Blood pressure, salivary cortisol, and inflammatory cytokine outcomes in senior female cancer survivors enrolled in a tai chi chih randomized controlled trial.

PURPOSE: Older cancer survivors are a vulnerable population due to an increased risk for chronic diseases (e.g., cardiovascular disease) compounded with treatment late-effects and declines in physical functioning. Therefore, interventions that reduce chronic disease risk factors (i.e., blood pressure, chronic inflammation, and cortisol) are important in this population. Tai chi chih (TCC) is a mind-body exercise associated with reductions in chronic disease risk factors, but has not been examined with older cancer survivors. In a feasibility randomized controlled trial of TCC, we examined secondary outcomes of blood pressure, salivary cortisol, and inflammatory cytokines (interleukin (IL)-6, IL-12, tumor necrosis factor-α, IL-10, IL-4) due to their implications in chronic diseases. METHODS: Sixty-three senior female cancer survivors (M age = 67 years, SD = 7.15) with physical functioning limitations (SF-12 physical functioning ≤80 or role-physical ≤72) were randomized to 12-weeks (60-min, three times a week) of TCC or Health Education control (HEC) classes. Resting blood pressure, 1-day salivary cortisol samples, and fasting plasma samples for cytokine multiplex assays were collected at baseline and 1-week post-intervention. RESULTS: Controlling for baseline values, the TCC group had significantly lower systolic blood pressure (SBP, p = 0.002) and cortisol area-under-curve (AUC, p = 0.02) at post-intervention than the HEC group. There was no intervention effect on inflammatory cytokines (p's > 0.05). CONCLUSIONS: This TCC feasibility trial was associated with significant reductions in SBP and cortisol AUC in senior female cancer survivors. Larger, definitive trials are needed to confirm these findings. IMPLICATIONS FOR CANCER SURVIVORS: Senior survivors' have an increased risk for chronic diseases; however, TCC interventions may help reduce associated risk factors.

Prognostic factors and risk stratification in early mycosis fungoides.

Available demographic, clinical, histologic, immunohistochemical and laboratory findings, including serum cytokine/cytokine receptor levels, obtained at initial evaluation in a cohort of 33 patients with mycosis fungoides (MF) at stages I-IIA who had subsequent progression of disease were compared against 70 stage-matched cases of MF without observed progression. Significant factors that correlated with both disease progression and overall survival were: (1) presence of large Pautrier microabscesses (10 or more atypical lymphocytes), (2) presence of atypical lymphocytes with hyperchromatic or vesicular nuclei in the dermal infiltrate, (3) less than 20% CD8 + cells in the dermal infiltrate and (4) above normal (> 122 U/mL) serum immunoglobulin E (IgE) level. Combination of these factors was used to construct prognostic groupings which, if validated, might be useful to identify patients with clinically early MF at highest risk for disease progression and poor outcome.

Circulating pro-inflammatory cytokines are elevated and peak power output correlates with 25-hydroxyvitamin D in vitamin D insufficient adults.

The purpose of this study was to identify circulating cytokines, skeletal muscle strength, and peak power output in young adults with contrasting serum 25-hydroxyvitamin D (25(OH)D) concentrations. Serum 25(OH)D, inflammatory cytokines, muscle strength, and peak power output were, therefore, measured in young adults (25-42 years). Data were collected during the winter to avoid the seasonal influence on serum 25(OH)D. After serum 25(OH)D concentration measurements, subjects were separated into one of two groups: (1) vitamin D insufficient [serum 25(OH)D ≤32 ng/mL, n = 14], or (2) vitamin D sufficient [serum 25(OH)D >32 ng/mL, n = 14]. Following group allocation, serum 25(OH)D concentrations were significantly (p < 0.05) lower and pro-inflammatory cytokines [interleukin (IL)-2, IL-1ß, tumor necrosis factor-α, and interferon-γ] were significantly (all p < 0.05) greater in vitamin D insufficient adults. An anti-inflammatory cytokine (i.e., IL-10; p > 0.05), peak isometric forces (p > 0.05), and peak power outputs (p > 0.05) were not significantly different between vitamin D groups. However, peak power outputs correlated with serum 25(OH)D concentrations in vitamin D insufficient (r = 0.55, p < 0.05) but not in vitamin D sufficient adults (r = -0.27, p = 0.36). Based on these data, we conclude that vitamin D insufficiency, in part, could result in pro-inflammatory stress without altering muscular strength or function in young adults. Future research investigating the causality of the correlation between low-serum 25(OH)D and peak power output in young adults is required.

Significance of C-reactive protein in osteoarthritis and total knee arthroplasty outcomes.

BACKGROUND: The relationship between systemic inflammatory processes to total knee arthroplasty (TKA) outcomes remains unclear. This study investigates the relationship between serum high-sensitivity C-reactive protein (hs-CRP) and functional outcomes post-TKA. METHODS: A total of 31 patients with osteoarthritis (OA) who underwent TKA were enrolled in the study; 15 with hs-CRP ≤1.0 mg/l (low hs-CRP group) and 16 subjects with hs-CRP ≥4.0 mg/l (high hs-CRP group). During surgery, synovium and bone sections were sequestered, formalin-fixed, and paraffin embedded for slide preparation. Tissue sections were stained with hematoxylin and eosin and analyzed using a light microscope. A total of 12 cytokines were measured in synovial fluid samples from the knee joint at time of surgery and analyzed using the Luminex Multi-Analyte Profiling System. Relationships between cytokines and hs-CRP were assessed using Spearman correlation coefficients. Student's t-tests were used to compare Short Form health outcomes survey (SF-12) health outcomes between high and low hs-CRP, and presurgical and postsurgical visits. RESULTS: Mean ± standard deviation (SD) baseline and 1-year hs-CRP values for the low hs-CRP group were 0.55 ± 0.23 mg/l and 1.22 ± 1.32 mg/l, respectively (n = 15; p = 0.051) and for the high hs-CRP group were 7.86 ± 5.98 mg/l and 14.11 ± 38.9 mg/l, respectively (n = 13; p = 0.54). Lymphocytes were present in 10 synovium and one bone sample (all but one from high hs-CRP group). Interleukin (IL)-5 and IL-10 were significantly correlated with hs-CRP (p = 0.0137 and p = 0.0029, respectively). The low hs-CRP group exhibited significant improvement in the physical component of SF-12 at 6 and 12 months compared with baseline, whereas the high hs-CRP group exhibited significant improvement only at 6 months. Body mass index (BMI) had a significant positive correlation with presurgical hs-CRP. CONCLUSIONS: The results of this study provide support for inflammatory mechanisms contributing to the OA progression, with hs-CRP being a possible predictive variable, combined with BMI and other comorbidities, of post-TKA function.

Circulating interferon-γ correlates with 1,25(OH)D and the 1,25(OH)D-to-25(OH)D ratio.

The mechanism responsible for the decrease in vitamin D status (i.e., plasma or serum 25-hydroxyvitamin D (25(OH)D) concentration) during inflammatory stress is unknown in humans. Interferon (IFN)-γ is an inflammatory cytokine that regulates vitamin D metabolism in isolated immune cells, but data suggesting this regulation exists in vivo is lacking. The purpose of this study, therefore, was to associate circulating IFN-γ perturbations with 25(OH)D and 1,25-dihydroxyvitamin D (1,25(OH)D) alterations during inflammatory stress in young adults recovering from anterior cruciate ligament (ACL) reconstruction. Plasma 25(OH)D, 1,25(OH)D and IFN-γ concentrations were measured in fasting blood draw samples obtained from twelve-male patients pre-surgery and 90-m, 3-d and 7-d post-surgery. 25(OH)D decreased significantly (p<0.05) after surgery, and strikingly, tended to inversely correlate (r=-0.32, p=0.058) with IFN-γ changes from pre- to post- (i.e., 90-m, 3-d, and 7-d) surgery. Additionally, 1,25(OH)D (r=0.37, p<0.05) and the 1,25(OH)D-to-25(OH)D ratio (r=0.52, p<0.05) changes from pre- to post- (i.e., 90-m, 3-d, and 7-d) surgery correlated with those of IFN-γ. These are the first reported in vivo findings suggesting that the 25(OH)D decrease and conversion to 1,25(OH)D increase with increasing IFN-γ in the circulation. We conclude that IFN-γ contributes to the decrease in vitamin D and the conversion of vitamin D to its active hormonal form in the circulation during inflammatory insult in humans.

Analysis of proinflammatory and anti-inflammatory cytokine serum concentrations in patients with multiple sclerosis by using a multiplexed immunoassay.

We examined cytokines and other inflammatory markers in serum samples from 833 patients with multiple sclerosis and 117 healthy control subjects. A multiplexed immunoassay was used to assess the concentrations of 13 cytokines/inflammatory markers: interferon (IFN)-γ; interleukins (ILs)-1ß, 2, 4, 5, 6, 8, 10, 12, and 13; tumor necrosis factor (TNF)-α; IL-2 receptor; and soluble CD40 ligand. Significant increases between patients and control subjects were found for IFN-γ (mean, 7.5 vs 0.4 pg/mL; P = .0002), IL-2 (mean 5.7 vs 1.0 pg/mL; P =.0002), IL-1ß (mean, 23.0 vs 11.3 pg/mL; P ≤ .0001), TNF-α (mean, 4.1 vs 1.2 pg/mL; P = .01), IL-4 (mean, 1.4 vs 0.1 pg/mL; P ≤ .0001), IL-10 (mean, 16.8 vs 7.5 pg/mL; P = .03), and IL-13 (mean, 4.5 vs 0.8 pg/mL; P ≤ .0001). Profiling cytokines in multiple sclerosis may help to identify mechanisms involved in the pathogenesis of the disease, aid in monitoring the disease course and in evaluating responses to specific therapies, and, potentially, lead to new therapies directed at cytokines or their receptors.

Vitamins E and C modulate the association between reciprocally regulated cytokines after an anterior cruciate ligament injury and surgery.

OBJECTIVE: : The purpose of this study was to identify the influence of vitamin E and C supplementation on inflammatory cytokines and the association between reciprocally regulated cytokines after anterior cruciate ligament surgery. DESIGN: : A double-blind, placebo-controlled study was conducted in men undergoing anterior cruciate ligament surgery who were randomly assigned to one of two groups (n = 10/group): (1) antioxidant (vitamins E and C) or (2) matching placebos starting ∼2 wks before (baseline) and concluding 3 mos after surgery. Plasma inflammatory cytokines were measured in fasting blood draw samples before and after anterior cruciate ligament surgery. RESULTS: : Plasma interleukin (IL) 1ß concentrations were double at 3 mos after surgery compared with baseline. Plasma IL-1ß increased to a greater (P < 0.05) extent relative to IL-4 in the placebo group (mean ± SE slope, 18.87 ± 0.68; r = 0.97) than in the antioxidant group (mean ± SE slope, 4.84 ± 0.42; r = 0.89). Similarly, the relative increase in IL-1ß to IL-2 was greater (P < 0.05) in the placebo (mean ± SE slope, 2.70 ± 0.21) than in the antioxidant (mean ± SE slope, 1.08 ± 0.23) group. CONCLUSIONS: : Vitamins E and C were ineffective in ameliorating the increases in IL-1ß but altered associations between reciprocally regulated cytokines after anterior cruciate ligament surgery.

Severity of symptom flare after moderate exercise is linked to cytokine activity in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) patients often report symptom flare (SF) for >24 h after moderate exercise (post-ex). We hypothesized that SF is linked to increases in circulating cytokines and CD40 Ligand (CD40L). In 19 CFS patients and 17 controls, mental and physical fatigue and pain symptom ratings were obtained together with serum for 11 cytokines and CD40L before and at 0.5, 8, 24, and 48 h post-ex. Before exercise, CFS had lower CD40L (p<.05) but similar cytokines versus controls. In subgroups based on SF at 48 h, high SF patients (n=11) increased in IL-1beta, IL-12, IL-6, IL-8, IL-10, and IL-13 (p<.05) 8 h post-ex. Low SF patients (n=8) showed post-ex decreases in IL-10, IL-13, and CD40L, and controls decreased in IL-10, CD40L, and TNFalpha (p<.05). Thus, in CFS, cytokine activity may vary directly with SF, which may explain prior inconsistent findings.

Development and validation of a fluorescent microsphere immunoassay for soluble CD30 testing.

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.

Binding of anti-HLA class I antibody to endothelial cells produce an inflammatory cytokine secretory pattern.

Current methods are inadequate for the diagnosis of early chronic allograft rejection. The goal of this study was to determine whether ligation of anti-HLA antibodies to endothelial cells is associated with a distinctive cytokine secretory pattern. Human iliac artery endothelial cells (HIAEC) cultured in vitro were incubated with w6/32, an anti-HLA class I mAb. Culture supernatants collected daily for up to 4 days were tested for secretion of 13 cytokines using a multiplexed fluorescent microsphere immunoassay. Culture of HIAEC with medium containing mAb w6/32 supported the growth of HIAEC during the 4-day study period. Levels of the pro-inflammatory cytokines IL-1beta, IL-6, IL-8, and TNF-alpha became significantly increased in supernatants of HIAEC incubated with the mAb w6/32. We conclude that ligation of anti-HLA class I antibodies to HLA class I antigens in endothelial cells initiates an acute inflammatory process and detecting an inflammatory cytokine secretory pattern might be useful to diagnose sub-clinical chronic allograft rejection.

Modulation of inflammation by vitamin E and C supplementation prior to anterior cruciate ligament surgery.

Muscle atrophy commonly follows anterior cruciate ligament (ACL) injury and surgery. Proinflammatory cytokines can induce and exacerbate oxidative stress, potentiating muscle atrophy. The purpose of this study was to evaluate the influence of prior antioxidant (AO) supplementation on circulating cytokines following ACL surgery. A randomized, double-blind, placebo-controlled trial was conducted in men undergoing ACL surgery, who were randomly assigned to either: (1) AO (200 IU of vitamin E (50% d-alpha-tocopheryl acetate and 50% d-alpha-tocopherol) and 500 mg ascorbic acid), or (2) matching placebos (PL). Subjects took supplements twice daily for 2 weeks prior to and up to 12 weeks after surgery. Each subject provided five blood samples: (1) baseline (Bsl, prior to supplementation and approximately 2 weeks prior to surgery), (2) presurgery (Pre), (3) 90 min, (4) 72 h, and (5) 7 days postsurgery. Following surgery, inflammation and muscle damage increased in both groups, as assessed by increased circulating IL-6, C-reactive protein, and creatine kinase. During AO supplementation, plasma alpha-T and AA increased while gamma-T concentrations decreased significantly (P< 0.05). At 90 min the AO group displayed a significant decrease in AA, an inverse correlation between AA and (interleukin) IL-8 (r(2)= 0.50, P< 0.05), and a significantly lower IL-10 response than that of the PL group. IL-10 was significantly elevated at 90 min and 72 h in the PL group. In summary, our findings show that circulating inflammatory cytokines increase and AO supplementation attenuated the increase in IL-10 in patients post-ACL surgery.

Elevated serum levels of soluble CD154 in children with juvenile idiopathic arthritis.

OBJECTIVE: Cytokines play important roles in mediating inflammation in autoimmunity. Several cytokines are elevated in serum and synovial fluid samples from children with Juvenile Idiopathic Arthritis (JIA). Soluble CD154 (sCD154) is elevated in other autoimmune disorders, but has not been characterized in JIA. Our objectives were to determine if sCD154 is elevated in JIA, and to examine correlations between sCD154 and other inflammatory cytokines. METHODS: Serum from 77 children with JIA and 81 pediatric controls was analyzed for interleukin (IL)1beta, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL13, sCD154, interferon-gamma (IFNgamma), soluble IL2 receptor (sIL2R), and tumor necrosis factor-alpha (TNFalpha), using the Luminex Multi-Analyte Profiling system. Differences in levels of cytokines between cases and controls were analyzed. Logistic regression was also performed. RESULTS: sCD154 was significantly elevated in cases compared to controls (p < 0.0001). IL1beta, IL5, IL6, IL8, IL13, IFNgamma, sIL2R, and TNFalpha were also significantly elevated in JIA. Levels of sCD154 were highly correlated with IL1beta, IL6, IL8, and TNFalpha (p < 0.0001). Logistic regression analysis suggested that IL6 (odds ratio (OR): 1.4, p < 0.0001), sCD154 (OR: 1.1, p < 0.0001), and TNFalpha (OR: 1.1, p < 0.005) were positively associated with JIA, while IL10 (OR: 0.5, p < 0.002) was protective. sCD154 was elevated in all JIA subtypes, with highest levels among more severe subtypes. IL1beta, IL6, IL8, sIL2R and TNFalpha were also elevated in several JIA subtypes. CONCLUSION: Serum levels of sCD154, IL1beta, IL6, IL8, sIL2R and TNFalpha are elevated in most JIA subtypes, suggesting a major role for sCD154, and these cytokines and cytokine receptors in the pathogenesis of JIA.

Evaluation of a multiplex fluorescent microsphere immunoassay for the determination of epstein-barr virus serologic status.

Epstein-Barr virus (EBV), a human herpesvirus, affects up to 95% of adults. Diagnosis of acute EBV infection can be challenging and often relies on the serologic antibody pattern to 3 distinct antigens, most often determined by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assays (ELISAs), and, more recently, multiplex assays. We compared a multiplex assay for the simultaneous detection of antibodies to viral capsid (VCA), nuclear (EBNA), and early (EA) EBV antigens with ELISAs using IFA for discrepancy resolution. Concordance of the multiplex assay was good for all 4 antigens: VCA IgM, 86.6% vs ELISA and 92.9% vs IFA; VCA IgG, 92.8% vs ELISA and 98.0% vs IFA; EBNA IgG, 90.3% vs ELISA and 98.1% vs IFA; and EA IgG, 83.8% vs ELISA and 92.8% vs IFA. After IFA resolution, correlation between the multiplex assay and ELISA for serologic disease stage, based on the antibody profile of all 4 analytes, was 90%. The multiplex assay showed good correlation with an established ELISA and even better correlation with the "gold standard" IFA. Advantages of the multiplex assay over traditional methods include multiple results per assay, inclusion of internal controls for each assay, and well-to-well monitoring of assay drift.

Performance characteristics of six IMMULITE 2000 TORCH assays.

TORCH is an acronym for Toxoplasma gondii (Toxo), other microorganisms (eg, syphilis), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV) that are associated with congenital abnormalities from maternal infection. We evaluated linearity, imprecision, and comparison with commercially available methods of the IMMULITE 2000 (Diagnostic Products, Los Angeles, CA) Toxo IgG, Toxo IgM, RV IgG, RV IgM, CMV IgG, and HSV IgG assays. Linearity and imprecision results were acceptable. The IMMULITE 2000 assays show good concordance with other commercially available methods except for Toxo IgM and RV IgM. Toxo IgM showed better concordance with a consensus of 3 of 4 (Access, Beckman Coulter, Fullerton, CA; IMMULITE 2000; Platelia, Bio-Rad Laboratories Diagnostics Group, Redmond, WA; and Vidas, bioMerieux, Hazelwood, MO) assays than with Access alone. The RV IgM assay showed better concordance with the Zeus method than with the Diamedix method (Diamedix, Miami, FL). The IMMULITE 2000 TORCH assays studied show acceptable performance and are suitable for routine clinical use. Some commercial assays for Toxo IgM and RV IgM show rather poor concordance.

Risk factor analysis of plasma cytokines in patients with coronary artery disease by a multiplexed fluorescent immunoassay.

Coronary artery disease (CAD) is the leading cause of death in the United States. Increasing evidence suggests involvement of inflammation in the atherosclerotic process. We examined cytokines and other inflammatory markers in 865 patients with chest pain in whom coronary angiography revealed no evidence of CAD or CAD with or without concomitant myocardial infarction (MI). We developed a multiplexed immunoassay to simultaneously assess the plasma concentrations of 8 cytokines (interferon gamma, interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha), IL-2r, and soluble CD40 ligand in the patient groups. Concentrations of C-reactive protein (CRP) and IL-18 also were determined. Significant differences (P < .05) between no CAD and combined CAD groups were found for IL-2, IL-4, IL-6, IL-12, and IL-18. When the no CAD group was compared with the group with CAD with subsequent MI, significant differences were found for proinflammatory markers IL-6 (P pound .001), IL-8 (P = .017), and CRP (P pound .001). Cytokine profiles may have a role in differentiating patients with CAD with MI from those with chest pain due to other disorders and in deciphering the role of inflammation in the pathogenesis of CAD.

Comparison of complement fixation with two enzyme-linked immunosorbent assays for the detection of antibodies to respiratory viral antigens.

We compared complement fixation (CF) for the measurement of antibodies against influenza A, influenza B, respiratory syncytial virus (RSV), human adenovirus, and parainfluenza viruses 1, 2, and 3 (para-1, para-2, and para-3) with 2 enzyme-linked immunosorbent assays (ELISA kits, A and B). The IgG ELISA kits compared very well with each other except for the influenza A and B IgG ELISAs. The IgG ELISAs, in general, did not agree with CF In contrast, the IgM ELISAs compared well with CF and each other except for the consensus parainfluenza panel from ELISA B. The poor agreement of the IgG ELISAs with the CF test can be explained by the increased sensitivity of the ELISAs and differences between CF antigens and the ELISA antigens. The influenza A and influenza B ELISA antigens differed between both kits, which may explain their poor agreement. The ELISA is a suitable replacement for CF, providing greater sensitivity, isotype specificity, and ease of use.

The Bartonella henselae sucB gene encodes a dihydrolipoamide succinyltransferase protein reactive with sera from patients with cat-scratch disease.

Bartonella henselae is a recently recognized pathogenic bacterium associated with cat-scratch disease, bacillary angiomatosis and bacillary peliosis. A recombinant clone expressing an immunoreactive antigen of B. henselae was isolated by screening a genomic DNA cosmid library by Western blotting with sera pooled from patients positive for B. henselae IgG antibodies by indirect immunofluorescence (IFA). The deduced amino acid sequence of the 43.7 kDa encoded protein was found to be 76.3 % identical to the dihydrolipoamide succinyltransferase enzyme (SucB) of Brucella melitensis. SucB has been shown to be an immunogenic protein during infections by Brucella melitensis, Coxiella burnetii and Bartonella vinsonii. The agreement between reactivity with a recombinant SucB fusion protein on immunoblot analysis and the results obtained by IFA was 55 % for IFA-positive sera and 88 % for IFA-negative sera. Cross-reactivity was observed with sera from patients with antibodies against Brucella melitensis, Mycoplasma pneumoniae, Francisella tularensis, Coxiella burnetii and Rickettsia typhi.

Comparison of four enzyme immunoassays for the detection of immunoglobulin G antibody against glomerular basement membrane.

Goodpasture syndrome is a life-threatening autoimmune kidney and pulmonary disease that is characterized by pulmonary hemorrhage, renal failure, and the presence of autoantibodies against glomerular basement membrane (GBM) by indirect fluorescent antibody (IFA) techniques. In 1988, these antibodies were found to be specific for the noncollagen region of the alpha3 collagen IV chain. The antigen is characterized by a restricted tissue distribution occurring mainly in the kidneys and lungs. Specific enzyme immunoassays (EIAs) for anti-GBM have now been developed for in vitro diagnostic use in the laboratory. Our objective in this study was to compare the results obtained using four different EIAs for detecting anti-GBM IgG antibody with those using the standard IFA method using tissue from human kidney. Thirty-two patients with suspected Goodpasture syndrome, and 10 control sera were included in the study. GBM EIAs were purchased from or donated by the following vendors: Scimedx Corporation (Denville, NJ), INOVA Diagnostics (San Diego, CA), The Binding Site (Birmingham, England), and Wieslab (distributed by DiaSorin, Inc., Stillwater, MN). Percent agreement, sensitivity, and specificity were calculated for each EIA as compared to IFA. The results were as follows: Binding Site: 83.3, 100.0, and 72.0%; Wieslab: 95.2, 94.1, and 96.0%; INOVA: 96.2, 100.0, and 92.3%; and Scimedx: 81.0, 100.0, and 68.0%. We conclude that the INOVA and Wieslab GBM IgG EIAs compared well with the standard GBM IFA method using tissue from human kidney. The Scimedx and Binding Site EIAs had eight and seven false-positive results, respectively, when compared to GBM IFA using human kidney. The authors recommend conducting thorough evaluations of EIAs that screen for anti-GBM antibody before their implementation in the clinical laboratory. We also recommend confirming GBM EIA-positive sera by the standard IFA method using tissue from human kidney.

Development of internal controls for the Luminex instrument as part of a multiplex seven-analyte viral respiratory antibody profile.

The ability of the Luminex system to simultaneously quantitate multiple analytes from a single sample source has proven to be a feasible and cost-effective technology for assay development. In previous studies, my colleagues and I introduced two multiplex profiles consisting of 20 individual assays into the clinical laboratory. With the Luminex instrument's ability to classify up to 100 distinct microspheres, however, we have only begun to realize the enormous potential of this technology. By utilizing additional microspheres, it is now possible to add true internal controls to each individual sample. During the development of a seven-analyte serologic viral respiratory antibody profile, internal controls for detecting sample addition and interfering rheumatoid factor (RF) were investigated. To determine if the correct sample was added, distinct microspheres were developed for measuring the presence of sufficient quantities of immunoglobulin G (IgG) or IgM in the diluted patient sample. In a multiplex assay of 82 samples, the IgM verification control correctly identified 23 out of 23 samples with low levels (<20 mg/dl) of this antibody isotype. An internal control microsphere for RF detected 30 out of 30 samples with significant levels (>10 IU/ml) of IgM RF. Additionally, RF-positive samples causing false-positive adenovirus and influenza A virus IgM results were correctly identified. By exploiting the Luminex instrument's multiplexing capabilities, I have developed true internal controls to ensure correct sample addition and identify interfering RF as part of a respiratory viral serologic profile that includes influenza A and B viruses, adenovirus, parainfluenza viruses 1, 2, and 3, and respiratory syncytial virus. Since these controls are not assay specific, they can be incorporated into any serologic multiplex assay.