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Summary Background Tissue transglutaminase (TTG) antibodies and newly developed deamidated gliadin peptide (DGP) antibodies have better accuracy than native gliadin antibodies. Multiplex immunoassay (MIA) measures multiple antibodies simultaneously providing a complete antibody phenotype with reduced turnaround time and cost. Aim To evaluate the agreement between MIA and enzyme-linked immunosorbent assay (ELISA) test results for coeliac antibodies in biopsy-proven coeliac patients and controls and to model the diagnostic utility of combination testing. Methods We compared the sensitivity, specificity and accuracy of MIA and ELISA methods for TTG and DGP antibodies in mainly adult untreated coeliac patients (n = 92) and controls (n = 124). Results There was excellent agreement and a significant correlation between the results of MIA and ELISA methods (κ > 0.8, r > 0.7) for all tests, except TTG IgG. Diagnostic indices of individual and combination tests measured by the MIA method did not differ significantly from those measured by ELISA. The combination tests slightly increased sensitivity (if any test was positive) and specificity (if all tests were positive) compared to the individual tests. Conclusions Multiplex immunoassay testing for antibodies is as accurate as ELISA for coeliac disease diagnosis and has practical advantages over ELISA method. Rational combination testing can help identify patients who need intestinal biopsy and may reduce unnecessary biopsies.

Summary Serum and plasma are used interchangeably to measure anti-neutrophil cytoplasmic antibodies (ANCA), even though the release of ANCA target antigens during the preparation of serum could affect ANCA assays and cause discrepancies between the results obtained from serum and plasma. To what extent ANCA test results obtained from serum agree and correlate with results from plasma remains unknown. Therefore, a comprehensive comparison was performed using serum and plasma samples which were collected in 175 patients with active Wegener's granulomatosis at enrolment of a recent randomized trial. These paired serum and plasma samples were subjected to parallel ANCA testing by standard indirect immunofluoresence on ethanol-fixed neutrophils, a direct enzyme-linked immunoassay (ELISA) for proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, and two different capture ELISAs for PR3-ANCA. The concordance of categorical serum and plasma ANCA results was assessed using κ-coefficients. These were > 0·8 for all assays, indicating a very good concordance between positive and negative serum and plasma results. Spearman's correlation coefficients for serum and plasma PR3-ANCA values obtained by direct ELISA and both capture ELISAs were ≥ 0·95 (P

Systemic lupus erythematosus (SLE) rarely presents with a negative antinuclear antibody (ANA). Although antibodies to extractable nuclear antigens (ENA) are sometimes ordered despite a negative ANA, it is unclear if this contributes to the diagnosis of SLE or other forms of connective tissue disease (CTD). We reviewed 39 patients with anti-ENA antibodies despite a negative ANA during a 1-year period to determine the presence of SLE or other CTD. Several patients had clinical features suggestive of CTD, including 1 with possible SLE. A number of patients had neurologic disorders, especially peripheral neuropathy. In this study, the finding of anti-ENA despite negative ANA was associated with neurologic disorders and CTD. This may represent test bias or false-positive anti-ENA assays or false-negative ANA assays, or may imply immune-related mechanisms not previously described.

Chronic rhinosinusitis (CRS) is one of the most common long-term illnesses in the United States. The etiology of CRS is unknown, and no effective treatment has been established.We investigated the hypothesis that abnormal immunologic responses to ubiquitous airborne fungi contribute to the pathogenesis of this disease.The proliferative and cytokine responses of PBMCs to extracts from 4 common airborne fungi-including Alternaria , Aspergillus , Cladosporium , and Penicillium -were examined by in vitro culture. Serum specimens were tested for specific IgE and IgG to these fungi.PBMCs from approximately 90% of the patients with CRS, but not those from normal individuals, produced both IL-5 and IL-13 when exposed to Alternaria. Furthermore, PBMCs from patients with CRS produced significantly more IFN-gamma than PBMCs from normal individuals in response to Alternaria (median, 553 pg/mL vs 98 pg/mL; P.01). Levels of serum IgG antibodies to Alternaria and Cladosporium were clearly increased in patients with CRS compared with normal individuals ( P.01). Less than 30% of the patients with CRS had specific IgE antibodies to Alternaria or Cladosporium. The increased humoral (serum IgG) response strongly correlated with the increased cellular (IL-5 production) response to Alternaria ( r = 0.619; P.01).Patients with CRS show exaggerated humoral and cellular responses, both T(H)1 and T(H)2 types, to common airborne fungi, particularly Alternaria. The anomalous immune and inflammatory responses to ubiquitous fungi may explain the chronicity of airway inflammation in CRS.

In the environment of managed care, children with allergic diseases are increasingly likely to be evaluated by nonallergist physicians. While the presumptive diagnosis of an allergic disease can often be suspected on clinical grounds, signs and symptoms are not reliable for establishing a definitive diagnosis or for deciding on management options. This article discusses the use of immunoglobulin E antibody tests to classify children with allergic symptoms. The information is intended for pathologists who offer consultative laboratory services to nonallergist physicians. Emphasis is placed on defining the minimum number of tests necessary to identify allergic children at different ages.

To assess the performance of 4 clotting assays for lupus anticoagulant (LA) detection, to determine the prevalence of LA and anticardiolipin antibodies (aCL), and to correlate LA and aCL prevalence with systemic disease and thrombosis.We studied 664 consecutive patients at the Mayo Clinic in Rochester, Minn, who were referred for laboratory testing because of a clinical suspicion of LA or thrombophilia between June 25, 1990, and July 1, 1991.Of 664 patients tested for LA, 584 also were tested for aCL. Of patients tested for both LA and aCL, 137 (235%) had positive results for one or both tests (13 [95%], LA-positive only; 76 [555%], aCL-positive only; and 48 [35.0%], positive for both). The dilute Russell viper venom time (DRVVT) was the most frequently positive LA assay (74% of the 61 patients with positive results for LA). Twenty-two patients (36.1% of the 61) had positive results for all 4 LA assays, whereas 21 (34.4% of the 61) had positive results for only 1 LA assay: activated partial thromboplastin time (3 patients [4.9%]), plasma clot time (5 patients [8.2%]), kaolin clot time (5 patients [8.2%]), or DRVVT (8 patients [13.1%]). Thromboembolism prevalence was not definitely associated with positive test results (LA only, aCL only, or LA plus aCL), nor was it strongly associated with aCL isotype or titer. Furthermore, thromboembolism prevalence was not increased when all LA assays were positive, although a history of deep venous thrombosis or pulmonary embolism was nonsignificantly associated with positive results for all 4 LA tests. The likelihood of having both LA- and aCL-positive test results was higher among patients with systemic lupus erythematosus (26 [19.0%] of 137 patients with positive results for one or both tests), but they had no more thrombotic events or fetal loss than other patients in our study group.The DRVVT identified more patients with LA than the other LA tests, but more than 1 LA test was required to identify all patients with LA. Positive results were much more common for aCL than for LA. No single LA test or anticardiolipin isotype correlated with thrombosis or systemic disease in this population.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a useful diagnostic tool for Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). To maximize diagnostic utility, current guidelines recommend dual testing by standard indirect immunofluorescence (IIF) and target antigen-specific assays. Most published data come from specialized research laboratories, not reflecting the performance of assays under routine clinical conditions. Therefore, we compared the performance of standard IIF, PR3-, and MPO-ANCA-specific direct ELISA, and a PR3-ANCA-specific capture ELISA used alone and in combination under routine clinical conditions. Consecutive serum samples (615) submitted for routine ANCA testing over a 10-month period were assayed. Diagnoses were WG/MPA (n = 86), other autoimmune disease (n = 118), and various others (n = 411). The combination of PR3-ANCA and MPO-ANCA ELISA had the highest sensitivity (72.1%), and C-ANCA determination using IIF, the highest specificity (99.6%). While maintaining maximal diagnostic accuracy, significant labor savings are achieved by screening for WG/MPA by ELISA followed by confirmatory IIF.

Autoantibodies are immunoglobulins that react against normal host proteins and may be natural or pathologic.1 Natural autoantibodies are germline responses that develop from genetically programmed clones of B cells and are typically present in low serum titers as isotypes of immunoglobulin M.2–5 Natural autoantibodies do not fix complement and usually have weak antigen-binding affinities, multiple reactivities, and no disease associations.6,7 They occur more commonly in women than in men,8 the repertoires remain stable with aging,9,10 and they may bind products of natural cell death or foreign antigens that resemble selfantigens.11,12 In this fashion, natural autoantibodies may prevent the emergence of autoreactive immunocytes and have a protective function.2,4,6,11,12 Pathologic autoantibodies are produced by antigenspecific B-cell activation and subsequent clonal expansion of dedicated plasma cells.1,2 These autoantibodies typically are present in high serum titers as isotypes of immunoglobulin G. Pathologic autoantibodies fix complement, have high antigen-binding affinities, inhibit autoantigen activity in vitro, and have disease associations.2,6 They are rarely pathogenic, but their presence in serum does imply the existence of an antigen-driven immunopathic process. This process may be crucial for disease expression, a consequence of other cytodestructive mechanisms, or coincidental with the main disorder. Natural and pathologic autoantibodies can be distinguished from each other by class-specific attributes (Table 1). The presence of low-titer autoantibodies in the absence of hypergammaglobulinemia or a compatible clinical liver disease usually indicates the presence of natural autoantibodies. Any component of the liver cell can trigger the production of pathologic autoantibodies, and reactivities have been described against nuclear, microsomal, cytosolic, and mitochondrial proteins.13 Expression of the antigen on the surface of professional antigen-presenting cells is the principal mechanism for activation of B cells, and antigen presentation to CD4 T-helper cells is restricted by the class II molecules of the major histocompatibility complex.14–16 Intracellular antigens can be expressed on the surface of the liver cell membrane, and the hepatocyte can present autoantigens that generate autoantibodies.17,18 Mechanisms for pathologic autoantibody production include the discovery of self-antigens that had been poorly recognized or sequestered during ontogeny (cryptic or sequestered epitopes) or the generation of new antigens by the degradation of viruses, xenobiotics, and chemicals within the hepatocyte (neoepitopes).19,20 Selfantigens can be targeted by an exuberant immune response to an infectious agent (superantigen effect), and nonspecific mediators of inflammation, including cytokines, chemotactic substances, macrophages, and natural killer cells, can extend the immune response (bystander effects). Epitopes within the same antigen or between different antigens can become involved (epitope spread); autologous antigens can combine with infectious or environmental agents (adjuvant effects); or foreign antigens can mimic self-antigens (molecular mimicry).19,20 Autoantibodies may lack disease specificity or pathogenic importance, and bystander effects, adjuvant effects, and molecular mimicry may explain in part the occurrence of autoantibodies in nonautoimmune diseases. Importantly, autoantibodies are not found in all forms of hepatocellular injury and are not simply the trappings of liver cell destruction. Autoantibody titers do not correlate closely with the severity of liver cell damage, and changes in autoantibody expression do not reflect disease behavior.21

The following guideline presents a series of recommendations based on published medical literature for use of the antinuclear antibody (ANA) test and tests for specific autoantibodies to nuclear antigens in the diagnostic evaluation, prognostic assessment, and monitoring of patients with systemic rheumatic diseases. The guideline emphasizes the need for clinical evaluation to improve the usefulness of test results in patient management. Consideration is given to appropriate use of the generic ANA test in the initial evaluation of patients with signs and symptoms of a systemic rheumatic disease, the evaluation of patients suspected of having lupus erythematosus, use in clinical situations in which the ANA test is required to establish a disease diagnosis, and identification of clinical situations in which the ANA test has little value. Sections are also devoted to recommendations aimed at improving the analytic methods used to detect and measure ANA and specific autoantibodies to nuclear antigens and to the appropriate use of tests for specific autoantibodies in several disease situations that commonly occur in patients with suspected or documented systemic rheumatic diseases. Emphasis is placed on the use of these tests only in situations in which the test results can be expected to provide information necessary for clinical decision making. Those tests of limited medical usefulness and situations in which test results are likely to be misleading are also identified.

SUMMARY We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjögren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P

We examined the clinical usefulness of measurements of antimitochondrial autoantibodies (AMA) in predicting disease progression in patients with primary biliary cirrhosis (PBC). We determined the relationships between AMA levels measured by indirect immunofluorescence (IF) and those measured by quantitative enzyme immunoassays (EIAs) using recombinant 2-oxo-acid dehydrogenase complex (2-OADC) proteins and the Mayo Risk Score, an established indicator of disease progression in primary biliary cirrhosis (PBC). Results of tests for AMA by either method correlated weakly (r = .24 to .30) with disease progression as indicated by Mayo Risk Scores. The levels of AMA to 2-OADC proteins varied by more than 200-fold between patients but remained relatively constant over time in individual patients. Despite being positively correlated with Mayo Risk Score results, the levels of AMA to 2-OADC proteins were not useful for predicting disease progression in individual patients with PBC. In addition, we found no significant differences in the levels of autoantibodies to 2-OADC proteins among patients with different histological stages of disease. Our results show that measurements of AMA by IF or by quantitative EIA methods with recombinant 2-OADC proteins are not useful parameters for predicting disease progression in patients with PBC.

Hepatic dysfunction following liver transplantation is often caused by cellular rejection or infection with cytomegalovirus. These etiologies can at times be difficult to differentiate. We measured the levels of soluble T lymphocyte markers sIL2R, sCD4, and sCD8 in serum as possible diagnostic indicators of cellular rejection and cytomegalovirus hepatitis. Pretransplant levels, and serial post-transplant levels of soluble T lymphocyte markers were measured in five control patients without cellular rejection or cytomegalovirus infection, ten patients with cellular rejection, and six patients with cytomegalovirus hepatitis. In all cases, cellular rejection and cytomegalovirus hepatitis were documented with liver histology. For each group of patients, we calculated the mean ratio of the post-transplant marker level divided by the pre-transplant level. We found an elevation in the mean ratio of sIL2R in patients with cellular rejection shortly before or at the time of diagnosis of rejection as compared to the transplant control group. Levels of sCD8 were not significantly increased in patients with cellular rejection. We found a more pronounced elevation in the mean marker ratios of both sIL2R and sCD8 in patients with cytomegalovirus hepatitis which were higher compared not only to the transplant control group but also compared to the cellular rejection group. The rise of serum levels preceded the histologic diagnosis of cytomegalovirus hepatitis and detection of cytomegalovirus in blood cultures. Increased serum levels of sIL2R with concomitant elevation of sCD8 suggest the diagnosis of cytomegalovirus hepatitis over cellular rejection. We conclude that serial measurements of sIL2R and sCD8 may be useful to differentiate cytomegalovirus hepatitis from episodes of acute cellular allograft rejection, particularly those rejection episodes occurring late (>20 days posttransplantation).

To determine if the hepatitis viruses are important etiologic factors in autoimmune hepatitis, the clinical, immunoserologic, virologic and HLA phenotypes of 105 of the latter patients were assessed prospectively and compared to 45 patients with chronic viral hepatitis. Patients with autoimmune hepatitis were more often women with higher serum aspartate aminotransferase and immunoglobulin levels than patients with viral disease. Only eight patients (8%) were seropositive for anti-HBc and anti-HBs (four patients) or anti-HCV (four patients) and none with anti-HCV were reactive by second generation immunoassay or recombinant immunoblot assay. Smooth muscle (90 vs. 22%, P0.001) and antinuclear (70 vs. 22%, P0.001) antibodies were more common in patients with autoimmune hepatitis and the titers more frequently exceeded 1:80 (84 vs. 11%, P0.0001). Patients with autoimmune hepatitis were more often positive for HLA B8 (48 vs. 20%, P0.01) and DR3 (49 vs. 20%, P0.003) and they more frequently had the HLA A1-B8-DR3 phenotype (38 vs. 10%, P0.003). Only one of the 120 patients tested for anti-LKM1 was seropositive. We conclude that in an American referral population autoimmune hepatitis usually lacks virologic markers and has a distinctive clinical, immunoserologic and HLA phenotype. Hepatitis viruses are not important immunogenic stimuli for non-organ specific antibodies and they are unlikely to be important causes of this form of autoimmune hepatitis.

To assess the frequency of antibodies to liver/kidney microsome type 1 (anti-LKM1) in patients with chronic active hepatitis, 131 such patients were tested by an indirect immunofluorescence assay. Of 62 patients with type 1 autoimmune hepatitis, none were seropositive. In contrast, 3 of 11 patients with autoimmune hepatitis and antimitochondrial antibodies (27%) were seropositive for anti-LKM1. Each had responded to corticosteroid therapy, and retesting of sera confirmed that each had been misclassified as antimitochondrial antibody positive. None of the patients with chronic active hepatitis B (14 patients) or C (24 patients) had anti-LKM1. Similarly, none of the 20 patients with cryptogenic disease had these antibodies. It is concluded that anti-LKM1 is specific for type 2 autoimmune hepatitis and is infrequent in adult patients seen at a referral center in the United States for chronic active hepatitis. Anti-LKM1 reactivity may be misinterpreted as antimitochondrial antibody reactivity by indirect immunofluorescence. Chronic hepatitis B and C virus infections are not important stimuli for the production of anti-LKM1, and testing for anti-LKM 1 is unlikely to clarify the nature of cryptogenic disease.

The final part of this series describes the measurement methods for allergenspecific immunoglobulin E (IgE) antibodies. The IgE antibody test is discussed and evaluated.

Background: In adult patients, autoantibodies targeting the water channel aquaporin-4 (AQP4) are a biomarker for a spectrum of CNS inflammatory demyelinating disorders with predilection for optic nerves and spinal cord (neuromyelitis optica [NMO]). Here we describe the neurologic, serologic, and radiographic findings associated with CNS AQP4 autoimmunity in childhood. Methods: A total of 88 consecutive seropositive children were identified through service evaluation for NMO-IgG. Sera of 75 were tested for coexisting autoantibodies. Clinical information was available for 58. Results: Forty-two patients (73%) were non-Caucasian, and 20 (34%) had African ethnicity. Median age at symptom onset was 12 years (range 4–18). Fifty-seven (98%) had attacks of either optic neuritis (n = 48; 83%) or transverse myelitis (n = 45; 78%), or both. Twenty-six (45%) had episodic cerebral symptoms (encephalopathy, ophthalmoparesis, ataxia, seizures, intractable vomiting, or hiccups). Thirty-eight (68%) had brain MRI abnormalities, predominantly involving periventricular areas (in descending order of frequency): the medulla, supratentorial and infratentorial white matter, midbrain, cerebellum, thalamus, and hypothalamus. Additional autoantibodies were detected in 57 of 75 patients (76%), and 16 of 38 (42%) had a coexisting autoimmune disorder recorded (systemic lupus erythematosus, Sjogren syndrome, juvenile rheumatoid arthritis, Graves disease). Attacks were recurrent in 54 patients (93%; median follow-up, 12 months). Forty-three of 48 patients (90%) had residual disability: 26 (54%) visual impairment and 21 (44%) motor deficits (median Expanded Disability Status Scale 4.0 at 12 months). Conclusions: Aquaporin-4 autoimmunity is a distinctive recurrent and widespread inflammatory CNS disease in children.

Serologic tests are used frequently in celiac disease diagnosis. Gliadin antibodies generally lack the accuracy required for proper diagnosis. We evaluated the value of deamidated gliadin antibody measurements in the diagnosis and follow-up evaluation of celiac disease and compared their potential usefulness with that of gliadin and tissue-transglutaminase antibodies.We tested deamidated gliadin, gliadin, and tissue-transglutaminase-immunoglobulin (Ig)A and -IgG in 216 biopsy-selected subjects including 92 biopsy-proven untreated celiac patients (46% with total villous atrophy and 54% with partial villous atrophy) and 124 biopsy-proven nonceliac controls. Fifty-nine celiac patients also were tested after treatment with a gluten-free diet. Antibodies were measured by commercial enzyme-linked immunosorbent assays. Deamidated gliadin-IgA+G was detected using a conjugate reactive to both isotypes, which gives a positive if either isotype is present.The sensitivity, specificity, and accuracy of deamidated gliadin-IgA (74%, 95%, and 86%), deamidated gliadin-IgG (65%, 98%, and 84%), and deamidated gliadin-IgA+G (75%, 94%, and 86%) were superior to gliadin-IgA (63%, 90%, and 79%) (P.05) and gliadin-IgG (42%, 90%, and 69%) (P.01), and were similar to tissue-transglutaminase-IgA (78%, 98%, and 90%) before treatment. The sensitivity of IgA isotype for all tests was significantly greater in celiac patients with total villous atrophy compared with those with partial villous atrophy (P.05). The proportion of positive test results for all tests decreased significantly after treatment (P.0001).Deamidated gliadin antibody is a better diagnostic test for celiac disease than the conventional gliadin antibody testing; although histopathology remains the gold standard test for diagnosis of celiac patients.

We developed rapid 24-h immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum. In comparative studies, we evaluated the analytical sensitivity of the immunoblot assays and commercial immunodiffusion assays for anti-Scl 70 and anti-Jo 1 autoantibodies with the use of positive control sera, and compared the frequencies of positive and negative results in a group of 116 sera, including specimens from 34 healthy controls and 82 patients with various connective-tissue diseases. The immunoblot assays were greater than 100-fold more sensitive than immunodiffusion for detecting both autoantibodies. Despite greater analytical sensitivity, there were no false-positive results by the immunoblot assay for anti-Scl 70 or anti-Jo 1 autoantibodies in sera from either the controls or the patients. The diagnostic sensitivity of the immunoblot assay for anti-Scl 70 autoantibodies in patients with scleroderma was greater than that of the immunodiffusion assay, 70% vs 20%, and was equivalent in patients with polymyositis, 43%. We conclude that rapid immunoblot assays for anti-Scl 70 and anti-Jo 1 autoantibodies are superior to immunodiffusion assays for clinical use and are suitable for routine use in the clinical laboratory.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disorder that can progress to cirrhosis, shortening life expectancy. PBC patients are often asymptomatic, present with biochemical cholestasis, and test positive (≥90%) for antimitochondrial antibodies (AMAs) in serum. Although AMA positivity without biochemical cholestasis may indicate increased risk of future PBC development, the contribution of these antibodies to pathogenesis remains enigmatic. Environmental risks and genetic determinants are likely implicated in PBC etiology. Given the familial aggregation of PBC, we hypothesized that AMAs also aggregate among relatives of PBC probands. We investigated the prevalence of AMAs in first-degree relatives (FDRs) of PBC probands to examine whether AMAs aggregate in such pedigrees. Using a PBC family registry, we prospectively screened for AMAs in the serum of 306 FDRs in 145 pedigrees, 350 PBC probands, and 196 controls who were age-matched, sex-matched, race-matched, and residence-matched to probands. The prevalence of AMA in FDRs and controls was 13.1% and 1%, respectively. Greater prevalence of AMA was found in female FDRs of PBC probands [sisters (20.7%), mothers (15.1%), and daughters (9.8%)] than in male FDRs [brothers (7.8%), fathers (3.7%), and sons (0%)]. Conclusions: AMAs aggregate among FDRs of PBC probands. Our data have clinical implications for FDRs of PBC probands because AMA positivity may suggest susceptibility to PBC. Thus, the identification and follow-up of these relatives may lead to earlier disease diagnosis and treatment. Furthermore, if AMA development is heritable, this trait will provide a basis to dissect the genetic predisposition to PBC. (HEPATOLOGY 2007.)

During the past several years, immunoassays for specific IgE antibodies have been refined to permit reporting results in mass units. Thus quantitative immunoassays for IgE antibodies may be an adjunct to skin tests. In cases of food allergy among children with atopic dermatitis, cutoff values for IgE antibody concentrations to egg, milk, peanut, and fish have been derived to provide 95% positive and 90% negative predictive values. Food-specific IgE antibody determinations can also be used to predict which food allergies are resolving spontaneously. Elevated egg-specific IgE antibody levels in infancy are associated with significantly increased risk for development of inhalant allergies later in childhood. In cases of inhalant allergy, specific IgE antibody levels correlate closely with results of inhalation challenge studies in cat-sensitive persons. Also, mite-specific IgE antibody levels correlate significantly with the mite allergen contents of reservoir dust in the homes of mite-sensitive persons. Immunoassays for quantitation of specific IgE antibodies may be used to document allergen sensitization over time and to evaluate the risk of reaction on allergen exposure. However, immunoassays and skin tests are not entirely interchangeable, and neither will replace the other in appropriate circumstances.

The use of laboratory testing in the diagnosis of allergic diseases is discussed in this article. This is the first part of a two-part series.

Objectives To reevaluate the current criteria for diagnosing allergic fungal sinusitis (AFS) and determine the incidence of AFS in patients with chronic rhinosinusitis (CRS). Methods This prospective study evaluated the incidence of AFS in 210 consecutive patients with CRS with or without polyposis, of whom 101 were treated surgically. Collecting and culturing fungi from nasal mucus require special handling, and novel methods are described. Surgical specimen handling emphasizes histologie examination to visualize fungi and eosinophils in the mucin. The value of allergy testing in the diagnosis of AFS is examined. Results Fungal cultures of nasal secretions were positive in 202 (96%) of 210 consecutive CRS patients. Allergic mucin was found in 97 (96%) of 101 consecutive surgical cases of CRS. Allergic fungal sinusitis was diagnosed in 94 (93%) of 101 consecutive surgical cases with CRS, based on histopathologic findings and culture results. Immunoglobulin E-mediated hypersensitivity to fungal allergens was not evident in the majority of AFS patients. Conclusion The data presented indicate that the diagnostic criteria for AFS are present in the majority of patients with CRS with or without polyposis. Since the presence of eosinophils in the allergic mucin, and not a type I hypersensitivity, is likely the common denominator in the pathophysiology of AFS, we propose a change in terminology from AFS to eosinophilic fungal rhinosinusitis.

SUMMARYANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ-rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With δ-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils (polymorphonuclear cells, PMNs), is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener's granulomatosis (WG). We have recently developed an expression system for recombinant PR3 (rPR3) that is recognized by c-ANCA. Here, we report on the development and characterization of two monoclonal antibodies (moABs) and a rabbit polyclonal antiserum generated against this rPR3. Epitope competition analysis indicates that the moABs MCPR3-1 and MCPR3-2 recognize overlapping epitopes on the PR3 molecule that are distinct from the ones recognized by moABs 4A5 and 6A6 developed by others. Since MCPR3-2 does not appear to compete for epitopes recognized by a sizable proportion of PR3-ANCA, we used it to develop a sensitive capture enzyme linked immunosorbent assay (ELISA) for clinical PR3-ANCA testing. Both purified PMN PR3 and crude human mast cell line (HMC-1)/PR3-S176A cell lysates were used as sources of PR3 target antigen in this assay with equal analytical sensitivity and specificity. Of 109 patients with ANCA-associated disease, 91 (83.5%) and 90 (82.6%) were PR3-ANCA positive by capture ELISA when PMN-PR3 and HMC-1/PR3-S176A cell lysates were used as antigen, respectively. When HMC-1/PR3 and HMC-1/PR3-S176A cells were used as indirect immunofluorescence (IIF) substrate, 88 (80.7%) and 92 (84.4%) were PR3-ANCA positive, respectively. These differences were not statistically significant. Only 1 of 151 controls without defined ANCA-associated disease tested positive by capture ELISA with either target antigen (both negative by PR3-ANCA specific IIF). The capture ELISA can also be used to detect of PR3-ANCA immunecomplexes and, in combination with the rabbit antiserum, for the quantitative measurement of PR3 in biological fluids.

Background: Cryptogenic chronic active hepatitis may be an autoimmune or viral disease. Our aims were to determine the clinical features, human leukocyte antigen phenotype, and response to corticosteroid therapy of severe Cryptogenic chronic active hepatitis and to compare it with these other diseases. Methods: Twelve patients with Cryptogenic hepatitis were compared with 94 patients with autoimmune hepatitis and 30 patients with chronic viral hepatitis. Results: Patients with cryptogenic hepatitis were indistinguishable from those with autoimmune hepatitis by age, gender, and individual laboratory and histological findings. HLA B8 (75% vs. 49%, P = 0.2), DR3 (71% vs. 51%, P = 0.5), and A1-B8-DR3 (57% vs. 38%, P = 0.6) occurred as commonly in each group. Patients with cryptogenic hepatitis entered remission (83% vs. 78%, P > 0.9) and failed treatment (9% vs. 11%, P > 0.8) as frequently as those with autoimmune hepatitis during corticosteroid therapy. In contrast, patients with chronic viral hepatitis had lower biochemical abnormalities, less frequent multilobular necrosis at presentation, and different human leukocyte phenotypes than those with cryptogenic or autoimmune disease. Conclusions: Severe cryptogenic hepatitis has a clinical expression, genetic phenotype, and corticosteroid responsiveness that is similar to autoimmune hepatitis. It may be an autoimmune disorder that has escaped detection by conventional immunoserological markers.

Serologic tests are frequently ordered as the initial step in the diagnosis of celiac disease. Newly developed tests for deamidated gliadin antibodies and tissue-transglutaminase antibodies measured by enzyme linked immunosorbent assay (ELISA) have better accuracy than native gliadin antibodies for celiac disease. In contrast to ELISA, multiplex immunoassay measures multiple autoantibodies simultaneously thereby decreasing processing time and facilitating panel testing. In this study, we compared multiplex immunoassay and ELISA methods for diagnostic accuracy of deamidated gliadin antibodies and tissue transglutaminase antibodies. Serum samples from 92 biopsy-proven, untreated celiac patients (46% with total villous atrophy and 54% with partial villous atrophy) and 124 biopsy-proven non-celiac controls were tested by multiplex immunoassay (QUANTA Plex Celiac IgA and IgG Profile, INOVA Diagnostics, Inc) and ELISA methods (QUANTA Lite, INOVA Diagnostics, Inc) for deamidated gliadin IgA and IgG and tissue-transglutaminase IgA and IgG antibodies. The sensitivity, specificity and accuracy of each single test and the combination of tests are summarized in the table below. The combination tests did not increase the sensitivity dramatically. Diagnostic indices of the tests and the combination tests measured by multiplex methods did not differ significantly from those measured by ELISA. The two methods showed excellent agreement for all tests except tissue-transglutaminase IgG. In conclusion, the multiplex immunoassay method is as accurate as the combination of ELISAs for celiac disease diagnosis and has practical advantages for testing in the clinical laboratory. Diagnostic indices of serologic tests for celiac disease diagnosis by Multiplex versus ELISA methods

Background The pathogenic significance of antineutrophilic cytoplasmic antibodies (ANCA) in Wegener's granulomatosis is controversial. Their presence is influenced by the extent, severity, and activity of the disease at the time of sampling. The objective of this study was to determine the frequency of ANCA in patients with active Wegener's granulomatosis and to assess the influence of disease severity on test results. Methods Baseline serum samples from the 180 participants in a multicentric prospective trial were tested for ANCA by indirect immunofluorescence, direct enzyme-linked immunosorbent assay (ELISA), and capture ELISA. Disease activity was measured using the Birmingham Vasculitis Activity Score for Wegener's granulomatosis. All patients had active disease at enrollment. Patients were categorized as having severe (n=128) or limited (n=52) Wegener's granulomatosis. Results When all ANCA detection methods were combined, 166 patients (92%) were ANCA positive, including 96% with severe disease and 83% with limited disease. Conclusion ANCA are detectable in nearly all patients with active severe Wegener's granulomatosis, but approximately 1 of 5 patients with active limited disease are ANCA negative. Immunofluorescence and both direct and capture ELISAs are required for optimal detection, suggesting that ANCA are not recognized equally well by all testing methods.

Rationale We developed a quantitative immunoassay using microsphere photometry to enable simultaneous, quantitative measurement of IgG antibodies to 24 Streptococcus pneumoniae serotypes as an aid in evaluating the immune response to pneumococcal vaccination in children and adults. Methods Pneumococcal polysaccharide antigens (24 serotypes, ATCC, Rockville, MD) were coupled covalently to polystyrene microspheres (Luminex Corp., Austin, TX). Each polysaccharide was coupled to a different fluorescent microsphere, and the microspheres were mixed to create a single immunosorbent reagent. This reagent was used to capture antibodies to S. pneumoniae in patient's sera, standards and controls. After incubating with the immunosorbent, bound antibodies were detected with R-phycoerythrin conjugated, goat anti-human IgG antibody using a flow microfluorometer (Luminex Corp.). The concentrations of IgG antibodies to each serotype were determined from standard curves using a primary reference material (10 serotypes, US Reference Pneumococcal Antiserum, FDA, Bethesda, MD)or a secondary reference material (14 serotypes, in-house preparation). Testing for antibodies to all 24 serotypes was completed in 2 hours. Results Measurements of anti-pneumococcal antibodies in sera from 80 healthy adults agreed with published data for the 10 standardized serotypes. The immunoassay detected concentrations ranging from 0.005 to 7.8 micrograms/ml. Interassay reproducibility across different serotypes ranged from 4.8 to 11.9%. Conclusions The microsphere immunoassay we describe is accurate and reproducible, and is suitable for clinical evaluation of patients treated with pneumococcal vaccines. Quantitative results are available for all serotypes in current vaccines.

We describe the development of specific immunoassays for IgG4 protein and for allergen-specific IgG4 antibodies. We also measured the concentrations of IgG4 protein and determined the frequencies of detectable IgG4 antibodies to several common allergens in sera from adults and children with asthma and from nonallergic subjects. Serum concentrations of IgG4 protein increase with age but are not different in children with asthma and nonallergic children, nor does a raised serum concentration predict a severe clinical course in childhood asthma. IgG4 antibodies to milk and egg are common in children and adults and are more common in children with asthma than in nonallergic children less than 3 years of age. The presence of detectable IgG4 antibodies or a raised concentration of IgG4 protein in serum is not useful empirically as a diagnostic indicator of asthma but more likely results from antigen exposure occurring at mucosal surfaces.

A new method to measure the concentrations of IgG antibodies to phospholipase A 2 in sera from patients treated with honeybee venom immunotherapy is described. This method utilizes a microcentrifugal analyzer to detect inhibition of PLA 2 enzymatic activity by antibodies in serum standards and unknowns. Sera from radkeepers with known concentrations of specific antibodies, measured by radioimmunoprecipitation, were used to construct a logit-log standard curve for the immunoenzyme assay. The standard curve was linear for concentrations between 2.3 and 20.0 μg/ml. The concentrations of specific antibodies measured by enzyme inhibition correlated well with the concentrations as measured by radioimmunoprecipitation, r = 0.959 (least squares linear regression), n = 32. Interassay variation was 10.1% at 10 μg/ml. The immunoenzyme method is rapid and does not require radiolabeled reagents.

We have developed an immunoradiometric assay for IgE antibodies to Staphylococcus aureus (Staph IgE-Ab) which uses purified cell walls (PCW) from the Wood 46 strain of S. aureus as an immunosorbent. We compared Wood 46 PCW and whole organisms (WO) as immunosorbents for Staph IgE-Ab by performing tests on sera from patients with atopic dermatitis (AD) or the hyperimmunoglobulin E syndrome (hyper IgE syndrome). Sera with Staph IgE-Ab demonstrated dose-dependent binding to PCW and WO, but the ratio of specific to non-specific binding was much greater with PCW. Mean non-specific binding to WO was greater than to PCW, 5% versus 2%; and non-specific binding to WO varied directly with the serum concentration of IgE. Results of tests on patients' sera indicated that PCW are required in screening assays for Staph IgE-Ab to avoid false positive results caused by high levels of non-specific binding to WO.

A 57-year-old woman with IgA deficiency and Still's disease was the proband in a 20-member, three-generation kindred in which we studied the possible linkage of IgA deficiency with her HLA-A1-B8 haplotype. The presence of paternal A1-B8 haplotype complicated the analysis. Known maternal HLA-A1-B8 haplotype, present in three of the children of the proband, was associated with IgA deficiency, whereas all five family members with exclusively paternal A1-B8 had normal IgA. Of three third-generation family members whose A1-B8 haplotype was of indeterminate origin--that is, potentially either maternally or paternally derived--two had IgA deficiency and one did not.

Two employees developed allergic rhinitis and bronchial asthma which was occupationally related to raising crickets. Skin tests, bronchial challenge, radioallergosorbent test (RAST), in vitro histamine release and a passive transfer test supported the presence of type I hypersensitivity to cricket allergens. Skin tests of other employees and patients of an allergy clinic suggested that cricket emanations are potent allergens.

To assess the etiologic role of cockroach antigen in bronchial asthma, 46 asthmatic subjects were studied using in vitro assays for total and cockroach-specific IgE antibodies (IgEcr) and the responsiveness of the skin and bronchial tree to the antigen challenge in vivo. Asthmatic subjects were divided into skin test-positive (PCR) and skin test-negative (NCR) groups according to immediate skin response to cockroach antigen. The 28 in the PCR group showed high total IgE (1,901 ng/ml) and a high cockroach-specific IgE antibody level (329%) in the serum compared to the 10 in the NCR group (IgE: 915 ng/ml, IgEcr: 84%) (p < 0.001). Bronchial challenge with the antigen revealed immediate asthmatic reaction (3033) and late asthmatic reaction (1633) in the PCR asthmatics, whereas the NCR asthmatics showed neither immediate asthmatic reaction (213 showed questionable decrease in FEV1) nor late asthmatic reaction (p < 0.001). A marked increase in peripheral eosinophils (758% vs 121%) was noted following antigen inhalation in the skin test-positive asthmatics (p < 0.025). The results indicate that cockroach antigen causes antigen-specific IgE-mediated bronchial asthma and peripheral eosinophilia in specifically sensitized asthmatic subjects.

On epidemiologic grounds, respirable particles from chilled-water air-conditioning systems in textile production plants have been implicated as a cause of hypersensitivity pneumonitis. We have purified the antigen from scum growing in this chilled water by antibody-affinity chromatography by use of IgG isolated from a pool of serum obtained from three workers with disease. Two patients with the disease, three coworkers without the disease, and two unexposed control subjects inhaled a dose of the purified antigen approximately equivalent to that amount calculated to be inhaled during an 8-hour work shift. Both workers with disease experienced fever, malaise, cough, and dyspnea 6 to 8 hours after the aerosol challenge. In these two patients the exposure evoked a transient decrease in circulating lymphocytes, predominately T cells. Before challenge the patients' peripheral blood mononuclear cells demonstrated a blastogenic response to the antigen. The responding cells had disappeared from the circulation 24 hours after the challenge. Seventy-two hours after the challenge, mononuclear cells that were producing large amounts of specific IgG antibody appeared in the circulation. We conclude that the same antigen(s) that react with IgG antibody produced an acute episode of the disease, that specific antigen-recognition cells disappear from the peripheral blood after exposure to the antigen (presumably because they are attracted to the lung), and that antibody-forming cells appear in the peripheral blood approximately 2 days after a challenge. These antigen-specific reactions of circulating mononuclear cells may be specific for the disease, but studies on a larger number of cases are needed to be certain.

T-cell chronic lymphocytic leukemia (T-CLL) accounts for about 2% of the various types of CLL and can be subtyped into helper/inducer (h/i) and cytotoxic/suppressor (c/s) cell membrane phenotypes. Seven patients with CLL were shown to have T-CLL with a h/i cell membrane phenotype; four with monoclonal antibody reagents and three by demonstration of the E-rosette receptor and focal acid alpha naphthyl acetate esterase activity. The clinical courses, treatment responses, and laboratory findings of these seven patients were reviewed to determine the prognosis and unique clinicopathologic features of this subtype. Two patients presented with skin rashes, and five were diagnosed during evaluation for other medical problems. Initially, four patients had splenomegaly and two had lymphadenopathy, but none of the patients had hepatomegaly. Morphologic examination revealed uniform, small lymphocytes in three patients, and the lymphocytes had nuclear indentations in four patients. Sera from the three patients tested were negative for antibody to the human T-cell leukemia/lymphoma virus I. Peripheral blood mononuclear cells from one patient showed normal interleukin-2 production and lacked antibody-dependent cell-mediated cellular cytotoxicity and natural killer activity. Cytogenetic analysis was done on one patient, revealing an abnormal clone with several chromosomal abnormalities, including an X; 14 translocation with a break point at 14q11. All patients required chemotherapy, and all died a median of 21 months from the time of diagnosis. The findings in these patients, in addition to those in 31 patients described in the literature, indicate that h/i T-CLL is associated with a poor prognosis and has distinct clinical and pathologic features that separate it from c/s T-CLL, adult T-cell leukemia/lymphoma, the cutaneous T-cell lymphomas, and B-CLL.

Blastogenesis of autoreactive T lymphocytes in the autologous mixed lymphocyte reaction has been shown to be decreased in patients with primary biliary cirrhosis, but the mechanisms underlying this abnormality are not known. To investigate further the abnormal autologous mixed lymphocyte reaction in primary biliary cirrhosis, we measured the activation of autoreactive helper/inducer and suppressor/cytotoxic T lymphocytes concurrently with blastogenesis in 35 patients with primary biliary cirrhosis and 18 healthy controls. Blastogenesis was diminished in autologous mixed lymphocyte reaction cultures from primary biliary cirrhosis patients compared to the control subjects (25,273 +/- 20,259 dpm vs. 36,004 +/- 14,951 dpm, p less than 0.02), but cultures from patients with primary biliary cirrhosis contained significantly increased percentages of activated helper/inducer and suppressor/cytotoxic T lymphocytes: 20.6 +/- 7.9 vs. 15.5 +/- 5.3%, p less than 0.008, and 9.8 +/- 7.8 vs. 5.4 +/- 3.0%, p less than 0.02, respectively. These findings were not related to the histologic stage of liver disease or to the serum bilirubin concentration and were not associated with abnormalities of lymphocyte subsets in fresh peripheral blood specimens. We conclude that the percentage of autoreactive T lymphocytes in autologous mixed lymphocyte reaction cultures from peripheral blood is increased in patients with primary biliary cirrhosis and diminished blastogenesis in autologous mixed lymphocyte reaction cultures is not due to the loss of autoreactive T lymphocytes from peripheral blood. Diminished blastogenesis reflects a diminished proliferative response of activated, autoreactive T lymphocytes in primary biliary cirrhosis. Possible mechanisms that may account for the paradoxical findings of decreased blastogenesis and increased activation of autoreactive T lymphocytes in primary biliary cirrhosis are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytic variability affects the accuracy of measurements of specific IgE antibodies, but the frequency of false results attributable to analytic variability is not well documented. We have monitored the accuracy of the results generated in our laboratory by testing aliquots of positive serum pools and a negative serum pool submitted blindly for the measurement of IgE antibodies to 16 different allergens, including foods; weed, grass, and tree pollens; mites, molds, and epithelia; and Hymenoptera venoms. Positive serum pools were prepared to contain modest amounts of IgE antibodies. Tests were performed by immunometric assays with microcrystalline cellulose allergen immunosorbents. Frank false-positive and false-negative results were very uncommon when binding levels were classified by a ratio-based reporting scheme. False-borderline results were more common. A borderline result is truly equivocal and may or may not indicate the presence of low levels of IgE antibodies. Analytic variability adds uncertainty to the measurement of small quantities of IgE antibodies regardless of the classification scheme used to report test results.

Relatively few laboratory tests are of proven value in the differential diagnosis and management of allergic diseases. Immunoassays for IgE and for IgE antibodies are the mainstays. Measurement of IgE in serum is advocated as a first-order laboratory test in the differential diagnosis of allergic disease in children and adults. The usefulness of laboratory tests for IgE antibodies in serum, once a subject of debate in the clinical allergy literature, is now firmly established. Confusion, in respect to the use of these tests, is most evident in clinical situations which have been the subject of limited clinical investigation, e.g., the use of tests for IgE antibodies to screen for allergic disease, the indications for their use in patients treated with allergen immunotherapy, and the diagnostic specificity of IgE antibodies to foods as an indicator of food-induced allergic symptoms. Confusion is also apparent in the interpretation of borderline test results, i.e., results which may indicate the presence of low titers of IgE antibodies, and in defining the optimum format for reporting results to maximize the analytical sensitivity of the test method. This review addresses the ambiguities noted above in the interpretation of results. The paragraphs that follow also consider the possible uses of laboratory tests for inflammatory mediators of immediate hypersensitivity, for IgG antibodies to allergens, and of tests designed to evaluate the in vitro functions of lymphocytes in patients with allergic disease.

Primary sclerosing cholangitis is a chronic, cholestatic liver disease in which immune mechanisms are thought to play a role in the pathogenesis. We have determined the frequencies and functional phenotypes of autoreactive T-lymphocytes in peripheral blood specimens from patients with primary sclerosing cholangitis and healthy adults as an indicator of autoreactivity in primary sclerosing cholangitis. We found a significant increase in the percentage of autoreactive suppressor/cytotoxic T-lymphocytes that become activated in the T-, non-T-autologous mixed lymphocyte reaction in primary sclerosing cholangitis patients (p less than 0.002). Blastogenesis in autologous mixed lymphocyte reaction cultures from primary sclerosing cholangitis patients was significantly decreased (p less than 0.02), and the magnitude of the decrease correlated directly with the percentage of activated suppressor/cytotoxic T-lymphocytes (r = 0.56, p less than 0.01). The percentage of autoreactive, suppressor T-lymphocytes was increased in autologous mixed lymphocyte reaction cultures from primary sclerosing cholangitis patients (p less than 0.0001); and suppression of mitogen-induced blastogenesis by autologous concanavalin A-treated mononuclear cells was significantly enhanced. These results, which were unrelated to the histologic stage of liver disease and the presence or absence of active colitis, document the presence of an expanded population of T-lymphocytes in the peripheral blood of patients with primary sclerosing cholangitis that became activated on exposure to autologous major histocompatibility antigens in the autologous mixed lymphocyte reaction. We hypothesize that these autoreactive cells may be a marker or may participate as effector cells in cell-mediated autoimmunity in primary sclerosing cholangitis.