Evaluation of an automated chemiluminescent immunoassay kit for antinuclear antibodies in autoimmune diseases

The identification of antinuclear antibodies (ANA) is an essential step in the diagnosis of different autoimmune diseases. The gold standard method for their detection is immunofluorescence assay. However, this is a subjective and laborious method, thus a need for simplified objective methods has aroused. In the current work, we evaluated such automated method, the LIAISON^® (DiaSorin, Italy) for the detection of ANA. A total of 242 sera were analyzed including 67 from healthy subjects, 107 from primary biliary cirrhosis (PBC) patients, 20 from scleroderma patients and 48 from patients with Sjögren’s syndrome. All sera were analyzed using the automated chemiluminescent immunoassays, LIAISON^® for the presence of ANA (kit No. 310300). Positive samples were further analyzed for the presence of antidouble-stranded DNA (dsDNA) and autoantibodies to 6 extractable nuclear antigens (ENA) of the LIAISON^® (kits No. 310330 and 310331). Negative samples were further analyzed by Blueblot ANA assay (D-TEK, Belgium) or BlueDot Liver (D-TEK, Belgium) as appropriate. The LIAISON^® specificity for ANA screening was 97 %. ANA positivity was determined in 80 % of all patients. The sensitivity was 95.5 % in scleroderma, 83 % in PBC and 72.9 % in Sjogren’s syndrome. ENA was positive in all ANA-positive scleroderma and Sjögren’s sera and in 27 % of ANA-positive PBC sera. Among scleroderma or Sjögren patients that were ANA negative, 4 samples were positive for anti-SSA and 2 for RNP-68 utilizing Blueblot assays. M2 protein was found in 1 out of the ANA-negative PBC patients. The LIAISON^® ANA screen is specific and sensitive for the evaluation of ANA in patients with primary biliary cirrhosis, scleroderma and Sjögren’s syndrome.     

Comparison of commercial diagnostic tests for Helicobacter pylori antibodies.

A number of serological tests measuring the presence of Helicobacter pylori-specific serum immunoglobulin G (IgG) are now commercially available. The aim of this study was to evaluate the clinical accuracy of five commercial H. pylori antibody tests: GAP-IgG (Biomerica), HELpTEST (AMRAD, Kew, Victoria, Australia), HELICO-G (Porton Cambridge), Pyloriset (Orion Diagnostica), and ROCHE (Roche Diagnostics). A total of 162 subjects presenting for routine upper endoscopy were studied. H. pylori was diagnosed if culture, histology, or both were positive. Ten milliliters of venous blood was collected at the time of endoscopy for serological assessment. The sensitivity and specificity of each test (GAP-IgG, HELpTEST, HELICO-G, Pyloriset, and ROCHE) were as follows: 83 and 79%, 92 and 77%, 86 and 65%, 89 and 56%, and 98 and 69%, respectively. Positive and negative predictive values were 97 and 83%, 90 and 91%, 76 and 83%, 68 and 84%, and 86 and 97%, respectively. The specificity of most tests increased by approximately 10% when sera from subjects less than 45 years old were examined. The number of sera falling into the grey zone for each test (an indeterminate result with respect to H. pylori status) varied between 2.5 and 19%. This study highlights the need for all serological kits to be independently evaluated on the population to be studied by testing against a microbiologically defined panel of H. pylori-positive and -negative sera.     

Evaluation of rapid diagnostic tests for typhoid fever

Laboratory diagnosis of typhoid fever requires isolation and identification of Salmonella enterica serotype Typhi. In many areas where this disease is endemic, laboratory capability is limited. Recent advances in molecular immunology have led to the identification of sensitive and specific markers for typhoid fever and technology to manufacture practical and inexpensive kits for their rapid detection. We evaluated three commercial kits for serologic diagnosis of typhoid fever. Patients presenting with > or = 4 days of fever were enrolled at two hospitals in Southern Vietnam. Cases were patients with serotype Typhi isolated from blood samples, and controls were patients with other laboratory-confirmed illnesses. Serotype Typhi isolates were confirmed and tested for antimicrobial susceptibility at the Pasteur Institute in Ho Chi Minh City. The Widal test was run at the hospitals and the Pasteur Institute. Sera were shipped frozen to the Centers for Disease Control and Prevention and tested by using Multi-Test Dip-S-Ticks, TyphiDot, and TUBEX to detect immunoglobulin G (IgG), IgG and IgM, and IgM, respectively. Package insert protocol instructions were followed. We enrolled 59 patients and 21 controls. The sensitivity and specificity findings were as follows: 89 and 53% for Multi-Test Dip-S-Ticks, 79 and 89% for TyphiDot, 78 and 89% for TUBEX, and 64 and 76% for Widal testing in hospitals and 61% and 100% for Widal testing at the Pasteur Institute. For all assays, the sensitivity was highest in the second week of illness. The Widal test was insensitive and displayed interoperator variability. Two rapid kits, TyphiDot and TUBEX, demonstrated promising results.     

Evaluation of rapid diagnostic tests for malaria in Swedish travellers

Rapid diagnostic tests (RDTs) for malaria have become valuable tools for the diagnosis of malaria in both endemic and non-endemic areas. During a 7-year period, first the MalaQuick rapid test and then the NOW Malaria test, were evaluated by well-trained laboratory technicians in a university hospital laboratory of parasitology. A total of 635 blood samples were selected from 4731 blood specimens obtained from travellers at the emergency department, at wards and at out-patient clinics. The samples were analysed by microscopy and RDT. Malaria parasites were detected in the blood films of 134 (21%) samples. The sensitivity of the RDT for Plasmodium falciparum was 97.7% (84 of 86 samples) with a negative predictive value of 99.6%. The two false-negative results were associated with low levels of parasitaemia. For non-falciparum species the sensitivity was only 58.3% (28 of 48 samples). Based on the excellent ability of the RDTs to detect P. falciparum infections, we recommend the use of the NOW Malaria test as a complement to microscopy in the laboratory.     

Offering prenatal diagnostic tests: European guidelines for clinical practice

For over four decades, it has been possible to offer prenatal diagnostic testing for fetal abnormalities. Prenatal testing is now available for a wide range of monogenic disorders as well as chromosomal abnormalities and should be provided within the ethical framework of informed consent and autonomous choice. However, there are no published guidelines for health professionals from varied disciplines who offer prenatal diagnosis (PND) in a range of possible settings including departments of maternity, obstetrics and clinical genetics. We used an Expert Group technique to develop a set of guidelines for provision of prenatal diagnostic services. Thirteen European health professionals, all experts in PND, participated in a workshop to develop the guidelines, which were then subjected to a wide consultation process. The objective of PND was defined as providing prenatal diagnostic testing services (for genetic conditions) that enable families to make informed choices consistent with their individual needs and values and which support them in dealing with the outcome of such testing. General principles, logistical considerations, clinical care and counselling topics are all described and are equally applicable to invasive and non-invasive testing. These guidelines provide a framework for ethical clinical care; however, they are flexible enough to enable practitioners to adapt them to their particular setting. Ideally, an individualised approach to each family is required to ensure autonomous choice and informed consent regarding prenatal diagnostic testing within the local ethical and legal framework.     

Evaluation of Rh monoclonal antibodies for flow cytometry tests

As participants of the “III International Workshop on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens”, we tested 43 RH monoclonal antibodies by the flow cytometry technique. Besides the anti-D antibodies (not included in this paper), we tested the following antibodies: three (3) anti-C; one (1) anti-C^w; six (6) anti-c; eight (8) anti-E; three (3) anti-e; two (2) anti-G; one (1) anti-CcEe (total = 24 antibodies). These antibodies (from different lab sources) were tested against antigen positive cells (homozygous or heterozygous) and antigen negative cells. When available, some of them were tested against “rare” phenotypes like r_yr, r″^Gr, r^Gr, R₂R_z. All the three anti-C tested, showed poor discrimination between positive and negative cells; from the six anti-c tested, only three had good results (Workshop n° 18, 20, 21) with a superior performance of one of them (Workshop n° 18). From the eight anti-E tested, we found two (Workshop n° 139 and 153) with good performance; all the three anti-e were non reactive; the two anti-G failed to react with r′r red cells; the anti-C^w reacted better with R₁^wr cells than R₁^wR₁; and the anti-CcEe antibody showed good results with all the phenotypes tested. From the 24 antibodies tested, we found six (25%) antibodies with a good performance.     

Evaluation of three commercial screening tests for AIDS virus antibodies

Three commercial enzyme-linked immunosorbent assays (ELISA) for acquired immune deficiency syndrome (AIDS) virus antibodies were evaluated using serum that had been characterized by an ELISA and western blot procedure developed at the University of California at Davis (UCD). Each of the commercial tests was more specific than the UCD ELISA, but the UCD ELISA was more sensitive in the detection of sera that lacked reactivity by western blot to the envelope glycoprotein (gp-41). The HTLV-III Bio-EnzaBead (Litton Bionetics, Charleston, SC) was less sensitive and specific than the Abbott HTLV-III EIA (Abbott Laboratories, North Chicago, IL) or the Virgo HTLV-III ELISA (Electro-Nucleonics Inc., Columbia, MD). Overall, 22.9% (57 of 250) and 51.0% of sera that were repeatedly (X2) positive by commercial screening kits tested at blood donor centers and clinical laboratories, respectively, were confirmed by western blot. These results indicate that the screening assays for AIDS virus antibodies are not equal in performance and that positive screening test results must be confirmed by a more specific test like western blot before results are released.     

Antigenic specificity of chlorpromazine-induced antinuclear antibodies

A few patients have been reported who developed systemic lupus erythematosus (SLE) in the course of prolonged treatment with chlorpromazine. Patients on this drug have also been found to have antinuclear antibodies (ANAs) although they do not develop lupus.

We have studied the antigenic specificity of ANAs in fifty-four patients on longterm chlorpromazine treatment and compared our findings with those on 175 patients on anticonvulsants, 215 patients on isoniazid, 109 SLE patients and fifty-four healthy subjects, sex and age matched to the chlorpromazine patients.

Thirty-nine per cent of patients on chlorpromazine had ANAs which were most frequently directed to single stranded (s)DNA. In contrast, patients on anticonvulsants as well as those on isoniazid had ANA directed to soluble nucleoprotein (sNP) most frequently and none of the patients on isoniazid had ANA to sDNA.

The mechanisms by which chlorpromazine, isoniazid or anticonvulsant intake results in ANAs probably differ. Our findings suggest that development of ANAs in patients on chlorpromazine may be initiated by interaction of the drug with denatured DNA.

     

An immunoenzymatic technique for the detection of antinuclear antibodies

In the present work, we propose, for the detection of the anti-nuclear antibodies (ANA) an immunoenzymatic test in simple sandwich in heterogeneous phase (ANA-EIA) allowing the screening of the antinuclear antibodies, and its assessment in comparison with the indirect immunofluorescent assay (IFA) for 292 sera of patients affected by rheumatic diseases and 40 sera of individuals presenting no pathology. The ANA-EIA test proved to have a sensitivity of 97% and a specificity of 96%. Moreover, it gives a global concordance of 96.6% and a discordance of 3.4% compared to IFA. Also, the analysis of correlation between this test and IFA for the positive individual gives a very good concordance according to homogeneous, speckled and nucleolar aspects. On the other hand, the results obtained for cytoplasmic aspect (74%), although acceptable, needs to be confirmed a second time with a more important sampling. According to the results obtained, this test could have an interesting routine application in laboratory of immunopathology given its analytic characteristics similar to those of IFA, and its great easiness of realisation.     

Evaluation of screening tests for the detection of antistreptolysin O antibodies

The accuracy of two screening tests, one utilizing serum and the other utilizing whole blood, was compared with the accuracy of the conventional macrotitration method for the detection of antistreptolysin O antibodies. Of the 569 specimens tested with the serum screening procedure and the macrotitration method, 235 and 282, respectively, were positive for antistreptolysin O antibodies. Comparative testing of 200 specimens with the peripheral blood screening test and the macrotitration method gave 60.5% by the macrotitration procedure. Discrepant results with both procedures were obtained with specimens having 166 Todd units. The results suggest that these screening procedures can be used to screen specimens for the presence of significant antistreptolysin O antibodies.     

Tissue specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guinea pig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate. Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.     

Prevalence of positive antinuclear antibodies in healthy children

Antinuclear antibodies (ANA) frequently arise in the sera of children with connective tissue disease and is used in the diagnosis of these diseases. Therefore it is also important to know the prevalence of ANA in normal children. The main objective of the present study was to determine the prevalence of antinuclear antibody (ANA) in healthy children. Ninety-nine serum samples from a serum bank and 108 samples from patients who had attended elective surgery and whose blood had been withdrawn for other investigations, were tested for ANA by indirect immunofluorescence method using HEp-2 cells as substrate. Sera from 52 children with SLE were also tested during the same period. It was found that antinuclear antibodies were present in 32 (15%) of the 207 sera of healthy children at a dilution of 1:40 or higher. ANA were positive in 9% at a serum dilution of 1:40, in 3% at 1:80 and in 3% at 1:160. The patterns of immunofluorescence staining were as follows: homogeneous in 46.7%, speckled in 20%, and nucleolar in 10%. In SLE patients, ANA were positive in 91%; 13% at a serum dilution of 1:40, 7% at 1:80, 20% at 1:160, 15% at 1:320, 9% at 1:640, 20% at 1:1,280 and 9% at > or = 1:2,560. It was concluded that the prevalence of positive ANA using the HEp-2 cells as substrate was 15% in healthy children at dilutions of 1:40 or higher. Using the cutoff serum dilution of 1:40, the sensitivity of this test was 91%, the specificity was 85%, the positive predictive value was 57% and the negative predictive value was 97%.     

Incidence of circulating antinuclear antibodies in cancer patients

The following study was conducted to know the incidence of antinuclear antibody [ANA] in various types of cancers in different age groups of both sexes. Results revealed that an overall level of ANA in female is higher than males. This study also showed that out of 50 only 20 cancer patients had raised level of ANA. Whereas in control group only one was positive for antinuclear antibody. Of these 20 positive cases, 4 were having very high titer of ANA, 13 showed high titer and 3 samples were moderately high titer of ANA. The high prevalence of autoantibodies found in aged cancer subjects could be attributed to several cellular and humoral immunological aberrations, which occur with the aging process. The results of this study confirm the earlier observation of necrosis of tumour tissue could be an important contributing factor for production of autoantibodies.     

Evaluation of the automatic fluorescent image analyzer, Image Titer, for quantitative analysis of antinuclear antibodies

By making comparisons with the usual manual method, we evaluated an automatic fluorescent image analyzer (Image Titer, Tripath Imaging, Burlington NC), the software for which was developed to simplify measuring indirect immunofluorescent antinuclear antibodies (FANAs). In this new system, images of the stained sample are displayed, and it measures the FANA titer and staining pattern using only 1 slide per subject and does not required the staining of a series of diluted samples as does the manual method. This system showed good reproducibility and linearity for 4 types of control serum samples (with homogeneous, speckled, discrete speckled, and nucleolar staining patterns). In 132 serum samples, consistency between the methods was 100% for the FANA staining pattern and 93.9% for the FANA titer. The Image Titer system detected each pattern in samples with 2 mixed patterns. This system should partly reduce labor and lead to results with minimum differences among individuals, including newly trained persons.     

Immunoglobulin G subclass of human antinuclear antibodies

Fourteen sera containing antinuclear antibodies were studied to determine whether the immunoglobulin G subclass of these antibodies correlated with the relative complement fixing activity of the antibodies or the presence or absence of lupus nephritis. Sera studied included eight with high complement fixing activity, all from patients with active lupus nephritis and six with low complement fixing activity, three from patients with lupus without nephritis and three from patients with antinuclear antibodies induced by procainamide. Purified antinuclear antibodies reactive with desoxyribonucleoprotein and desoxyribonuclease were prepared by elution, and subclass typing was carried out by radial immunoassay with specific antisera.In general, subclasses represented were similar in frequency to the occurrence of the subclasses in normal immunoglobulin G. There was no striking predominance or absence of any single subclass which might correlate with presence of nephritis, or differing complement fixing activity of sera. There was a trend to a predominance of subclasses high in complement fixing activity in antinuclear antibodies from lupus patients with active nephritis and subclasses low in complement fixing activity from those without nephritis.     

`Nuclear' antigens and antinuclear antibodies in mink sera

Aleutian disease of mink is transferrable by cell-free extracts and is characterized by hepatitis, vasculitis, nephritis, and hypergammaglobulinaemia. Because of increasing evidence incriminating antigen—antibody complexes in vasculitis disorders, the presence of nuclear antigens and antinuclear antibodies in mink sera was investigated.

Serum pools as well as individual serum specimens were obtained from uninfected mink, mink with experimentally induced Aleutian disease, mink with spontaneous Aleutian Disease, all of genotype Aa as well as from `normal' mink of similar age from colonies without Aleutian disease.

The serum pool from mink before and after experimentally induced Aleutian disease appeared to contain `nuclear' antigens detectable by rabbit anti-DNA antibodies in complement fixation and precipitin tests. The protein-free extracts of these serum pools gave strong reactions for deoxypentose in the diphenylamine tests. These serum pools also were shown to contain antinuclear antibodies by the immunofluorescence tests on human leucocyte nuclei and in precipitin tests against single strand calf thymus DNA. Sera from individual mink were similarly shown to contain `nuclear' antigens and antinuclear antibodies. The incidence and quantity of antigens and antibody detected were much greater in sera from mink after experimentally induced disease than in sera taken from mink before inoculation. The presence of `nuclear' antigens and antinuclear antibodies did not correlate with the degree of hypergammaglobulinaemia. Sera from `normal' mink in colonies without overt disease had neither antigens nor antibodies detectable in precipitin tests. Sera from mink with spontaneously acquired Aleutian disease had a high incidence of `nuclear' antigens and anti-DNA antibodies detectable in precipitin tests.

The `nuclear' antigens were detectable in Ouchterlony precipitin tests by specific rabbit anti-DNA antibodies. The precipitins formed lines of partial identity with those between the rabbit anti-DNA antibodies and single strand calf thymus DNA. However, the antigens in mink sera were not destroyed by prior incubation with DNAse which had been the case with DNA antigens detected in some human and mouse sera.

The antinuclear antibodies were detected in immunofluorescence tests using specific antibodies to mink γ-globulins, were shown to fix complement with single strand calf thymus DNA, but not with DNA that had been digested with DNAse, and formed precipitins with single strand calf thymus DNA which showed complete identity with precipitins formed by rabbit anti-DNA antibodies. Evidence for the simultaneous presence of `nuclear' antigens with antinuclear antibodies in the serum from mink with Aleutian disease was frequently evident. This observation is consistent with the hypothesis for the pathogenetic role of antigen-antibody complexes.

Aleutian disease of mink has certain clinical pathological and serological similarities with disease in New Zealand Black mice and in man with systemic lupus erythematosus. Since Aleutian disease of mink and disease of New Zealand black mice may both be examples of `slow virus' infections, a similar aetiology should be considered for certain autoimmune diseases of man, e.g. systemic lupus erythematosus.