme-linked immunosorbent assay (ELISA) plate are coated with PR3 or MPO antigens. Antibodies present in serum sample bind to the respective coated antigens. Non-specific and unbound serum component are removed by washing of the ELISA plate. Subsequently added enzyme conjugate binds to with antibody-antigen complex present in the microwell to form conjugate-antigen-antibody complex. Second washing step removed unbound conjugate. After addition of substrate, the bound enzyme conjugate hydrolyses the substrate to form a blue colour. Addition of an acid stops the reaction by forming a yellow colour end-product.The intensity of colour is directly proportional to the concentration of antibodies present in the serum sample and measured by ELISA reader at 450 nm.

Procedure of the ANCA assay: Ten ml of serum was taken in a clean test tube and diluted with 990 ml of sample diluents. Then 100ml of diluted serum, 100ml of calibrators and 100 ml of control was added to micro well of the ELISA plate and kept for 30 minutes incubation at room temperature. After incubation, contents of the micro well were discarded and washed with 300ml of wash solution for three times. Then enzyme conjugates of 100 ml wasdispensed into micro well and plate was kept for 15 minutes incubation at room temperature. Again, after incubation contents of the micro well was discarded and washed with 300 ml of wash solution for three times. After washing, tetramethyl benzidine (TBM) solution of 100ml was added to the micro well and kept for 15 minutes incubation. In final step, stop solution of 100 ml was added and then kept for 5 minutes incubation. After that optical density was measured at 450 nm in ELISA reader. For quantitative estimation of MPO and PR3-ANCA values, the optical density of each standard was plotted in y-axis and corresponding manufacturer provided concentration of each standard was plotted in x-axis. Then calibration curve was drawn, which was used to determine the value of patient samples. Serum level of ≥ 5 U/ml was taken as positive for PR-3 and MPO antibodies.

Screening for antinuclear antibodies was done by an indirect immunofluorescence ANA Kit. (Aesku, Germany) Quantitative estimation of Anti dsDNA antibody was done by an indirect non-competitive enzyme immunoassay dsDNA Kit (Aesku ,Germany) Detection of other ANAs antibodies like anti-Sm, anti-RNP, anti-SSA, anti-SSB, anti-Scl 70, anti-centromere, anti-Jo 1 was done by D-tekBlueDriverDot ANA8 IgG Immunodot kit (Diagnostic technology, Sydney, Australia). This test is based on the principle of enzyme Immunoassay. Serum Complement C3 and C4 estimation was done by nephelometer. All the patients of SLE were divided into ANCA positive and ANCA negative group, bases on results of ANCA immunoassay. The clinical presentation, autoantibody profile and hematological parameters of both groups were compared.

Statistical analysis:

Statistical package for social sciences (SPSS) version 20 was used for statistical analysis. Quantitative variable expressed as means and standard deviation. Categorical variable expressed as frequency. Chi-square test was used to compare categorical data of two groups. Unpaired t test was used to detect significant difference in mean values of ANCA positive SLE patients and ANCA negative SLE patients. P-value of less than 0.05 (P<0.05) was considered as statistically significant.

RESULTS:

Out of total 92 cases, 19 cases (20.65%) were positive of ANCA. Among 19 positive ANCA cases, ten cases (10.87%) were positive for anti- PR-3, seven cases (7.61%) positive for anti-MPO and two cases positive for both anti-PR3 and anti-MPO antibodies. The mean age of presentation of SLE patient was 31.39 (SD- 9.27) years with age range from 16 years to 55 years. ANCA positive SLE patients had younger mean age of presentation (28.21, SD-8.1) as compared ANCA negative patients (32.22, SD-9.49). However, the difference between the mean age of presentation of both groups was not statistically significant (t=1.687, P=0.095). Most of the SLE patients were female (85.8%) with female: male ratio of 6.08:1. The frequency of involvement of males was more in ANCA positive SLE patient group (18.75%, F:M ratio-5.3:1) compared to ANCA negative group (13.69%, F:M ratio-6.3:1). The clinical manifestation of both ANCA positive SLE patients and ANCA negative SLE patients are shown in table no-1. Arthritis is the most common clinical presentation in both ANCA positive (84.21%) and negative group (90.41%). Nephritis is the second frequent (73.68%) clinical manifestation in ANCA positive SLE patients, however alopecia is the second frequent (56.16%) in ANCA negative group. Fever, malar rash alopecia, photophobia, nephritis, Raynaud’s phenomenon was more frequent in ANCA positive SLE patients. Discoid rash, oral ulcer, arthritis and neurological manifestations were more frequent in ANCA negative patients. Nephritis was significantly more common in ANCA positive patients (P<0.05). Rest all the clinical parameters did not show any significant difference between ANCA positive and ANCA negative groups. Table No 2 show complement levels and hematological abnormalities in SLE patients. Reduced serum level of C3 and C4 were more common in ANCA positive patients (36.84%) as compared to ANCA negative patients (34.24%). Leucopenia, lymphopenia, thrombocytopenia occurred more frequently in ANCA positive patients but did not show any significant statistical difference from ANCA negative patients. Autoantibody profile of SLE patients is shown in table no 3. All the SLE patient showed positive ANA screening test.  Anti-dsDNA was the most frequent autoantibody positive in 55(59.78%) cases of SLE, followed by anti-SSA antibody, which was detected in 40 cases (43.48%). Anti-dsDNA, anti-Sm, anti-RNP, anti-SSB antibody were more frequently detected in ANCA positive patients but did not show any significant difference from ANCA negative group. Anti-SSA antibody was noted more commonly in ANCA negative patients.

Table No 1 shows clinical manifestation in ANCA positive and ANCA negative SLE patient groups.

Parameters	All SLE patients (n=92), number, %	ANCA positive group (n=19), Number, %	ANCA negative group (n=73) Number, %	X^2	P value
Fever	50(54.34%)	11(57.89%)	39(53.42%)	0.121	0.727
Malar rash	38(41.30%)	8(42.1%)	30 (41.09%)	0.006	0.936
Discoid rash	17(18.47%)	3(15.78%)	14(19.17%)	0.115	0.735
Alopecia	55(59.78%)	14(73.68%)	41(56.16%)	1.925	0.165
Oral ulcer	35(38.04%)	7(36.8%)	28(38.36%)	2.670	0.102
Photophobia	50(54.34%)	11(57.89%)	39(53.42%)	0.121	0.727
Arthritis	82(89.13)	16(84.21%)	66(90.41%)	0.598	0.439
Pleuritis	19(20.65%)	05(26.31%)	14(19.1%)	0.469	0.493
Pericarditis	3(3.26%)	00(0%)	3(3.26%)	0.807	0.369
Nephritis	40(43.48%)	14(73.68%)	26(35.6%)	8.890	0.0028*
Neurological manifestation	15(16.3%)	3(15.79%)	12(16.44%)	0.005	0.945
Raynaud’s phenomenon	14(15.21%)	3(15.79%)	11(15.07%)	0.006	0.937

*- Statistically significant (P<0.05)

Table No 2 shows reduced serum complement level and haematological profile in ANCA negative SLE patients and ANCA positive SLE patients.

Parameters	All SLE patients (n=92), number, %	ANCA positive group (n=19), Number, %	ANCA negative group (n=73) Number, %	X^2	P value
Reduced C3, C4 level	32(34.78%)	7(36.84%),	25(34.24%)	0.045	0.832
Leukopenia	26(28.26%)	8(42.10%)	18(24.65%)	2.263	0.132
Lymphopenia	23(25%)	7(36.84%)	16(21.91%)	1.790	0.180
Thrombocytopenia	17(18.47%)	4(21.05%)	13(14.1%)	0.105	0.745
Haemolytic Anaemia	4(4.39%)	1(5.26%)	3(3.26%)	0.048	0.826

Table No 3 shows autoantibody profile in ANCA negative SLE patient group and ANCA positive SLE patient group.

Autoantibody type	All SLE patients (n=92),  number, %	ANCA positive group (n=19), Number, %	ANCA negative group (n=73)  Number, %	X^2	P value
Anti-dsDNA	55(59.78%)	13(68.42%)	42(57.53%)	1.641	0.200
Anti-Sm	30(32.60%)	8(42.10%)	22(30.14%)	0.983	0.321
Anti-RNP	39(42.39%)	10(52.63%)	29(39.72%)	1.028	0.310
Anti-SSA	40(43.48%)	8(42.10%)	32(43.83%)	0.184	0.892
Anti-SSB	20(21.74%)	5(26.32%)	15(20.54%)	0.295	0.587
Anti-centromere	5(5.37%)	1(5.26%)	4(5.48%)	0.001	0.970
Anti-Scl-70	2(2.17%)	1(5.26%)	1(1.36%)	1.074	0.299
Anti-Jo-1	2(2.17%)	0(0%)	2(2.17%)	0.532	0.466

Table no 4 Shows incidence of various clinical manifestation of SLE in different Indian studies.

Clinical presentation	Malaviya et al, 1985^15 (n=101)	Paul et al, 2003^14 (n=75)	Saigal et al, 2011^13 (n=60)	Talukdaret al, 2020 ^16 (n=145)	Present study (n=92)
Arthritis	66%	89.3%	53.3%	46.9%	89.13%
Fever	44%	4.0%	6.7%	NA	54.3%
Photosensitivity	67%	32%	75%	55.17%	54.34%
Malar rash	68%	40.0%	13.3%	46.21%	41.3%
Oral ulcer	64%	64.0%	61%	25.52%	38.4%
Pleuritis	17%	8%	11.7%	8.28%	20.65%
Pericarditis	5%	8%	11.7%	10.34%	3.26%
Alopecia	82%	60%	65%	67.58%	59.78
Raynaud phenomenon	32%	2.7%	21.7%	NA	16.3%
Nephritis	73%	33.3%	56.7%	58.03%	43.48%
Neurological manifestation	15%	13.3%	13.3%	20%	16.3%

DISCUSSION:

In the present study, prevalence of ANCA positivity in SLE patient is 20.6%. Fauzi et al reported prevalence of ANCA positivity in 24% of SLE patients.⁷ High prevalence of 42% was detected by Suet al.⁶ Another study reported low ANCA positivity rate (16.4%) as compared to present study.⁴ The difference of prevalence in various studies may be due to demographic variation in study populations, ethnicity and different laboratory techniques used for detection of ANCA. Effect of immunosuppressive treatment and steroids may also influence the results. The present study excludes those patients who took immunosuppressive treatment thus minimising the bias due to treatment. PR3-ANCA was more commonly found than MPO-ANCA in our study. Manolova et al also noted PR3-ANCA in 12.7% of SLE cases and MPO-ANCA in 10.9% 0f cases, which is close to our study findings.⁹ Prevalence of PR3-ANCA in SLE was more common than MPO-ANCA in study done by Mohammed et al.¹⁰ In contrast to our findings, many earlier studies noted higher rate of MPO-ANCA positivity than PR3-ANCA in SLE patients.^4,6 Pradhan et al and Wang et al noted MPO-ANCA was more prevalent than PR3 ANCA in patients of lupus nephritis.^11,12 Fauzi et al detected MPO and PR3-ANCA in equal number of SLE patients.⁷ In the present study arthritis was the most common clinical manifestation of SLE, which was present in 82 cases (89.13%). Paul et al and Saigal et al reported arthritis in 89.3% and 86.7% of cases.^13,14 Renal involvement was found in 43.3% of cases. Various Indian studies found renal involvement varying in frequency from 33.3% to 73%.^13-16 Incidence of various clinical manifestations of SLE in different Indian studies are shown in table no-4. Results of the previous studies show a variable association between different clinical manifestations and ANCA positivity in SLE patients. In the present study, only renal system involvement was significantly more in ANCA positive patients as compared to ANCA negative group. Su et al reported association between presence of ANCA with renal involvement, rash and Raynaud phenomenon. ⁷Chin et al found that ANCA, particularly p-ANCA was associated with nephritis.¹⁷ Pan et al found that, the incidence of renal involvement, serositis, myocarditis and neurological abnormalities was significantly higher in ANCA positive patients.¹⁸ They also noted positive correlation between serum ANCA level and disease activity. Pradhan et al reported high serum titres of ANCA in lupus nephritis as compared to SLE without nephritis.¹¹ In the present study, rest of the clinical manifestations did not show any significant correlation with of presence ANCA in SLE patients. Many previous studies did not find out any association between presence of ANCA and different organ system involvement.^5,7,8,19 Wang et al found no significant association between extrarenal manifestation and ANCA detection in lupus nephritis patients.¹²Galeazzi et al found a positive correlation between ANCA and clinical manifestations like serositis, livedo reticularis, venous thrombosis and arthritis.⁵

In our study, there was no significant difference in reduction of serum complement levels in ANCA positive and ANCA negative patients. Earlier studies found an association between reduction in complement C3/C4 level and ANCA positivity.^6,18 However, study by Wang et al did not found significant difference in complement levels in ANCA positive and ANCA negative group in lupus nephritis patients.¹² Haematological abnormalities like leukopenia, thrombocytopenia and lymphopenia are more frequent in ANCA positive patients, however no statistically significant difference exists between the ANCA positive and ANCA negative group. Manolova et al and Pan et al also found no association between presence of ANCA and leucopoenia and anaemia.^9,18 One earlier study noted similar finding in cases of lupus nephritis.¹¹

Anti-ds DNA antibody was present in 55(59.78%) cases of SLE in the present study. The frequency of anti-dsDNA positivity varies from 55% to 76% in Indian studies.^13-16 Among extractable antigens (ENAs) antibody, anti-SSA antibody is the most prevalent antibody (43.48%) in our study. Anti-SSB was detected in 21.74% of SLE cases in our study. Faria et al reported presence  of anti-SSA in 47% of patients and anti-SSB in 7% of patient.²⁰ Study by Jallouli et al found anti-SSA psitivity of 58% and anti-SSB of 22% in SLE patient.²¹ Indian study by Talukdar et al detected anti-SSA in 49.66% of cases and anti-SSB in 13.1% of cases.¹⁶ Anti-Sm antibody positivity was noted in 32.60% cases of present study. Frequency of anti-Sm positivity in SLE varies from 21% to 39.15%.^13,16,20,21 Anti-RNP antibody positivity rate in SLE was higher (42.39%) in present study compared to previous studies.^20,21 Present study did not find a significant association between ANCA positivity and presence of anti-dsDNA, anti-SSA, anti-SSB, anti-Sm and anti-U1-RNP antibodies in SLE patients. Similar results were found in study by Manolovaet al.⁹Su et al and Fauzi et al also did not find association between serum anti-dsDNA positivity with presence of ANCA in SLE patients.^6,7 In contrast to our result, study by Pan et al found that positivity rate of anti-dsDNA and anti-Sm antibody was significantly higher in ANCA positive SLE patients.¹⁸

CONCLUSION:

Although the occurrence of ANCA in SLE is not uncommon, yet it is not sufficiently analyzed as a potential diagnostic or prognostic marker. Present study reveals renal involvement is significantly more frequent in ANCA positive SLE patients. It suggests ANCA have an important role in pathogenesis of lupus nephritis. Measurement of serum ANCA levels may be helpful to predict or diagnose renal involvement in SLE patients. In contrast to findings of many previous studies, PR3-ANCA is more prevalent as compared to MPO-ANCA in our study population.  Since present study involved patients from single institution with limited sample size, studies with larger sample size are needed to confirm or rule out the association. 

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Received on 19.03.2024            Modified on 22.05.2024

Accepted on 29.08.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(10):5065-5070.

DOI: 10.52711/0974-360X.2024.00779