As has been previously described (16), LUMINA is a longitudinal study of outcome of SLE patients from 3 ethnic groups (Hispanic, African American, and Caucasian) living in 3 distinct geographic areas of the US (Texas, Alabama, and Puerto Rico). The eligibility and enrollment of the patients, patients' evaluation and followup, and data collection have been previously described (16). Briefly, patients who meet the American College of Rheumatology (ACR) criteria for the classification of SLE (17), have a disease duration ≤5 years, are ≥16 years of age at the time of enrollment into the cohort, are of a defined ethnicity (all 4 grandparents of the same ethnicity as the patient), and live in the geographic catchment areas of the participating institutions are eligible to participate. The Institutional Review Board of each center approved the LUMINA study; written informed consent was obtained from each patient according to the Declaration of Helsinki.
Prior to enrollment, all medical records are reviewed. This is done to confirm the patient's eligibility and to gather information about the patient's socioeconomic/demographic and clinical features before disease onset and the enrollment visit. Every patient has a baseline visit (T0); followup visits are conducted every 6 months for the first year and yearly thereafter until the last visit (TL). Each LUMINA study visit consists of an interview, a physical examination, and laboratory tests. Data for missed study visits are obtained whenever possible by review of all available medical records.
As previously reported (18), the LUMINA database includes variables from the following domains: socioeconomic/demographic, clinical, immunologic, genetic, behavioral, and psychological. These variables are measured at T0 and at every subsequent visit except for genetic variables, which are obtained only at T0. Only the variables included in the present study are described here.
Variables from the socioeconomic/demographic domain include age, sex, ethnicity, education, poverty (as defined by the US Federal Government, adjusted for the number of subjects in the household) (19), health insurance, and marital status. Time variables are defined as follows: time of diagnosis of SLE (TD) as the time at which a patient meets 4 ACR criteria, disease duration at T0 as the interval between TD and T0, followup time as the time elapsing between T0 and TL, and total disease duration as the interval between TD and TL.
Hemolytic anemia secondary to lupus is defined, per the corresponding ACR criterion, as anemia with reticulocytosis (hemoglobin level <13 gm/dl in men or <11 gm/dl in women and corrected reticulocyte count >3%); patients were categorized as having severe hemolytic anemia if their hemoglobin level was ≤7 gm/dl and mild/moderate if >7 gm/dl. For the purpose of these analyses, the first episode of hemolytic anemia, which is usually the most severe, was the one categorized for those patients who had experienced >1 episode of hemolytic anemia.
Clinical variables included the number of ACR criteria at TD, disease manifestations, disease activity, damage accrual, laboratory tests, medications, and death. Cumulative organ system manifestations were recorded by categories as follows: neurologic involvement (seizures, psychosis, headaches, cerebrovascular accidents, neuropathies, transverse myelitis, myelopathy, and/or radiculopathy), renal involvement (nephritis by histopathology [World Health Organization class II–VI] and/or 2 and 3+ proteinuria), hematologic involvement (thrombocytopenia [<100,000/mm3], leucopenia [<4,000/mm3], and/or lymphopenia [<1,500/mm3]), cardiopulmonary involvement (pericarditis, endocarditis, cardiomyopathy, congestive heart failure, pneumonitis, pulmonary hemorrhage, pulmonary hypertension, and/or interstitial lung disease), arterial thrombosis (myocardial infarction, definite or classic angina, and/or vascular procedure for myocardial infarction [coronary artery bypass graft], stroke, intermittent claudication, and/or peripheral arterial thrombosis), and venous thrombosis (peripheral and/or visceral).
Disease activity was assessed using the revised Systemic Lupus Activity Measure (SLAM-R) (20) at all visits. A weighted average SLAM-R score was calculated, from TD to TL as a measure of disease activity over time, by multiplying the SLAM-R score at each individual visit by the number of months in the preceding interval; these scores were then summed and averaged over the total disease duration. This measure was chosen in order to better capture the undulant course of the disease. Because hemolytic anemia is one of the parameters included in the SLAM-R, it was excluded from the total SLAM-R score so that no activity could be attributed to it. Quantification of disease activity was also assessed at all study visits using the physician's and the patient's global assessments. To this end, 0–10-cm visual analog scales were used, where 0 represents completely inactive disease and 10 represents most disease activity possible. Damage was measured with the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI) (21) at T0 (or first computed for those patients with disease duration <6 months at T0) and TL.
Cumulative exposure to glucocorticoids (as prednisone equivalent), hydroxychloroquine, azathioprine, cyclophosphamide, methotrexate, aspirin, nonsteroidal antiinflammatory drugs, hormones (oral contraceptives and hormone replacement therapy), and warfarin were ascertained. The average dose of glucocorticoids was computed at each study visit, taking into account the dose taken every month; a weighted average from T0 to TL was then calculated by multiplying the average glucocorticoid dose for each individual visit by the number of months in the interval between visits and dividing it by the total followup time (T0 to TL).
The presence of autoantibodies (obtained at T0) included anti–double-stranded DNA (anti-dsDNA; by immunofluorescence against Crithidia luciliae [normal titer <1:10]), anti-Sm, anti-Ro, and anti-La (by counterimmunoelectrophoresis against human spleen and calf thymus extract) (22, 23), IgG and/or IgM antiphospholipid antibodies (aPL; abnormal value >13 IgG phospholipid units/ml and/or >13 IgM phospholipid units/ml, respectively) by enzyme-linked immunosorbent assay technique (24), and the lupus anticoagulant (StaClot test; Diagnostica Stago 92600, Asnières-Sur-Seine, France) (25).
In the genetic domain, FCGR functionally important promoter single-nucleotide polymorphisms (SNPs; FCGR2A-H131/R131 [rs1801274], FCGR2B-I131T [rs1050501], FCGR2B-120 [rs34701572], FCGR2B-386 [rs3219018], FCGR3A-F176V [rs396991], FCGR3B-NA1/NA2, and FCRH3-169 [rs7528684]) and select promoter SNPs in Fas and FasL (Fas-670 [rs1800682], Fas-690 [rs2234768], FasL-205 [no rs number currently assigned], and FasL-844 [rs763110]) were included. Genomic DNA was extracted using the PureGene kit (Gentra Systems, Minneapolis, MN) following the manufacturer's recommendations. FCGR and Fas/FasL SNPs were genotyped using Pyrosequencing (Biotage, Charlottesville, VA) as previously described (26, 27). Primer sequences are available from the authors.
Self-reported physical and mental functioning were assessed using the Medical Outcomes Study Short Form 36 (SF-36) (28), a 36-item questionnaire with 8 scales that can be aggregated into physical and mental component summary measures. Higher scores on the SF-36 indicate better functioning.
Psychosocial and behavioral domain variables include social support (ascertained with the Interpersonal Support Evaluation List, where higher scores indicate better social support) (29), learned helplessness (assessed with the Rheumatology Attitudes Index) (30, 31), and abnormal illness-related behaviors (ascertained with the Illness Behavior Questionnaire , where higher scores indicate either greater degree of helplessness or of abnormal illness-related behaviors).
Allele and genotype frequencies from FCGR, Fas, and FasL were calculated for all patients and for those with and without hemolytic anemia. Each marker was tested for departure from Hardy-Weinberg equilibrium expectations using a chi-square goodness-of-fit test. To identify any association between the individual polymorphism and hemolytic anemia status, we performed a 2 degrees of freedom (3 genotypes) test of association, tests of the dominant, additive, and recessive genetic models (Cochran-Armitage trend tests), and the corresponding test for lack of fit to additivity. Ethnic-specific analyses were computed with careful attention to potential population substructure.
To compare features from the nongenetic domains between patients with none, mild/moderate, and severe hemolytic anemia (3 groups), standard statistical tests, a one-way analysis of variance for continuous variables, and the Mantel-Haenszel chi-square test for categorical variables were used. Variables with a P value less than or equal to 0.10 in the univariable analyses were included in multivariable analyses of association; age, sex, and ethnicity were entered into this model regardless of their level of significance in the univariable analyses. A proportional odds model was used to evaluate factors potentially associated with degrees of hemolytic anemia. The proportional odds model assumes that the odds ratio (OR) associated with severe hemolytic anemia versus none or mild/moderate is the same as the OR associated with severe and mild/moderate hemolytic anemia versus none. To assess the validity of the underlying assumption, score tests were used.
The role of hemolytic anemia in damage accrual, as measured by the SDI at TL, was examined by a multivariable linear regression analysis, adjusting for those variables previously found to be associated with damage accrual (age, sex, ethnicity, poverty, health insurance, total disease duration, disease activity over time, damage first computed, and glucocorticoid weighted average) (33). The contribution of hemolytic anemia to mortality was examined by multivariable Cox proportional hazards regression model, also adjusting for variables previously found to be associated with mortality (age, sex, ethnicity, poverty, disease activity, and damage at T0) (34). Statistical significance in all cases was defined as a P value less than or equal to 0.05. Statistical analyses were performed using the SPSS program, version 15.0 (SPSS, Chicago, IL) or SAS, version 9.1 (SAS Institute, Cary, NC); statistical analyses of the genotype data were done with SNPGWA software (Research IT Services, Winston-Salem, NC).