ANCA: serology in Wegener's granulomatosis.

BACKGROUND AND OBJECTIVES: Wegener's granulomatosis (WG) is being increasingly diagnosed in India, which exists in two forms, the 'limited Wegener's granulomatosis' (LWG) having upper respiratory tract (URT) and lower respiratory tract (LRT) involvement and the 'classical Wegener's granulomatosis' (CWG), with the triad of URT, LRT involvement along with kidney involvement. Cytoplasmic ANCA (C-ANCA) or anti-Proteinase3 (anti-PR3), which is highly diagnostic for WG, rarely perinuclear ANCA (P-ANCA) may exist. AIMS: To detect anti-neutrophil cytoplasmic antibodies (ANCA) and correlate it with serological, hematological parameters, and the Birmingham Vasculitis Activity Score (BVAS). SETTINGS AND DESIGN: Twenty-three clinically and histopathologically proven WG (16 CWG, 7 LWG) were studied. MATERIAL AND METHODS: C-ANCA and P-ANCA patterns were identified by immunofluorescence and specificities were confirmed by 'alpha granule' enzyme linked immunosorbent assay (ELISA), anti-PR3, anti-MPO (myeloperoxidase) and anti-Lactoferrin (anti-LF) by ELISA. RESULTS: LRT involvement was seen in 91.3%, URT in 78.3%, and renal manifestations in 69.6% cases. The BVAS in CWG was significantly higher than BVAS in the LWG. Decreased hemoglobin, increased WBC counts, ESR, CRP and Creatinine were seen in CWG as compared to LWG. The C-ANCA was present in 65.2% patients and P-ANCA in 13% cases. Anti-PR3 was seen in 69.6% patients and anti-LF in 17.4% cases. Severity of disease and ANCA was higher in CWG than in LWG. CONCLUSIONS: Vasculitis syndromes are known to overlap and many go undetected; therefore ANCA testing, along with the clinical and histopathological observations may be helpful in early detection and management of WG cases.

Anti-neutrophil cytoplasmic antibodies (ANCA) in systemic lupus erythematosus: prevalence, clinical associations and correlation with other autoantibodies.

AIM: This study was undertaken to clarify the nature of anti-neutrophil cytoplasmic antibodies (ANCA) along with other autoantibodies in lupus nephritis (LN) patients and in systemic lupus erythematosus (SLE) patients without nephritis and to know their correlation with clinical manifestations and presence of other autoantibodies. MATERIAL AND METHODS: Fourty one LN patients and 18 SLE patients without nephritis were studied. LN patients were subdivided into diffuse proliferative glomerulonephritis (DPGN), focal proliferative glomerulonephritis (FPGN), rapidly progressive glomerulonephritis (RPGN) and membranoproliferative glomerulonephritis (MPGN). Anti-neutrophil cytoplasmic antibodies (ANCA) were detected by indirect immunofluorescence and confocal laser scanning microscope using PMN and HL60 cells. ANCA specificities like anti-myeloperoxidase (anti-MPO), anti-proteinase 3 (anti-PR3), anti-lactoferrin (anti-LF) and anti-cathepsin G (anti-CG) were detected by ELISA. Other autoantibodies like anti-nuclear antibodies (ANA), anti-double stranded DNA (anti-dsDNA), anti-single stranded DNA(anti-ssDNA), anti-ribonucleoproteins (anti-nRNP), anti-Smith antibodies (anti-Sm) and rheumatoid factor (RF) were also tested. RESULTS: ANCA was detected in 37.3% patients. The predominant ANCA pattern was perinuclear (p-ANCA). ANCA positivity was higher in LN patients and when confirmed by ELISA, 54.5% ANCA positives had anti-myeloperoxidase (anti-MPO). The cytoplasmic ANCA (c-ANCA) pattern was not seen in any patient. Two patients having FPGN with crescents showed atypical 'X-ANCA' pattern with dual specificity to anti-MPO and anti-PR3 by ELISA. The titers of ANCA were more in LN as compared to SLE without nephritis. LN cases having DPGN, FPGN, RPGN with crescents had higher titer p-ANCA positivity with corresponding anti-MPO antibodies, along with ANA, anti-dsDNA, anti-ssDNA and anti-Sm + anti-nRNP and also high SLEDAI scores. CONCLUSION: ANCA in SLE may be used as a serological marker along with clinical and histopathological assessment to differentiate vasculitides in LN cases from SLE without nephritis.

Anti-myeloperoxidase antibodies in patients with microscopic polyangiitis.

BACKGROUND & OBJECTIVES: Anti-neutrophil cytoplasmic antibodies in active necrotizing and crescentic glomerulonephritis are associated with systemic vasculitides like Wegener's granulomatosis, Microscopic polyangitiis and Churg Strauss Syndrome. This study shows the incidence of ANCA with specificities to Myeloperoxidase and Proteinase3 in MPA cases and gives the correlation of ANCA with Birmingham Vasculitis Activity Score. MATERIAL & METHODS: Eighteen cases of MPA were diagnosed as per Chapel Hill Consensus Criteria. ANCA was detected by indirect immunofluorescence microscopy using fluorescence and Confocal Laser Scanning Microscopes. Anti-MPO and anti-PR3 were identified by commercial ELISAs and anti-MPO subclass and IgG isotypes were also detected. RESULTS: MPA patients showed a male preponderance with BVAS ranging from 17-30. Systemic involvement was seen in 88.9%, lower respiratory tract involvement in 77.8% and upper respiratory tract in only 33.3% cases. All these patients had perinuclear pattern on IIF, where titers ranged from 80-640 and ELISA showed anti-MPO; values ranging from 20-80 units/ml. IIF and ELISA showed a good correlation (r=0.77). Two patients having FPGN had dual specificities and had both anti-MPO and anti-PR3 which could be picked up only by ELISA. A good correlation (r=0.78) was observed between BVAS and ANCA levels as well. IgG ANCA was detected in 88.7% and 11.1% had IgG+IgM and IgG1+IgG4 ANCA was detected in 50% patients. CONCLUSION: p-ANCA with anti-MPO is highly specific for MPA; both IIF and ELISA should be carried out for true positivity and to identify rare cases of dual specificities. Confocal laser scanning microscopy is useful in identifying ANCA patterns especially when ANA is also positive. ANCA testing with BVAS assessment will surely help in early diagnosis and estimating the severity of this life threatening disease.

A study of anti-neutrophil cytoplasm antibodies in systemic vasculitis and other related disorders.

BACKGROUND: Anti-neutrophil cytoplasm antibodies (ANCA) play an important role as specific and sensitive markers for small vessel vasculitis and in some other systemic disorders. Indirect immunofluorescence test, known as the "Gold Standard" for screening of ANCA, can be further substantiated by ELISA for confirmation and for identifying sub-specificities like anti-Myeloperoxidase (anti-MPO), anti-Proteinase 3 (anti-PR3) and anti-Lactoferrin (anti-LF). AIMS: The present study was undertaken to investigate the incidence, specificities and strength of ANCA in suspected vasculitis cases and to correlate their presence with that of these auto-antibodies and with the disease. SUBJECTS AND METHODS: Sera from 130 clinically suspected vasculitis patients were studied. Indirect immunofluorescence microscopy (IIF) was used to identify cytoplasmic (c-ANCA), perinuclear (p-ANCA) and atypical (X-ANCA) patterns using ethanol and formalin fixed polymorphonuclear cells (PMN) and HL-60 cells from a human promyelocytic leukaemic cell line as substrates. ELISA was performed for identifying ANCA sub-specificities to anti-MPO and anti-PR3 and HEp-2 cells were used for detection of anti-nuclear antibodies (ANA). RESULTS: ANCA positivity was noted in 42.3% of these patients, wherein p-ANCA positivity rate was 34.6% and c-ANCA positivity was noted in 5.4% subjects. Three patients showed the unusual X-ANCA positivity. ELISA determined the sub-specificities: Out of 45 p-ANCA positive patients, 38 patients (84.4%) had anti-MPO and out of 7 c-ANCA positive patients, 5 patients (71.4%) had anti-PR3 antibodies. One patient with Class IV Lupus Nephritis, showed both anti-MPO and anti-PR3 antibodies and 17.8% p-ANCA positive patients had anti-Lactoferrin antibodies. CONCLUSIONS: Use of the Immunofluorescence method coupled with identification of ANCA sub-specificities by ELISA, is recommended for detection of ANCA in clinically suspected cases of small vessel and other systemic vasculitis.

Antibodies to Ro/SS-A and La/SS-B in systemic lupus erythematosus and other autoimmune disorders.

AIM: 1. To study the presence of anti-Ro/SS-A, anti-La/SS-B, anti-Sm and anti-nRNP in diagnosed antinuclear factor (ANF) positive systemic lupus erythematosus (SLE) cases and their association with various organ involvement. 2. To study autoantibodies in other autoimmune disorders. MATERIAL AND METHODS: A total of 4050 suspected cases of autoimmune disorders referred for serological work up were evaluated for ANF by indirect immunofluorescence technique, anti-dsDNA by PHA, autoantibodies to Ro-SS-A and La/SS-B by ELISA and rheumatoid factor was tested by latex agglutination using commercial kits. RESULTS: Out of 4050 patients 19.5% were ANF positive and 5% were anti-dsDNA positive. Out of these 50 diagnosed ANF positive cases of SLE, an incidence of anti-dsDNA 54%, anti-Sm 25.9%, anti-nRNP 29.6%, anti-Ro/SS-A 10% and anti-La/SS-B was 22% was observed. In rheumatoid arthritis, 17.4% positivity of anti-Ro/SS-A and 39.1% positivity for anti-La/SS-B was observed. In SLE with renal involvement, joint complaints and skin or malar rash were seen in 66%, 56% and 46%, respectively. CONCLUSION: Determining anti-Ro/SS-A and anti-La/SS-B antibody could be important in evaluating patients with suspected connective tissue disorders, who usually show diverse clinical presentations like skin, kidney and joint manifestations. The most prominent feature in anti-Ro/SS-A and anti-La/SS-B positive patients was skin involvement and sicca complex in 60% of SLE patients.

Autoantibodies, immunoglobulins, complement and circulating immune complexes in acute malaria.

BACKGROUND: Malaria caused by Plasmodium vivax and Plasmodium falciparum is common in the Indian subcontinent. Studies conducted elsewhere have suggested that malarial infection causes intense immunostimulation. We screened patients with malarial infection for autoantibodies and measured the immunoglobulin, circulating immune complex and complement levels to determine the extent of immunological alterations in these patients. METHODS: One hundred adults with acute malarial infection confirmed by examination of the peripheral blood smear and 25 age- and sex-matched controls were studied. An autoantibody screen and serum immunoglobulin complement (C3 and C4) and circulating immune complex levels were measured at the time of admission and 4 weeks after they became afebrile. A direct Coomb's test was also done. RESULTS: Anti-ssDNA, anti-dsDNA and rheumatoid factor were positive at the time of admission in 51, 30 and 38 patients respectively. None of the controls were positive for these autoantibodies except for one who was positive for rheumatoid factor. The IgM, IgG and IgA levels were raised in 16, 25 and 36 patients respectively. Circulating immune complex levels were raised in 32 patients and complement C3 and C4 were low in 8 and 31 patients. Follow up studies at 4 weeks in 19 patients showed that the autoantibodies were negative. However, the immunoglobulin, C4 and circulating immune complex levels remained elevated. Six per cent of patients had a positive direct Coomb's test with reticulocytosis at the time of presentation. CONCLUSION: Acute malarial infection can cause false-positive results for anti-ssDNA, anti-dsDNA and rheumatoid factor and may also cause a rise in the serum immunoglobulin, complement and circulating immune complex levels.

In-vitro inhibition of antiplatelet autoantibodies by intravenous immunoglobulins and Rh immunoglobulins.

Autoimmune thrombocytopenia (AITP) is caused by autoantibodies to platelet glycoprotein antigens. Intravenous immunoglobulin (i.v.IgG) and Rh immunoglobulin infusions have found great significance in the treatment of AITP patients not responding to corticosteroids and other modes of therapy. In our study, it was observed that immunoglobulins (i.v.IgG & Rh), and their Fab fragments inhibited the binding of antiplatelet autoantibodies to normal platelets, from 15.8 to 90.7% and 25.6 to 90.08% respectively; whereas, their Fc portion did not show any inhibition. The presence of specific anti-idiotypic antibodies to antiplatelet autoantibodies was established by using monoclonal antibodies to Glycoprotein IIb/IIa and Glycoprotein Ib/IX, as the specific idiotype source. The i.v.IgG and Rh immunoglobulin products reacted with the monoclonal antibodies, only through their Fab and not through the Fc portions, thereby confirming its specific anti-idiotype activity.

Anti-idiotype antibodies and circulating immune complexes after immunisation with tetanus-toxoid.

OBJECTIVES: To determine if anti-idiotype antibodies and circulating immune complexes in individuals before and after immunisation with tetanus toxoid play a role in the immune response. DESIGN: A study of individuals who were administered a single dose of tetanus toxoid (TT) and who were unimmunized. SETTING: Out patient departments of a large public hospital in Bombay, India. SUBJECTS: Thirty eight individuals pre-immunisation and forty five individuals post-immunisation with tetanus toxoid, tested at 1, 3, 6, and 12 months. MAIN OUTCOME MEASURE: Development of anti-tetanus anti-idiotype antibodies and circulating immune complexes. RESULTS: Pre-immunisation cases did show presence of anti-tetanus antibodies but in lower titres than post-immunisation up to six months, after which there was a reduction. Specific anti-idiotype antibodies were detected in 19 cases. One and three months after immunisation more cases had high titre antibodies and circulating immune complexes, though after six months, there was a fall in anti-tetanus antibody titres. Circulating immune complexes were seen in those samples having anti-idiotype antibodies. CONCLUSIONS: Though a significant rise in anti-tetanus antibody anti-idiotype antibodies, protective levels in mice and circulating immune complexes are seen after immunisation with TT it lasts for six months. When followed up for a period of one year it is observed that in cases having auto anti-idiotype antibodies, the anti-tetanus antibodies are maintained for a longer period.

Auto antibodies to nucleosides in systemic lupus erythematosus.

Serological studies in 110 patients with systemic lupus erythematosus (SLE) have shown that autoantibodies to DNA and RNA had subspecificity to adenosine (30.9%), cytidine (79%), guanosine (44.5%), thymidine (20%) and uracil (56.3%). It was also observed that DNA antibodies are heterogenous and that antibody with specificity for both the native confirmation as well as exposed nucleoside of the denatured molecule were present in sera of most of the patients with SLE. There was also alteration in the pattern of antibody to nucleoside in some patients who were treated with steroids or immunosuppressive drugs.

Systemic lupus erythematosus in Indian children.

Twenty cases of systemic lupus erythematosus (SLE) in prepubertal children (less than 14 years of age) were seen over a period of 14 years. The male:female ratio was 1:2.3, and the mean age at onset was 9.37 years. Fever with joint involvement was the commonest presenting manifestation (60%), followed by nephrotic syndrome (25%). Notable clinical features included a high incidence of renal involvement (75%), significant hypertension (45%) and reversibility of acute renal failure (2 cases). The other organs and systems involved included: mucocutaneous manifestations (60%), cardiovascular system (30%), respiratory system (25%), neuropsychiatric manifestations (45%), and anemia (75%). Raynaud's phenomenon and thrombocytopenia were rare while leucopenia was not seen in a single patient. Immunological abnormalities noted were 100% positivity for antinuclear antibodies, and 87.5 and 75% positivity for antibodies to double-stranded and single-stranded DNA, respectively. Hypocomplementemia was seen in 75% of patients tested.