Antiphospholipid Antibodies in Pediatric Lupus Nephritis Susan F. Massengiil, MD, Claire Hedrick, Elia M. Ayoub, MD, John W. Sleasman, MD, and Kuo Jang Kao, MD 0 Antiphospholipid antibodies (aPL) of various isotypes are known to occur in systemic lupus erythematosus (SLE), but the significance of this finding in the pediatric population remains unclear. Our aim was to determine whether children with lupus nephritis have an increased risk of thrombosis and whether antiphosphatidylserine (APS) or antiphosphaticlylinositol (API) antibodies were predictive of thrombotic complications. Thirty-six children (27 girls/Q boys; 44% black) with SLE nephritis (WHO II, 1; WHO Ill, 7; WHO IV, 21; WHO V, 7) were evaluated for antiphosphatidylserine, antiphosphatidylinositol, and anticardiolipin immunoglobulin (Ig) G and IgM isotypes, using a modified solid-phase enzyme-linked immunoassay (ELISA). Twenty-four patients (67%) had at least one positive aPL. Longitudinal data on 26 patients showed fluctuations in the degree of positivity. Eight patients experienced thrombotic complications, with equal distribution between arterial and venous events. Other clinical manifestations included thrombocytopenia in seven patients (lQ%), hemolytic anemia (44%), lupus anticoagulant (6%) and falsepositive Venereal Disease Research Laboratory (VDRL) test results (11%). Comparisons between those with and without a thrombotic event showed no detectable difference in the incidence of aPL positivii between the two groups. We conclude that neither APS, API, nor anticardiolipin (ACL) activity was predictive of thrombotic complications in our subset of patients with lupus nephritis. 0 1997 by the National Kidney Foundation, Inc. INDEX WORDS: Systemic lupus erythematosus; kidney; pediatrics; A NTIPHOSPHOLIPID antibody syndrome is a thrombophilic disorder defined as the association of antiphospholipid antibodies (aPL) with both arterial or venous thrombosis, thrombocytopenia, and recurrent fetal 10~s.“~Antiphospholipids are a heterogeneous group of autoantibodies directed against anionic phospholipids, namely, cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.4,5 p,-glycoprotein I (P2-GPI), a phospholipid-binding plasma protein, has recently been identified as a cofactor in the recognition of phospholipid antigens by aPLs.6F8The p,-GPI-phospholipid complex is the likely antigenic target of the aPL autoantibodies. In addition, lupus anticoagulant (LAC), present in approximately 7% of systemic lupus erythematosus (SLE) patients, is an aPL that prolongs phospholipid-dependent clotting assays and is more commonly associated with venous thrombosis.‘-” Antiphospholipid antibodies of various isotypes are associated with SLE and other rheumatic disorders and autoimmune diseases, but the significance of these aPLs with respect to clinical manifestations remains to be determined. Although common in pediatric SLE, the reported prevalence of aPLs vary from 38% to 87%.‘*-14 Some investigators have found that the degree of aPL positivity correlates with disease activbut this has not been a uniform finding. ity, 12~13~‘5 In patients with SLE, renal manifestations vary greatly and span the entire spectrum of glomeruAmerican Journal of Kidney Diseases, Vol 29, No 3 (March), antiphospholipid antibodies; thrombosis. lar, tubular, interstitial, and vascular diseases. Despite early and aggressive immunosuppressive therapy, progression to renal insufficiency is not uncommon, particularly in the face of diffuse proliferative glomerulonephritis. Although considerable interest has been recently generated concerning the prevalence of aPLs in the SLE population, few studies have focused on those patients with nephritis. Our objective was to prospectively determine whether patients with lupus nephritis have an increased incidence of thrombosis. We further sought to evaluate whether antiphosphatidylserine (APS) or antiphosphatidylinositol (API) antibody testing had additional predictive value for thrombotic complications. MATERIALS AND METHODS Study Population Thirty-six children (27 girls, 9 boys; mean age, 13.2 years) who met the criteria of the American Rheumatism Associa- From the Division of Nephrology, Department of Pediatrics: Decision Support Services, Shands Hospital: Division of Immunology and Infectious Diseases, Department of Pediatrics: and the Department of Pathology, Immunology, and Laboratory Medicine, Universiry of Florida, Gainesville, FL. Received August 20, 1996; accepted in revised form October 29, 1996. Address reprint requests to Susan F. Massengill, MD, Department of Pediatrics, PO Box 100296, University of Florida College of Medicine, Gainesville, FL 32610-0296. 0 1997 by the National Kidney Foundation, Inc. 0272-6386/97/2903-0004$3.00/O 1997: pp 355-361 355 356 MASSENGILL tion (ARA) for the classification of SLEr6 and manifested nephritis, as defined by hematuria, proteimtria, urinary casts, and histopathological parameters, were prospectively followed from June 1, 1990 to April 1, 1996. Renal tissue obtained by percutaneous renal biopsy was processed for light microscopy, immunofluorescence, and electron microscopy by standard techniques” and classified according to World Health Organization (WHO) morphologic criteria.” Renal biopsy specimens were classified as mesangial glomemlonephritis (WHO class II), focal proliferative glomerulonephritis (WHO class III), diffuse proliferative glomerulonephritis (WHO class IV), or membranous glomerulonephritis (WHO class V). Clinical and laboratory manifestations as defined by the ARA were evaluated. In addition, history was elicited for common clinical manifestations of antiphospholipid syndrome, such as thrombotic events, neuropsychiatric symptoms, livedo reticularis, recurrent fetal loss, easy bruising, or bleeding tendencies. Antiphospholipid Antibodies Antiphospholipid antibodies were defined as APS immunoglobulin (Ig) G and IgM isotypes, API IgG and IgM isotypes, and anticardiolipin (ACL) IgG and IgM isotypes, using a modified solid-phase enzyme-linked immunoassay (ELISA). Lupus anticoagulant was assessed by performing activated partial thromboplastin time and tissue thromboplastin inhibition assay, and results were confirmed by a platelet neutralization procedure. Serologic tests for syphilis were performed by Venereal Disease Research Laboratory (VDRL) test. Anticardiolipin Assay Each well of polystyrene Nunc-Immune microtiter plates (USA/Scientific Plastics, Ocala, FL) was coated with 30 PL of a 50-&mL cardiolipin solution (Sigma Chemical Co, St Louis, MO) in ethanol. The ethanol was rapidly evaporated in a chemical hood for 15 to 30 minutes. The wells were washed three times with 200 PL phosphate-buffered saline (PBS), pH 7.2, and then blocked with 100 PL of 10% newborn calf serum (NCS) (Life Technologies, Gaithersburg, MD) in PBS (NCS-PBS) for 1 hour at room temperature. After blocking, the plates were washed three times with PBS, then 50-PL aliquots of standards (Louisville APL Diagnostics, Inc. Louisville, KY) diluted 1:50 in 10% NCS-PBS were added to each of duplicate wells, and incubated 90 minutes at room temperature. After three washes with PBS, 50 PL affinity-purified, alkaline phosphatase-conjugated goat antihuman IgG (Sigma Chemical), IgM (Sigma Chemical), or IgA (Dako Corp, Carpinteria, CA) diluted 1:lOOO in 10% NCS-PBS was added to each well and incubated for 1 hour at room temperature. After three washes, 100~PL aliquots of p-nitrophenyl phosphate substrate (Sigma Chemical) in 0.05 mol/L carbonate buffer, pH 9.8, were added to each well and incubated for 30 minutes at room temperature. The reaction was stopped by adding 50 PL of 3 mol/L sodium hydroxide to each well. Absorbance was read at 410 nm in a Vmax ELISA plate reader (Molecular Devices, Menlo Park, CA). Data were analyzed using Assay Zap software (Biosoft, Ferguson, MD). Antibody levels were reported as units in GPL, MPL, or APL for IgG, IgM, or IgA, respectively. One PL ET AL unit was defined as the binding activity of 1 &mL antibody. Pooled normal plasma and a positive serum sample were included as negative and positive controls, respectively, with each assay. High positive samples were defined as those containing greater than 80 PL units; moderate positive as those containing 21 to 80 PL units; low positive containing 10 to 20 PL units; and negative, less than 10 PL units. For the purpose of our study, only samples that contained moderate or high positive concentrations of ACL were considered positive. Antiphosphatidylserine Antiphosphatidylinositol (APS) and (API) Assay APS and API antibodies were assayed by ELISA following the same procedure as described for ACL antibody. Phosphatidylserine (Sigma Chemical) and phosphatidylinositol (Sigma Chemical) were diluted to a concentration of 50 pg/ mL in 4:l methanol/chloroform. Pooled normal plasma diluted 1:50 in 10% NCS-PBS and pooled positive plasma diluted 1:50 for IgM and l:l,OOO for IgG in 10% NCS-PBS were used as controls. Anti-IgA antibody was not assayed. Because no standards are available for APS or API, results were reported as the number of standard deviations (SD) above the mean of normal pooled control plasma. Strong positive was defined as those with relative OD values 6 SD or more above the mean; positive was 4 to 6 SD above the mean, weak positive was 2 to 4 SD above the mean, and negative samples were less than 2 SD above the mean. A positive test was defined as any APS or API activity, except in cases of isolated weak positive (< 2-4 SD) APS or API. In our experience (K.J.K.), isolated aPL IgG or IgM activity is inconsequential, and subsequent evaluations are typically negative. Statistical Analysis Dam were entered into a Dell 486 computer (Dell Computer Corporation, Austin, TX), and data analysis was performed using the SAS version 6 (fourth generation language statistical analysis programming tool) developed by SAS Institute, Inc (Cary, NC). Chi-square analysis was used to examine differences between the groups, with a Fisher’s exact test correction where appropriate. RESULTS Between June 1, 1990 and January 31, 1996, 36 children with SLE nephritis were prospectively evaluated for the presence of antiphospholipid antibodies, namely APS, API, ACL; as well as LAC and false-positive VDRL. Clinical and laboratory characteristics of the patients are shown in Tables 1 and 2. Eight patients (22%) had a history of thrombotic episodes (Tables 3 and 4). The distribution between arterial and venous events were similar. Heavy proteinuria (> 1,000 mg), a known risk factor for thrombosis, was present in three patients. Otherwise, other predisposing risk factors ANTIPHOSPHOLIPID ANTIBODIES IN SLE Table 1. Clinical Characteristics had end-stage renal 357 of Study Patients Total no. Age (yrs) at diagnosis of SLE Range Mean 2 SD Median Sex (F:M) Disease duration (yrs) Race/ethnicity (%) African American Caucasian Other Renal WHO histopathology (%) Mesangial proliferative glomerulonephritis (WHO II) Focal proliferative glomerulonephritis (WHO Ill) Diffuse proliferative glomerulonephritis (WHO Iv) Membranous glomerulonephritis (WHO Urinary protein excretion Normal (< 150 mg) Mild (< 500 mg) Moderate (500-l 000 mg) Severe (2 1000 mg) * One patient of thrombosis. NEPHRITIS in Eight Patients No. Patients 0.12-20 13.2 ” 4.4 14 27:9 5.3 2 3.6 16 (44) 16 (44) 4 (12) History of thrombotic event Initial thrombosis First event venous Deep vein thrombosis Pulmonary embolus Renal allografi First event arterial Stroke Chorea Recurrence* Recurrent fetal loss (%) 8 (22) 2 (6) 1 (3) 1 (3) 4 1 1 1 (11) (3) (3) (3) 1 (3) 7 (19) 21 (58) 7 (19) VJ 4 12 3 17 disease at the time Manifestations No. Positive/No. Thrombosis CNS Livedo reticularis Fetal loss Thrombocytopenia Hemolytic anemia Prolonged PlT False + VDRL Complications 36 for hypercoagulable conditions were not evident. One patient, with recurrent thrombotic events, including a pulmonary embolus and recurrent deep vein thrombosis, has since been anticoagulated and has a functioning renal allograft. Another patient experienced renal allograft loss from renal vein thrombosis within 7 days of transplantation. On anticoagulant therapy, she has successfully undergone retransplantation and has a functioning allograft. Central nervous system thrombotic events included cerebral vascular accidents in four patients and chorea in another. Table 2. Clinical and Laboratory Table 3. Thrombotic 8136 7136 0 l/36 7/36 Tested (22) (19) (3) (19) 8/l 8 (44) 2136 (6) 2/l 5 (13) (%) * One patient recurrent deep had a history of a pulmonary vein thrombosis. embolus and Other neuropsychiatric manifestations included one patient with auditory hallucinations and another with a transient ischemic event. No patients had evidence of livedo reticularis. Within this patient population, two patients each experienced one successful pregnancy. One of these women had a history of recurrent fetal loss, and after the addition of heparin, she delivered at 34 weeks’ gestation. No other patients reported a history of either fetal loss or pregnancy. Thrombocytopenia was present in seven patients (19%) but was never considered life-threatening. Coombs positive hemolytic anemia occurred in 8 of 18 patients (44%) examined. Two patients each had either LAC or a prolonged par- Table 4. Association of Antiphospholipid Antibodies No Thrombosis (n = 8) Positive aPLs n (%) IgG only IgM only IgG and IgM Prolonged PlT Lupus anticoagulant False + VDRL (n = 28) P 0.649 0.679 7 (88) 21 (75) 2 1 4 2 0 (25) (11) (44) (25) 7 (25) 3 (11) 11 (39) 0 3 (60) 2 (8) 1 (8) 1.0 1.0 0.054 1.0 0.260 NOTE. Positivity defined as nonisolated APS or API values 2 4 SD from the mean of pooled normal sera or ACL levels with moderate or high positive concentration (2 21 PL units) of cardiolipin antibodies. * Only 5 of 8 and 14 of 28 patients had a test for VDRL in the thrombosis and non thrombosis groups, respectively. MASSENGILL Table 5. Prevalence in Patients of Antiphospholipid With Lupus Nephritis First Determination* (No. Positive/No. Tested) (%) Negative aPL Any positive aPL APS only API only APS + API APS and/or API ACL only APS + ACL APS + API + ACL Lupus anticoagulant Prolonged PlT False positive VDRL 12136 24136 3134 If34 3/34 7/34 1136 2734 15/34 2/34 2133 2l19 (33) (67) (9) (3) (9) (21) (3) (6) (44) (6) (7) (11) Antibodies Serial Determinationst (No. Positive/No. Tested) (%) 7126 21126 2126 l/26 5/26 8/26 l/28 l/26 17126 (27) (81) (8) (4) (19) (31) (4) (4) (65) NOTE. Positivity defined as nonisolated APS or API values z 4 SD from the mean of pooled normal sera or ACL levels with moderate or high positive concentration (2 21 PL units) of cardiolipin antibodies. Two patients were evaluated for ACL only. T Twelve (33%) patients evaluated on two occasions; 6 (17%) patients on three occasions; and 8 (22%) patients on 2 4 occasions. l tial thromboplastin time (PIT). Among 19 patients for whom data was available, 2 patients (11%) had false-positive VDRL results. As seen in Table 5, the incidence of at least one positive aPL within our population was 67% (24 of 36 patients). Positivity was defined as positive aPL (APS, API, or ACL) with the exclusion of those patients with isolated weak positive (< 4 SD) APS or API activity. Antiphospholipid antibody status was evaluated at disease onset in 20 patients (56%), at greater than 1 year after diagnosis in 14 patients (39%), and after progression to end-stage renal disease in two patients (5%). The distribution of the individual aPLs with respect to the number of positive values within each category is shown in Table 5. Seven (21%) patients had only APS or API antibody activity, and in this group, two patients experienced a thrombotic event. In comparison, ACL activity alone was seen in only 1 of the 36 patients, and she had experienced a deep vein thrombosis. Among those 18 children with newonset nephritis, 13 (73%) patients had positive aPLs of moderate to high activity. Serial determinations of APS, API, and ACL ET AL were available in 26 patients (Table 5) and suggest that sequential measurements are more likely to identify those patients with aPL activity, especially with respect to APS and API antibody activity. By evaluating longitudinal data, fluctuations in the degree of positivity were evident (data not shown). In 13 patients who had previously demonstrated markedly elevated aPL activity, longitudinal follow-up showed either lessening positivity or negative studies. Nine children in this group were receiving pulse cyclophosphamide therapy. In another six patients, fluctuation in aPL positivity was not evident. In these patients, the patterns of positive individual aPLs and respective isotypes were sustained throughout the study period. Seven patients showed increasing aPL activity; two of these patients had a thrombotic event. Six of the patients with negative aPLs on initial evaluation continued to have no detectable phospholipid antibodies. When evaluating aPL activity in those patients receiving pulse cyclophosphamide therapy, serial determinations were available in 16 of 17 patients. Within this group, four patients showed no fluctuation in aPL activity, nine patients had lessening aPL activity, and three patients had worsening aPL activity. Comparison between those patients with and without thrombosis is summarized in Table 4. There was no detectable difference in the incidence of aPL positivity between the two groups. An association between thrombosis and a predominant aPL isotype could not be identified, although IgG isotypes were more common in both groups. Prolonged P’TT, LAC, or false-positive VDRL was not associated with an increased risk of thrombosis. DISCUSSION The increased frequency of antiphospholipid antibodies in SLE patients and the known association with venous or arterial thromboses is of considerable importance when dealing with childhood SLE. The impact of thrombosis, particularly when involving the central nervous system, has far reaching consequences. Although most previous studies evaluating the prevalence of antiphospholipid antibodies in SLE patients with and without nephritis have primarily focused on cardiolipin antibodies, lupus anticoagu- ANTIPHOSPHOLIPID ANTIBODIES IN SLE NEPHRITIS lant, and VDRL, our study expands the evaluation of aPLs to include antibodies to phosphatidylserine and phosphatidylinositol in a subset of children with SLE nephritis. The reported prevalence of aPLs in pediatric SLE varies greatly and is likely affected by the disease activity, organ system involvement, and the specific antiphospholipid antibodies evaluated. Molta et alI4 reported that 38% of pediatric patients had at least one aPL (ACL, LAC, VDRL), but with the exception of LAC these determinations were made during periods of clinical remission. Among those children with positive aPLs, three (21%) experienced thrombotic episodes. In a study by Ravelli et a1,15the prevalence of positive ACL was 87%, and thrombosis was seen in only one patient at a time of disease exacerbation. More recently, Seaman et all3 found that 29% of pediatric SLE patients had evidence of either ACL, LAC, or false-positive VDRL, and thrombotic events were seen in 24%. A common theme within each of these studies is that disease manifestations of the patients encompassed multiple organ systems and were of variable degrees of severity. By comparison, our study was limited to only those children with biopsy-proven lupus nephritis. By evaluating simultaneous APS, API, and ACL in addition to LAC, 24 of our 36 patients (67%) showed at least one detectable aPL. In our analysis, we have excluded isolated low positive levels of APS or API as being significant and recognize that this bias may have led to an underestimation in the prevalence of aPLs in those with lupus nephritis. However, our experience has deemed that such activity is insignificant and infrequently reproducible (data not shown). The only other study to examine antiphospholipid antibodies, specifically ACL, in a group of patients with lupus nephritis was conducted by Frampton et a1.19In this study of 76 adult lupus patients with nephritis, 43% (33 patients) had elevated levels of at least one ACL isotype (IgG, IgM, or IgA) when compared with normal controls with an average of 4%. Both Frampton et all9 and Gleuck et al” have reported an association between the presence of intraglomerular capillary thrombi and aPLs and speculate that aPLs may contribute in some way to the pathogenesis of nephritis. Unlike these two studies, an association between intraglomerular capillary thrombi and aPLs (IgG ACL or LAC) could not be identified on review of histological dam within our study population. The low frequency of LAC in our study population is unexplained. Unlike the data in adults, in which the reported prevalence of LAC in SLE is between 35% and 80%, we found only two patients (5.6%) with positive LAC. These two patients have not experienced either thrombosis or obstetric complications. Frampton et all9 found that 80% of the 32 patients with elevated IgG ACL and lupus nephritis had positive lupus anticoagulant. The high frequency of LAC observed in this population may reflect the use of activated partial thromboplastin time (aPIT) as a screening test, which has been shown by Petri et al” to be an unreliable marker for LAC. In our hands (K.J.K., unpublished data), thromboplastin inhibition assay is as sensitive as the diluted Russell viper venom time, which has historically been regarded as more sensitive and specific for LAC.” It is not known whether the use of the less sensitive tissue thromboplastin contributed to the infrequency of LAC in our patients. The clinical relevance of IgG or IgM aPL isotypes in the pathogenesis of thrombosis remains to be established. In general, high-titer IgG antibody, particularly ACL, seems to best correlate with future thrombotic events, whereas IgM isotypes are a relatively nonspecific finding in both immune and non-immune-related conditions.2’*22 In our study, an association between individual aPLs or isotypes with thrombotic complications was not found. In fact, two patients with a thrombotic complication had only aPL IgM isotypes. Similar to findings by Alarcon-Segovia et a1,23 our study shows that immunosuppressive therapy during acute disease activity may be effective in lowering aPL titers and hence lessen the risks of thrombosis. However, not all patients with improved serological parameters showed improvement in aPL positivity. Longitudinal measurements of patients with variable degrees of disease severity and immunosuppressive therapy will assist in better defining any association between aPLs activity and disease-related manifestations. Although APS and API activity did not seem predictive of thrombotic complications in our patients, APS and API activity was more commonly present in patients with thrombotic complications than was ACL. Had patients only been evaluated 360 for ACL, seven (21%) patients would not have been identified as having aPLs. Among this group, two patients experienced thrombotic complications and would not have been appropriately anticoagulated had APL testing comprised solely ACL, LAC, or VDRL. In only one instance did a patient with thrombosis have moderate ACL activity alone without identifiable APS or API activity. Lack of prospective, randomized, controlled treatment trials has led to considerable debate on the appropriate management of patients with aPLs. However, several studies have served to clarify several issues in therapeutic management. First, there are no data to support the use of prophylactic therapy in SLE patients with detectable APLs without a history of associated thrombotic event(s). Second, for those patients with aPL-associated thrombotic events, long-term oral anticoagulation, probably lifelong, remains the cornerstone of therapy. Khamashta et alz4 found that the risk of recurrent thrombosis is greatest within the first 6 months of cessation of anticoagulant therapy. International normalized ratio levels of 3.0 or higher appear to afford the greatest protection against recurrence.24’25 Lastly, but perhaps more importantly, there is no evidence to support the benefit of immunosuppressive agents in the management of aPLs, and these agents should be reserved for clinically evident vasculitis or other disease manifestations warranting such therapy. The unreliability of predictive values of aPLs for thrombotic complications, as observed in our study, further supports the aforementioned recommendations. The pathological role of these antiphospholipid antibodies, either individually or incombination, needs to be further defined. Investigation as to what other as yet unknown susceptibility factors (ie, disease exacerbation) predispose to thrombosis remains to be identified. Similarly, analysis of longitudinal aPL activity will provide further information as to the clinical relevance of these aPLs with disease manifestations. REFERENCES 1. 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