Systemic Lupus Erythematosus (SLE) is a morbid autoimmune disease with multisystem organ involvement [
We performed a retrospective case-control serum bank study comparing MPO-ANCA and PR3-ANCA levels years prior to PLN diagnosis to matched healthy and SLE without LN disease controls. We previously reported preclinical dsDNAab and CRP levels for these same patients and serum samples.
As described previously, we identified 23 cases of biopsy proven PLN (WHO class III or WHO class IV) from the Walter Reed Army Medical Center renal biopsy database from 1993 to 2009. A comprehensive electronic data base review was performed for each PLN case to populate a clinical background data collection sheet. None of the cases had medications in their record that were associated with drug-induced lupus.
The Department of Defense Serum Repository (DoDSR) identified 23 age, sex, race, and age of serum sample matched healthy and 21 SLE without LN disease controls [
The DoDSR then pulled the oldest, the second most recent, and the most recent 0.5 mL serum samples prior to PLN or SLE without LN diagnosis and sent them to Quest Diagnostics Nichols Institute (Chantilly, VA).
Quantitative measurement of MPO-ANCA antibody serum concentration was performed using the Varelisa™ MPO-ANCA EIA kit (Phadia GmbH, Freiburg, Germany). Serum aliquots from the subjects were diluted 1 : 101 with sample diluent and run in duplicate. Microwells were precoated with purified human MPO. 100 microliters each of calibrators (0, 3, 7, 16, 40, and 100 U/ml), controls, and diluted subject serum samples were dispensed into the wells, and the assay was performed as described in the instructions for use. The secondary antibody was a horseradish peroxidase-labeled anti-human IgG conjugate, and detection was achieved using TMB stopped with phosphoric acid solution. Absorbance was determined at 450 nm. The results are reported in U/ml, with a measuring range of 1.0 to 100 U/ml, and a detection limit of 1.0 U/ml. A clinically negative result was indicated by <6.0 U/ml. Intra-assay variability was 4.8 to 9.5 CV, and interassay variability was 3.5 to 10.7 CV over the range of the standards. The frequency distribution in 432 healthy subjects was 1.5 U/ml (95th percentile, 3.4 U/ml).
Quantitative measurement of PR3-ANCA antibody serum concentration was performed using the Varelisa™ PR3 ANCA EIA kit (Phadia GmbH, Freiburg, Germany). Serum aliquots from the subjects were diluted 1 : 101 with sample diluent and run in duplicate. The microwells were precoated with purified human neutrophil PR3. 100 microliters each of calibrators (0, 3, 7, 16, 40, and 100 U/ml), controls, and diluted subject serum samples were dispensed into the wells, and the assay was performed as described in the instructions for use. The secondary antibody was a horseradish peroxidase-labeled anti-human IgG conjugate, and detection was achieved using TMB stopped with phosphoric acid solution. Absorbance was determined at 450 nm. The results are reported in U/ml, with a measuring range of 0.5 to 100 U/ml and a detection limit of 0.5 U/ml. A clinically negative result was indicated by <6.0 U/ml. Intra-assay variability was 4.8 to 5.9% CV, and interassay variability was 2.4 to 9.3% CV over the range of the standards. The frequency distribution in 432 healthy subjects was 0.7 U/ml (95th percentile, 1.2 U/ml). For all three assays, the results were rounded to and reported in whole numbers.
The percent of PLN subjects with MPO-ANCA above selected threshold values prior to diagnosis was compared to healthy and SLE without LN disease controls using the Fisher exact probability test. Odds ratios and 95% confidence intervals were also calculated. The same statistical analysis was used for all secondary outcomes and subgroup analysis. Conditional logistic regression and ROC curves were performed using STATA 12.0. Absolute MPO-ANCA change per year was calculated by dividing the difference between last MPO-ANCA (MPO-ANCA
The DoDSR was not able to assign a matching disease control for one case. Serum samples for a second disease control were accidently destroyed at Quest leaving two less disease control subjects for analysis of the entire time period prior to diagnosis. Not all subjects had samples available for each subgroup time period. If multiple serum samples were present for a subject in a specific subgroup analysis time period, the highest antibody level dictated group assignment. Only 20 study subjects had multiple serum samples for evaluation of change in MPO-ANCA levels over time. Antecedent rise in MPO-ANCA versus dsDNAab or CRP was established only for patients with a clear initial biomarker elevation. If both became elevated in the same sample or if neither was elevated in any sample prior to diagnosis, there was no antecedent elevation determined.
This study was approved by the Human Use Committee at Walter Reed National Military Medical Center and informed consent was waived.
As previously reported, this study population consisted of predominantly African American females less than 40 years old. Joint, hematologic, and dermatologic involvement was most common (Table
Proliferative lupus nephritis (PLN) background information on based on electronic medical record chart review. Medians presented with 25% and 75% values in parentheses for continuous data because they were not normally distributed. Some percentages are accompanied by
Average age (range) | 36 years old (18–60) |
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|
Race | |
Caucasian | 15% |
African American | 60% |
Other | 25% |
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|
Gender | |
Female | 65% |
|
|
History of HTN | 83 (19/23) |
|
|
History of DM | 0 (0/23) |
|
|
Arthralgia | 70 (16/23) |
|
|
Dermatologic | 48 (11/23) |
|
|
Hematologic | 53 (12/23) |
|
|
Cardiac involvement | 35 (8/23) |
|
|
CNS involvement | 9 (2/23) |
|
|
Lung involvement | 35 (8/23) |
|
|
Liver involvement | 9 (2/23) |
|
|
Hematuria (>3 RBC phf) | 100 (23/23) |
|
|
Proteinuria (>300 mg) | 100 (23/23) |
|
|
Nephrotic range proteinuria (>3.5 gm) | 35 (8/23) |
|
|
Proteinuria Quantification (average in gm) | 2.36 (0.380–4.61) |
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|
Serum Creatinine (average mg/dl) | 1.13 (0.6–2.4) |
|
|
ANA (% positive) | 96 (22/23) |
|
|
dsDNA antibody (% positive) | 82 (18/22) |
Average (titer) | 1 : 320 |
Average (U/mL peak; |
189 (130–228) |
|
|
dsDNA antibody OR ANA (% positive) | 100 (23/23) |
|
|
ANCA (% positive) | 0 (0/3) |
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|
Anti-phospholipid antibody (% positive) | 38 (8/23) |
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|
Anti-SM antibody (% positive) | 38 (6/16) |
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|
Anti-RNP antibody (% positive) | 50 (8/16) |
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|
CRP | |
(Average mg/dL) | 2.1 (0.1–6.6) |
(% >0.8 mg/dL) | 79 (11/14) |
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|
Biopsy (%) | |
WHO Class III | 22 (5/23) |
WHO Class IV | 78 (18/23) |
WHO Class IV + V | 35 (8/23) |
Crescent Formation | 23 (6/23) |
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|
Median NIH activity index | 8 |
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|
Median NIH chronicity index | 3 |
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|
Median SLE disease activity index | 16 |
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|
Immunosuppression before biopsy (%) | |
Prednisone | 22 (5/23) |
Hydroxychloroquine | 39 (9/23) |
Other (cyclophosphamide, mycophenolate mofetil, rituximab) | 9 (2/23) |
Systemic lupus erythematosus (SLE) without lupus nephritis (LN) disease control background information on based on ICD-9 codes provided by the Department of Defense Serum Repository (DoDSR). Laboratory data were not available for these disease control patients.
Average age (range) | 36 years old (18–60) |
|
|
Race | |
Caucasian | 15% |
African American | 60% |
Other | 25% |
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|
Gender | |
Female | 65% |
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|
History of HTN | 36% |
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|
History of DM | 9% |
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|
Renal involvement | 0% |
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|
Arthralgia | 73% |
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Dermatologic | 46% |
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|
Hematologic | 41% |
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|
Anti-phospholipid positive | 23% |
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|
Cardiac involvement | 9% |
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|
CNS involvement | 9% |
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|
Lung involvement | 18% |
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|
Liver involvement | 5% |
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|
Median SLE disease activity index | 10 |
A greater percentage of PLN cases had an elevated MPO-ANCA level (≥6 U/mL) compared to both matched healthy and SLE without LN disease controls at any time prior to diagnosis (57% versus 0%,
The percent PLN patients with MPO-ANCA above specific thresholds compared to matching healthy and SLE without LN disease controls. The Department of Defense Serum Repository could not assign a matching disease control for one patient. The samples for a second control were lost in processing. Not all patients had samples available for each subgroup time period. If multiple serum samples were present for a patient in a specific subgroup analysis time period, the highest antibody level dictated group assignment.
MPO-ANCA | Cases |
Healthy controls |
OR |
CI |
|
---|---|---|---|---|---|
(≥3 U/mL): | |||||
All | 91 (21/23) | 26 (6/23) | 30 | 5.3–167 | <0.001 |
<1 year | 88 (15/17) | 19 (3/16) | 33 | 4.7–226 | <0.001 |
1–4 years | 87 (13/15) | 13 (2/15) | 42 | 5.2–347 | <0.001 |
>4 years | 69 (11/16) | 25 (4/16) | 6.6 | 1.4–31 | 0.03 |
(≥6 U/mL) | |||||
All | 57 (13/23) | 0 (0/23) |
|
|
<0.001 |
<1 year | 59 (10/17) | 0 (0/16) |
|
|
<0.001 |
1–4 years | 7 (1/15) | 0 (0/15) |
|
|
1.0 |
>4 years | 19 (3/16) | 0 (0/16) |
|
|
0.23 |
MPO-ANCA | Cases |
Disease controls |
OR |
CI |
|
---|---|---|---|---|---|
(≥3 U/mL): | |||||
All | 91 (21/23) | 43 (9/21) | 14 | 2.6–76 | <0.001 |
<1 year | 88 (15/17) | 39 (5/13) | 12 | 1.9–76 | 0.007 |
1–4 years | 87 (13/15) | 38 (6/16) | 11 | 1.8–66 | 0.009 |
>4 years | 69 (11/16) | 44 (7/16) | 2.8 | 0.7–12 | 0.29 |
(≥6 U/mL) | |||||
All | 57 (13/23) | 5 (1/21) | 26 | 3.0–228 | <0.001 |
<1 year | 59 (10/17) | 8 (1/13) | 17 | 1.8–164 | 0.006 |
1–4 years | 7 (1/15) | 6 (1/16) | 1.1 | 0.1–19 | 1.0 |
>4 years | 19 (3/16) | 0 (0/16) |
|
0.5– |
0.23 |
Conditional logistic regression (CLR) for PLN MPO-ANCA by unit versus healthy controls, SLE without LN disease controls, and all controls together.
CLR for MPO-ANCA by unit: PLN versus controls | OR |
CI |
|
---|---|---|---|
Healthy controls | 9.1 | 3.4–24.5 | <0.001 |
Disease controls | 1.5 | 1.2–1.8 | <0.001 |
All controls | 7.1 | 1.2–44.2 | <0.001 |
The MPO-ANCA ROC area under the curve was greater than 0.7 at less than 1 year, 1–4 years, and greater than 4 years for analysis of PLN cases with both healthy controls (Figure
MPO-ANCA receiver operating curves for PLN cases versus healthy controls for <1 year, 1–4 years, and >4 years before PLN diagnosis.
MPO-ANCA receiver operating curves for PLN cases versus SLE without LN disease controls for <1 year, 1–4 years, and >4 years before PLN diagnosis.
Neither the PLN cases, nor the disease and healthy controls had an elevated PR3-ANCA level in any serum sample. A greater percentage of PLN cases had a PR3-ANCA level ≥ 2 U/ml compared to both matched healthy and disease controls at any time prior to diagnosis (30% versus 4%,
In the PLN patients, MPO-ANCA became elevated a median of 139 days (25%, 75%; 63, 258 days) prior to diagnosis. MPO-ANCA was ≥3 U/mL a median of 5.75 years prior to diagnosis (25%, 75%; 1.3, 7.5 years) which is an underestimation because this level was present oldest index sample in 81% of the patients. PR3-ANCA ≥ 2 U/mL was only present in serum samples with a concurrent elevated MPO-ANCA.
A greater percentage of PLN cases had a rise in MPO-ANCA over time in comparison to both healthy and SLE without LN disease controls (70% versus 10%,
The percent of PLN patients with MPO-ANCA absolute change and rate of rise over time above specific thresholds compared to matching healthy and SLE without LN disease controls. Only 20 cases and healthy controls and 19 SLE without LN disease controls had multiple samples to evaluate change over time.
Change in MPO-ANCA |
Cases |
Healthy controls |
OR |
CI |
|
---|---|---|---|---|---|
>0 U/mL | 70 (14/20) | 10 (2/20) | 21 | 3.7–120 | <0.001 |
>0.3 U/mL | 60 (12/20) | 0 (0/20) |
|
3.6– |
<0.001 |
>0.5 U/mL | 45 (9/20) | 0 (0/20) |
|
2.1– |
0.001 |
>1 U/mL | 20 (4/20) | 0 (0/20) |
|
0.7– |
0.10 |
Absolute rise in MPO-ANCA |
Cases |
Healthy controls |
OR |
CI |
|
---|---|---|---|---|---|
>0 U/mL | 70 (14/20) | 10 (2/20) | 21 | 3.7–120 | <0.001 |
>1 U/mL | 55 (11/20) | 0 (0/20) |
|
3.0– |
<0.001 |
>2 U/mL | 35 (7/20) | 0 (0/20) |
|
1.4– |
0.008 |
>3 U/mL | 30 (6/20) | 0 (0/20) |
|
1.1– |
0.02 |
Change in MPO-ANCA |
Cases |
Disease controls |
OR |
CI |
|
---|---|---|---|---|---|
>0 U/mL | 70 (14/20) | 16 (3/19) | 12 | 2.6–59 | 0.001 |
>0.3 U/mL | 60 (12/20) | 5 (1/19) | 27 | 3.0–49 | <0.001 |
>0.5 U/mL | 45 (9/20) | 0 (0/19) |
|
2.2–179 | 0.001 |
>1 U/mL | 20 (4/20) | 0 (0/19) |
|
0.5–49 | 0.10 |
Absolute rise in MPO-ANCA |
Cases |
Disease controls |
OR |
CI |
|
---|---|---|---|---|---|
>0 U/mL | 70 (14/20) | 16 (3/19) | 12 | 2.6–59 | 0.001 |
>1 U/mL | 55 (11/20) | 11 (2/19) | 10 | 1.9–57 | 0.006 |
>2 U/mL | 35 (7/20) | 5 (1/19) | 10 | 1.1–89 | 0.04 |
>3 U/mL | 30 (6/20) | 0 (0/30) |
|
1.1– |
0.02 |
The lowest statistically significant subclinical MPO-ANCA level (≥3 U/mL), 50% of the threshold for clinical disease, was present prior to the lowest statistically significant subclinical dsDNAab level (≥3 U/mL), as well as the dsDNAab level that is 50% of the threshold for clinical disease (≥5 U/mL) when it was possible to ascertain an antecedent antibody (89% versus 11%,
Temporal relationship of detectable autoantibodies. Patients without a clear antecedent antibody above the designated threshold were not included in analysis. OR, odds ratio; CI, confidence interval.
Comparison thresholds | Antecedent MPO-ANCA |
Antecedent dsDNAab |
OR | CI |
|
---|---|---|---|---|---|
MPO-ANCA ≥ 3U/ml |
89 (8/9) | 11 (1/9) | 64 | 3.4–1211 | 0.003 |
|
|||||
MPO-ANCA ≥ 3 U/ml versus |
100 (10/10) | 0 (0/10) |
|
5.5– |
<0.001 |
There was no association between elevated dsDNAab and elevated MPO-ANCA (Supplemental Table
More PLN patients had an MPO-ANCA level ≥ 3 U/mL prior to an elevated CRP of >0.8 mg/dL (100% versus 0%,
In addition to SLE, MPO-ANCA has been described at diagnosis in subpopulations of multiple autoimmune and inflammatory diseases to include anti-GBM disease, IgA nephropathy, and inflammatory bowel disease [
Improved knowledge about subclinical MPO-ANCA trends could have prognostic implications. Overall, even low but significant MPO-ANCA may portend future PLN in SLE patients without LN. And up to 35% of SLE patients will manifest LN after initial diagnosis [
Improved knowledge about subclinical MPO-ANCA trends could also broaden our understanding of PLN pathophysiology. There is a strong correlation between ANCA and dsDNAab at diagnosis, but the subclinical relationship was previously unknown [
MPO-ANCA seropositivity has been previously attributed to cross reactive dsDNAab, but there is a body of evidence which suggests that this is not the only contribution [
MPO-ANCA stimulation of NETosis, the externalization of cellular chromatin and cytoplasmic protein containing fibers that capture pathogens or stimulate autoantibody production, is one intriguing hypothesis for MPO-ANCA directed pathogenicity. While NETosis has not been specifically evaluated previously, previous literature supports the hypothesis. MPO-ANCA triggers neutrophil NET deployment in vitro. There is increased NET formation in both MPO-ANCA vasculitis and SLE. NET burden can serve as a nidus for dsDNAab, ANA, C1q, and additional MPO-ANCA production to induce a deleterious positive feedback cycle. Both NET and autoantibody containing immune complexes can then cause end organ damage to include LN [
DoDSR study limitations inherent to the retrospective case-control design have been reported previously [
Follow-up studies are required to more fully describe the subclinical pathophysiology of PLN. Preclinical presence, trajectory, and temporal relationship of anti-Smith, anti-RNP, anti-DNAase, anti-C1q, anti-lactoferin, anti-Cathepsin G, anti-elastase, and anti-NET antibody levels need to be evaluated in a larger cohort of PLN. Additional mesangial and membranous LN disease control comparison groups will strengthen analysis. Mesangial LN is a particularly important comparison group because it can have a similar clinical presentation to early PLN. In addition, we need to better characterize the prediagnostic MPO-ANCA to include epitope specificity, Fc glycosylation patterns, and avidity [
MPO-ANCA may help delineate the SLE patients that are at risk for future PLN and may also directly contribute to the PLN pathogenesis. A more precise understanding of preclinical SLE pathogenesis may provide more specific future therapeutic targets for research.
The views expressed in this manuscript are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or the US Government.
The authors declare that they have no conflicts of interest.
Supplemental Table 1: a comparison of the percent of PLN cases with an elevated dsDNAab that have a concurrent elevated MPO-ANCA versus those that have a normal MPO-ANCA.