Comparison of antinuclear antibody profiles obtained using line immunoassay and fluorescence enzyme immunoassay

ObjectiveLIA-ANA-Profile-17S is a multiplex line immunoassay that simultaneously detects 17 antinuclear antibodies (ANAs) against extractable nuclear antigens (ENAs). We evaluated the utility of LIA-ANA-Profile-17S as a supplement to ANA indirect immunofluorescence (IIF) and EliA ENA (a fluorescence enzyme immunoassay) for diagnosis of ANA-associated rheumatic diseases.MethodsSera were collected from 245 patients referred for an ANA IIF test. LIA-ANA-Profile-17S results were compared with those of EliA ENA. The kappa coefficients, agreement rates, and diagnostic performance of these tests were assessed for systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SjS).ResultsWe observed almost perfect interassay agreement for antibodies against Ro52/Ro60, CENP-B, and Scl-70 (kappa = 0.91, 0.97, and 1.00, respectively); strong agreement for anti-SS-B/La antibody (kappa = 0.81); and relatively low agreement for other antibodies, including those against dsDNA, Sm, RNP, and Jo-1. For SLE diagnosis, LIA-ANA-Profile-17S showed lower sensitivity and similar specificity compared with EliA ENA. The sensitivity and specificity of these two assays were similar for SjS diagnosis.ConclusionsThe specificity of LIA-ANA-Profile-17S was enhanced when combined with ANA IIF and was comparable with that of EliA ENA. LIA-ANA-Profile-17S showed relatively good agreement with EliA ENA. In combination with ANA IIF, these assays showed enhanced diagnostic performance.     

Diagnostic assays for Anti-PM/Scl IgG antibodies: Heterogeneity in antibody response or lack of standardization?

BackgroundThe aim of this study was to compare the correlation between new diagnostic methodologies for detecting anti-polymyositis/scleroderma (anti-PM/Scl) IgG antibodies associated with myositis and/or systemic scleroderma assays with existing platforms.MethodsSera from 164 samples previously tested for anti-PM/Scl IgG antibody by immunodiffusion, ID; 171 sera screened for anti-PM/Scl IgG by immunoprecipitation, IP; an additional group of 215 sera tested by ID and 46 healthy blood donor sera were retrospectively evaluated. Anti-PM/Scl IgG antibodies were measured using three PM/Scl-100 specific enzyme immunoassays (EIAs), PM1-alpha (PM1-α) EIA and a line immunoblot assay (LIA) for anti-PM/Scl-75 and ? 100 IgG antibodies. Selected samples were tested for the presence of antinuclear antibody (ANA) by indirect fluorescent antibody (IFA) assay.ResultsThe overall agreement between ID and all anti-PM/Scl IgG EIAs as determined by Crohnbach's alpha was unacceptable (α < 0.50). The concordance between the IP and either LIA or PM1-α EIA was greater than 90% however, the best agreement was seen between the IP and LIA PM/Scl-100 assays (98.3%). Compared to the LIA PM/Scl-75 and PM1-α tests, the LIA PM/Scl-100 IgG assay showed the best specificity in the healthy control group.ConclusionsOur results demonstrate considerable differences between assays for detecting anti-PM/Scl IgG antibodies which cannot be attributable to heterogeneity in antibody response alone. Further characterization and standardization of these assays are needed.     

3种检测方法在诊断自身免疫性疾病中的价值

目的通过对抗核抗体、可提取性核抗原(ENA)及抗核抗体谱3检测方法的分析比较,探讨有益于临床诊断的检测组合。方法采用人喉癌上皮细胞及猴肝作为基质的间接免疫荧光法检测抗核抗体;应用欧蒙斑点法检测可提取性核抗原中的如下抗原:nRNP/Sm、Sm、SSA、SSB、Scl-70、Jo-1;以绿蝇短膜虫为基质,应用间接免疫荧光法检测抗双链DNA抗体;通过欧蒙印迹法检测15种自身抗体。结果将3种稀释度(1:10、1:80、1:100)抗核抗体检测的阳性率进行比较,1:10及1:80稀释的血清抗核抗体阳性率差异有统计学意义(P0.01),其余2种比较差异无统计学意义;在核点型、胞浆颗粒型及均质型等多种核型的检测中抗核抗体谱3的阳性检出率高于ENA谱;在抗双链DNA抗体的检测中间接免疫荧光法的阳性检出率略高于欧蒙印迹法。结论在应用间接免疫荧光法的抗核抗体检测中,使用1:80的稀释度更有利于提高检出率,而且涵盖15种抗原的抗核抗体谱3也为临床诊断提供了有利的依据。     

An enzyme immunoassay screening test for the detection of total antinuclear antibodies.

The enzyme immunoassay antinuclear antibody (EIA ANA) screening test method is a new assay format for the qualitative determination of antinuclear antibodies (ANAs) in human serum or plasma. This assay method collectively detects ANAs against double stranded DNA (dsDNA), SS-A/Ro, SS-B/La, Scl-70, Sm, and Sm/RNP antigens, along with serum positive for peripheral, homogeneous, speckled, nucleolar, and centromere patterns. This assay correlated well with data obtained with hemagglutination tests for specific ANA antigens, with the indirect immunofluorescence (IFA) ANA HEp-2 test, and with the Crithidia luciliae IFA test for anti-dsDNA. This new test procedure is both highly specific and sensitive and substantially decreases the time involved when screening large numbers of patient samples.     

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA)

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep-2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement. For SS-B antibodies, there was a good concordance between all four methods used (Kappa 0.66–0.74). For anti RNP antibodies, the results for CIE/ELISA (Kappa 0.60) were consistent as were the two immunoblot methods (Kappa 0.69). For anti Scl-70 (topoisomerase I) antibody, results from the ELISA and CIE methods were totally consistent (Kappa 1.00). In spite of the high prevalence of anti SS-A/Ro antibodies, the agreement between the methods was poor, without statistical significance. Finally, for Sm antibodies, more consistent results were obtained between CIE/ELISA (Kappa 0.51) and between one of the immunoblotting methods and ELISA (Kappa 0.54). In conclusion, CIE concurs mostly with ELISA for anti- RNP, Scl-70, Sm and SS-B antibodies, but with some disagreement for SS-A antibodies. J. Clin. Lab. Anal. 11:388–392, 1997. © 1997 Wiley-Liss, Inc.     

125例SLE患者血清抗核抗体及抗核抗体谱检测结果分析

目的探讨抗核抗体（ANA）及抗核抗体谱对系统性红斑狼疮（sLE）的临床价值。方法对1525例确诊的SLE患者血清ANA及ANA谱进行检测分析。ANA和抗双链DNA抗体采用间接免疫荧光法（IIF）,ANA谱采用欧蒙印迹法。结果①125例SLE患者血清ANA阳性共121例,阳性率为96．8％。②患者血清ANA谱大多为抗体二联及以上共同出现阳性,各抗体阳性率分别为抗一ul—nRNP／Sm：48．8％、抗一Smtl5．2％、抗一SS—A：36％、抗一R0—52：54．4％、抗一SS—B：14．4％、抗一scl—70：O．8％、抗一dsDNA：55．2％、抗一Neukleosmone：39．2％、抗一Histone：37．6％、抗一Rib—Pro!：12％、抗一Jo一1和抗一CENP—B未检出。结论ANA和ANA谱联合检测在SLE诊断中具有互补性,可提高检出率,对SLE的诊断分型、治疗、预后判断和病情随访等均有重要的临床价值。     

Evaluation of adsorption selectivity dextran sulfate bound cellulose beads for the removal of anti-DNA antibodies.

The purpose of this study was to clarify the basic adsorption selectivity characteristics of dextran sulfate (DS) columns (Selesorb, Kaneka Corporation, Osaka, Japan). Recovery rates of blood chemical components, hormones, coagulation factors, and antinuclear antibodies (anti-SS-A, SS-B, Sm, Scl-70, and RNP antibody) in vitro were assessed by mixing normal volunteers' or patients' sera with DS bound cellulose beads. For tested blood chemical components other than triglyceride and total cholesterol, the recovery rate was not changed significantly by incubation. No significant changes in hormone levels resulted from incubation. Among coagulation factors, the activities of antithrombin III, plasminogen, and factors V, VIII, IX, XI, and XII were significantly reduced by incubation. Among antinuclear antibodies tested, anti-SS-A and anti-RNP were absorbed to some extent, but not anti-SS-B, Sm, or Scl-70 antibodies. Taking into account these characteristics, apheresis therapy using a DS column should be performed.     

Detection and identification of significant ANAs in previously determined ANA negative samples

BACKGROUND: An antinuclear antibody (ANA) substrate transfected with the cDNA to hyperexpress the 60 kD SS-A/Ro antigen (HEp-2000) has been shown to detect anti-SS-A/Ro antibodies missed by standard HEp-2 and other immunoassays. Despite this evidence, many laboratories remain convinced that with experienced technicians, standard HEp-2 is acceptable for ANA detection. AIM: To challenge the ability of HEp-2000 to detect anti-SS-A/Ro antibodies in samples previously determined to be ANA negative using standard HEp-2. METHODS: Three hundred and seventy-one pre-screened "negative" ANA samples were provided by a university hospital laboratory in Germany. These samples were tested on the HEp-2000 substrate at a dilution of 1:40 by indirect immunofluorescence (IIF). Samples that screened positive for a nuclear pattern were titered (range of 1:40-1:640) and all ANA-positive patterns were identified. Samples containing at least one positive ANA pattern at a dilution greater than or equal to 1:160 were further tested. Samples that produced a speckled pattern were tested for antibodies to the extractable nuclear antigens (ENA) and samples that showed homogeneous staining were tested for antibodies to dsDNA, and if negative, were then tested for anti-histone antibodies. RESULTS: Ninety-one patient samples were positive with titers > or =1:160. Speckled patterns were the most common finding (30 samples) followed by speckled/homogeneous mixed patterns (19 samples) and samples demonstrating the SS-A/Ro pattern (16 samples) either alone or in combination with other ANA patterns. The remaining 26 positive samples consisted of various other ANA patterns. The most commonly identified ENAs were SS-A/Ro (14 samples), Scl-70 (11 samples) and SSB (6 samples). No antibodies to dsDNA were identified in 23 positive samples with homogeneous staining patterns, though 17 of these samples tested positive for antibodies to histone. CONCLUSIONS: HEp-2000 detected anti-SS-A/Ro antibodies in 16 (4%) of the "ANA negative" samples. In addition to improved sensitivity for anti-SS-A antibodies, HEp-2000 demonstrated improved sensitivity over standard HEp-2 substrate for other significant ANAs including anti-Scl-70, anti-histone, and anti-SS-B antibodies.     

定量酶联免疫吸附试验检测各种结缔组织病患者血清中的抗可提取核抗原抗体

目的　定量检测各种结缔组织疾病 (CTD)患者血清中抗可提取核抗原 (ENA)自身抗体滴度 ,并探索其临床意义。方法　自制抗各种ENA单克隆抗体 ,以夹心酶联免疫吸附试验 (ELISA)定量检测 1 2 8例CTD患者、5 0例非CTD患者及 6 0名健康人血清的各种抗ENA自身抗体滴度。用健康人血清各ENA抗体滴度的均数加上 3倍标准差 ( x +3s)为cutoff值 ,界定各种ENA抗体检出率。结果　与健康对照组和非CTD患者组比较 ,抗Sm抗体在系统性红斑狼疮 (SLE)、抗ul RNP抗体在SLE和混合性结缔组织病 (MCTD)、抗SSA和SSB在干燥综合征 (SS)、抗Scl 70抗体在系统性硬化病 (PSS)、抗Jo 1抗体在多发性肌炎 /皮肤炎 (PM/DM)患者中的水平和阳性率都明显升高 (P <0 .0 1 )。结论　定量检测CTD患者血清中各种ENA抗体滴度 ,对CTD疾病的鉴别、诊断和分型具有重要价值     

Cellular immune response to hepatitis C virus (HCV) in nonviremic blood donors with indeterminate anti-HCV reactivity

BACKGROUND: Blood donors with indeterminate hepatitis C virus antibody (anti-HCV) reactivity are rejected from blood donation. As they are mostly nonviremic, the source of these reactions remains unclear. Reasons for such findings can be resolved HCV infections as well as unspecific antibody reactions. The aim of this study was to investigate HCV-specific T-cell response in blood donors to determine the reason for the weak antibody detection.
STUDY DESIGN AND METHODS: Anti-HCV reactivity was tested in 72 blood donors initially diagnosed with an indeterminate HCV result by enzyme-linked immunosorbent assay and immunoblot. Cellular immune response was measured by proliferation assay and enzyme-linked immunosorbent spot analysis after stimulation with viral proteins core, NS3, and NS4.
RESULTS: In 56% of donors anti-HCV reactivity was detectable in the screening assay whereas 72% had a reaction in the confirmation immunoblot. Forty-six percent of donors had a cellular immune response against HCV proteins. The response was most frequent to NS3 protein.
CONCLUSION: In almost half of donors the indeterminate result in serologic testings could be explained by a previous resolved HCV infection as the pattern of T-cell response was similar to these patients. These findings indicate that HCV-specific antibodies disappear more rapidly after resolved infection than HCV-specific T cells. These results are important for counseling blood donors and patients with indeterminate serologic results.     

Laboratory screening of connective tissue diseases by a new automated ENA screening assay (EliA Symphony) in clinically defined patients

BACKGROUND: The measurement of antinuclear antibodies (ANA) is used in the autoimmune laboratory for the screening of connective tissue diseases (CTD). ANA measurements are mainly performed by indirect immunofluorescence (IIF) on HEp-2 cells or by enzyme immunoassay (EIA). The objective of this study was to clinically evaluate an automated EIA for extractable nuclear antigens (ENA) which lacks anti-dsDNA for the screening of CTD. METHODS: The study involved a total of 170 serum samples, 54 from patients with CTD, 26 from patients with other autoimmune diseases, and 90 from patients with non-autoimmune diseases. For all sera, ANA detection was performed by IIF and by EliA Symphony (Pharmacia Diagnostics, Freiburg, Germany), an ENA screening which detects the following autoantibodies: SSA/Ro, SSB/La, U1RNP (70 kDa, A, C), Scl-70, JO-1, centromere B and Sm. Also, anti-dsDNA (EliA dsDNA, Pharmacia Diagnostics, Freiburg, Germany) was measured on all samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), efficiency, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were calculated. RESULTS: Diagnostic efficiency was similar for IIF (82.6%) and EliA Symphony (82.3%), as well as PLR (6.5 for IIF, and 7.3 for Eli Symphony), and NLR (0.35 for IIF, and 0.41 for EliA Symphony). The combined measurement of EliA Symphony and dsDNA increased sensitivity but not PLR. Area under receiver operator characteristic (ROC) curve was similar for IIF (0.847) and EliA Symphony (0.823). CONCLUSIONS: The results of the study demonstrate that EliA Symphony solely or combined with anti-dsDNA detection has an efficiency similar to HEp-2 cells IIF with a cut-off of 1:160 for the diagnosis of CTD.     

Evaluation of the Bio-Rad syphilis IgG test performed on the CODA system for serologic diagnosis of syphilis

The performance of the Bio-Rad Syphilis IgG EIA test as a "screen for syphilis" [testing first by EIA and then by the rapid plasma reagin (RPR) assay if the EIA was positive or equivocal] and as a confirmatory test was evaluated by comparing results to those obtained by CAPTIA Syphilis-G. Discrepancies were resolved by repeating both EIAs and/or the SeroDia TP-PA (a particle agglutination assay that replaced the microhemagglutination Treponema pallidum test). Both EIAs were totally automated, the Bio-Rad test using the AutoPrep instrument for pipetting and the CODA system to perform all of the steps required to complete the EIA and interpret results, and the CAPTIA test using the LabOTech(R) to accomplish both functions. Of 449 unselected sera submitted to "screen for syphilis," both EIAs agreed for 432 (96.2%) specimens: 395 negative, 36 positive, and one equivocal. Fifty-four specimens were positive or equivocal by one or both EIAs; 41 of these were RPR reactive. Three of these 41 were incorrectly called negative by Bio-Rad (sensitivity 92.7%), and there was 1 false-negative result by CAPTIA (sensitivity, 97.6%) (P, not significant). To further evaluate the Bio-Rad assay as a confirmatory test, 144 known RPR-reactive specimens were tested by both EIAs. Results agreed for 134 (93.1%): 123 positive, 11 negative. After resolving discrepancies, there were 3 false-negative and no false-positive results by Bio-Rad (sensitivity 97.8%, specificity 100%), and with CAPTIA there were no false-negative results and 1 false-positive (sensitivity 100%, specificity 91.7%) (P, not significant). The sensitivity of the Bio-Rad assay could be improved, without altering specificity, by lowering the cut-off value for equivocal results. In summary, the Bio-Rad Syphilis IgG EIA performed using the AutoPrep instrument and CODA system is a reliable, efficient method of syphilis testing.     

A comparison of two supplemental procedures for confirmation of antibody to hepatitis C virus c100-3 antigen in Louisiana blood donors

In a pilot study designed to evaluate the performance of supplemental hepatitis C virus (HCV) tests, 146 consecutive HCV enzyme immunoassay (EIA)-reactive samples (0.98% of 14,949 donors) were comparatively evaluated with two sets of supplemental tests: HCV antibody neutralization/c100-3 peptide EIA and the first-generation HCV recombinant immunoblot assay (RIBA). Of these samples, 68.5 percent were positive and 17.8 percent were negative on both supplemental assays. Nineteen samples were discordant. Eleven samples were positive on one assay (9 on neutralization/peptide, 2 on RIBA) and negative or indeterminate on the alternate supplemental test, but reacted with two additional HCV antigens outside the c100-3 region in a second-generation dot immunoblot assay. The dot immunoblot assay was used as a reference and reactive samples were considered confirmed. The remaining eight discordant samples were indeterminate or negative on either assay and did not react on the dot immunoblot assay. These data indicate a 0.74-percent prevalence of HCV exposure detected by reactivity with the c100-3 antigen in blood donors in southern Louisiana.     

Evaluation of the RheumaStrip ANA profile test: a rapid test strip procedure for simultaneously determining antibodies to autoantigens U1-ribonucleoprotein (U1-RNP), Sm, SS-A/Ro, SS-B/La, and to native DNA

The "LipoGen RheumaStrip ANA Profile" test method (LipoGen, Inc.) is a new assay format for autoantibody detection in which recombinant autoantigens are used. This enzyme immunoassay, in test-strip format, detects antibodies to autoantigens U1-ribonucleoprotein (U1-RNP), Sm, SS-A/Ro, SS-B/La, and to native DNA (nDNA). We evaluated 200 antinuclear antibody (ANA)-positive and 100 ANA-negative sera for the presence of antibodies to U1-RNP, Sm, SS-A/Ro, SS-B/La, and nDNA by the new test-strip procedure. These data correlated well with those obtained with either Ouchterlony double immunodiffusion for U1-RNP, Sm, SS-A/Ro, and SS-B/La or with Crithidia luciliae indirect immunofluorescence for anti-nDNA. Assay sensitivity and assay specificity of the ANA Profile method as compared with those of established procedures were respectively as follows: 89.8% and 98.8% for U1-RNP, 86.4% and 95.3% for Sm, 97.9% and 89.3% for SS-A/Ro, 98.3% and 86.3% for SS-B/La, and 97.5% and 93.1% for nDNA. Agreement between the ANA Profile test and these other test methodologies ranged from 88.7% for the SS-B/La test to 97.3% for the U1-RNP test. This new test procedure substantially decreases the time and effort required to perform these assays. Total hands-on time and overall assay time were decreased by 72% and 97%, respectively.     

Anti-PM/Scl-100 and anti-RNA-polymerase III antibodies in scleroderma

BackgroundSystemic sclerosis (SSc) is a rare autoimmune disease characterized by the presence of various autoantibodies, including anti-centromere, anti-topoisomerase (Scl-70), anti-PM/Scl-100, and anti-RNA-polymerase III (RNA Pol-III) antibodies. Recently, new ELISA based immunoassays have become available for the detection of anti-PM/Scl and anti-RNA Pol-lII antibodies.ObjectiveWe studied the prevalence and clinical association of anti-PM/Scl-100 (PM1-Alpha) and anti-RNA Pol-III antibodies.MethodsAntibodies to PM1-Alpha and RNA Pol-III were measured by ELISA (DR. Fooke Laboratories and Inova Diagnostics, respectively) in 242 patients with various connective tissue diseases (CTD) (including 70 SSc patients) and in 36 non-CTD controls.ResultsLow levels of PM1-Alpha antibodies were found in various CTDs, whereas high levels were exclusively found in SSc, dermatomyositis and polymyositis, albeit at low frequency (4.7%). Anti-RNA Pol-III antibodies were found in 7% of SSc and in 1% of non-CTD and CTD controls. Anti-centromere and anti-Scl-70 antibodies were found in 37% and 21% of SSc patients, respectively. Anti-centromere antibodies were associated with limited cutaneous SSc and anti-Scl-70 antibodies with diffuse cutaneous SSc and interstitial lung disease. Because of the low number of samples positive for anti-PM/Scl-100 or RNA Pol-III antibodies, no clinical feature was statistically correlated with the presence of either reactivity, but taken together the presence of either antibody was correlated with interstitial lung disease. Anti-PM1-Alpha and anti-RNA Pol-III antibodies were mutually exclusive with anti-Scl-70 antibodies.ConclusionsAt high levels, anti-PM/Scl-100 antibodies were associated with SSc, PM, and DM, albeit at low frequency. Anti-RNA Pol-III antibodies were associated with SSc (in 7%) with high specificity.     

蛋白芯片技术及免疫印迹技术检测抗核抗体的比对研究

目的 通过比对免疫印迹法与蛋白芯片法对血清标本中抗核抗体的检测结果,验证这两种方法是否具有等效性. 方法 分别用免疫印迹技术和蛋白芯片技术检测70例临床标本的抗核抗体,记录检验结果,并对检查结果进行统计学分析. 结果 经配对资料卡方检验分析,两种方法测定SSA、SSB、SM、Scl-70、Jo-1和CENP时结果差异无...     

抗核抗体谱检测对弥漫性结缔组织病的临床诊断价值

目的通过抗核抗体谱检测结果对弥漫性结缔组织病患者临床诊断进行评价。方法回顾分析246例弥漫性结缔组织病和160例非弥漫性结缔组织病住院患者自身抗体检测结果。结果在各种弥漫性结缔组织病中抗核抗体谱阳性检出率间差异有统计学意义,在系统性红斑狼疮患者中,抗dsDNA抗体阳性率为44.1%,抗Sm抗体阳性率为47%;在混合性结缔组织病中抗UIRNP抗体阳性率达96.4%;在干燥综合征患者中抗SSA和抗SSB抗体阳性率为68.2%和59.1%;在系统性硬化病患者中抗Scl-70抗体阳性率为41.4%;重叠综合征抗dsDNA、抗Sm、抗UIRNP、抗SSA抗体的阳性率为45.4%、18.2%、45.4%、18.2%。结论抗核抗体谱检测对各种弥漫性结缔组织病有重要的鉴别诊断意义。      

Laboratory testing for the antibodies that cause heparin-induced thrombocytopenia: how much class do we need?

Heparin-induced thrombocytopenia (HIT) is usually caused by platelet-activating antibodies of immunoglobulin G class that recognize platelet factor-4 (PF4) bound to heparin or certain other polyanions. Commercial enzyme immunoassays (EIAs) for PF4/polyanion-reactive antibodies detect two immunoglobulin classes (IgA and IgM) besides IgG. To investigate whether the additional detection of these antibody classes improves or worsens assay operating characteristics, we compared the sensitivity and specificity of EIAs that detect these 3 immunoglobulin classes individually with that of a commercial EIA (Genetic Testing Institute, GTI), as well as a platelet-activation assay, the serotonin-release assay (SRA). We compared the operating characteristics of these 5 assays by evaluating 448 patients, in 14 of whom clinical HIT developed, who received either unfractionated or low molecular weight heparin in prospective studies that included systematic platelet-count monitoring and serologic evaluation for anti-PF4/polyanion antibodies. We found that the SRA and IgG and commercial EIAs had similar high sensitivity for HIT; however, diagnostic specificity (for unfractionated and low molecular weight heparin, respectively) varied considerably, as follows: SRA (95.1%, 97.2%) > IgG EIA (89.0%, 93.7%) > GTI EIA (74.2%, 87.6%). Additional detection of IgA and IgM antibodies by the GTI EIA worsened test specificity by detecting numerous nonpathogenic antibodies. Moreover, the frequency and magnitude of IgA and IgM antibody formation in non-HIT immune responses did not differ from that exhibited by patients in whom clinical HIT developed. We conclude that an EIA that detects anti-PF4/polyanion antibodies of only the IgG class has greater diagnostic usefulness in revealing clinical HIT than does an assay that also detects IgA and IgM class antibodies.     

Scl-86, a marker antigen for diffuse scleroderma.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.     

IgA and IgM human immunodeficiency virus antibodies in weakly reactive or false-negative blood donors

Detection of antibodies to the human immunodeficiency virus (HIV) in recently infected donors is crucial to prevent the transmission of HIV infection via blood products. To determine whether specific antibodies of the IgA or IgM class are present as markers of recent infection in donor specimens that have borderline reactivity on routine enzyme immunoassay (EIA) screening, 15 specimens that were positive by immunoblot were tested for IgA and IgM HIV antibodies. All 15 had detectable IgA HIV antibodies, and 14 had IgM HIV antibodies. The 15 specimens were tested further, each by two independent laboratories, with nine licensed EIAs. Two of the nine EIAs found all 15 units positive in both laboratories; seven EIAs found 1 to 5 of the 15 units negative, for a total of 31 false-negative results. The results indicated a difference between the sensitivity of EIA kits using only anti-IgG reagents and of kits using multispecific reagents that react with IgG and other classes of antibody. In a modified procedure, the addition of enzyme-conjugated anti-IgA or anti-IgM to the kit's enzyme-conjugated reagent increased the optical density values of most false-negative specimens to the positive range. It was concluded that licensed kits vary in reactivity with IgA and IgM HIV antibodies and that sensitivity could be increased by improved detection of these classes of antibody.