Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

Immunochemical measurement of serum prostatic acid phosphatase (PAP). Clinical evaluation of radioimmunoassay and counter immunoelectrophoresis.

Two radioimmunoassay procedures (RIA-1 and RIA-2) were evaluated for the quantitation of prostatic acid phosphatase in serum and compared with the enzymatic method and counter immunoelectrophoresis method for their specificity and sensitivity. Sera from 168 patients were analyzed and these included: normals, 27; untreated prostatic cancer patients Stage A, 2; Stage C, 3; Stage D, 17; cancer of prostate treated with different modalities, 42; sarcoma of prostate, 1; prostatitis, 3; nonprostatic carcinoma, 17; and benign prostatic hyperplasia (BPH), 56. RIA-1 procedure appeared more sensitive (82% sensitivity) and specific (94.5% specificity) than the RIA-2 procedure (68% sensitivity and 91.8% specificity), but the differences were not statistically significant. The enzymatic method was found to be least sensitive (63.6% sensitivity) but also the most specific (100% specificity). Only 69 of the specimens were analyzed by counter immunoelectrophoresis, which showed sensitivity of 87% and specificity of 51.4%. False positives were observed more often in patients with nonprostatic cancer and BPH. The variations in diagnostic specificity of immunologic assays suggest the need of characterization of each antibody specificity.

The role of counterimmunoelectrophoresis in the identification of antibodies to extractable nuclear antigens.

Antibodies to saline-extractable nuclear antigens are hallmarks of autoimmune diseases including systemic lupus erythematosus (SLE), mixed connective tissue disease, progressive systemic sclerosis and Sjogren's syndrome. In our laboratory, we use counterimmunoelectrophoresis as a screening test and immunodiffusion as a confirmatory test to identify these autoantibodies. This study examines the drawbacks of such an approach. Though 17 out of 19 sera that formed ribonuclease sensitive lines with rabbit thymus extract on counterimmunoelectrophoresis were confirmed to have anti-RNP by immunodiffusion, sera of several different autoantibody specificities were seen to form ribonuclease resistant precipitin lines with the thymus extract on counterimmunoelectrophoresis. Having screened sera to have autoantibodies by counterimmunoelectrophoresis, the identity of some of these autoantibodies were not confirmed because of the poor sensitivity of immunodiffusion or because inappropriate controls had been used for the confirmatory immunodiffusion test. To check these drawbacks and to obviate the need for a confirmatory test, a modification of the current approach is suggested.

Evaluation of co-agglutination (COA), counter immunoelectrophoresis (CIE), culture and direct microscopic (Dm) examination of cere-brospinal fluid (CSF) for detection of meningitis caused by common bacterial pathogens.

Cerebrospinal fluid from 260 children clinically diagnosed as meningitis were examined by Dm, culture, COA and CIE test. Dm revealed the presence of bacteria in 41 (15.8%) whereas culture showed growth of organism in 52 (20%) cases. COA and CIE test were done for the detection of antigen of H. influenzae, S. pneumoniae and N. meningitidis. Among the 3 methods viz. culture, COA and CIE test which were used for the detection of the above three organisms COA detected the maximum numbers (23.5%). COA test could detect antigen in both culture positive and culture negative CSF samples. COA test detected 100% of pneumococcal, 88.5% of H. influenzae and 66.7% of N. meningitidis antigens from CSF. Diagnosis by CIE in detecting H. influenzae and N. meningitidis antigens is inferior to culture and COA, whereas in detecting pneumococcal antigens CIE is superior to culture. So COA is a valuable, cheap, rapid and sensitive method for the diagnosis of meningitis caused by the above three organisms and when used along with culture 100% of cases can be diagnosed.

Diagnosis of cystic echinococcosis: ultrasound imaging or countercurrent immunoelectrophoresis?

Cystic echinococcosis is a major zoonotic diseases in the Islamic Republic of Iran. This study was carried out in 3 general hospitals in Shiraz. We examined the records of all 1227 surgical patients with a surgically-proven diagnosis of cystic echinococcosis for the 20-year period 1978-98. The results of countercurrent immunoelectrophoresis were compared with pathology and ultrasound reports to determine whether serological tests could be helpful for diagnosis. Countercurrent immunoelectrophoresis could detect only 62.0% of cases, whereas the pathology and ultrasound results were positive for 96.3% of cases. This study confirms the usefulness of ultrasound and suggests that only in doubtful cases would countercurrent immunoelectrophoresis be useful for diagnosing cystic echinococcosis.

Evaluation of crude and purified Toxocara canis antigens in serodiagnosis of human toxocariasis.

Three serological tests: Immunodiffusion (ID), Counterimmunoelectrophoresis (CIEP) and Enzyme-linked immunosorbent assay (ELISA) were used to study the role of crude adult worm antigen (CAWA) of Toxocara canis and each of its purified fractions in the serodiagnosis of human toxocariasis. Sensitivities of the three tests were lower in the ocular than in the visceral group, using different antigens. Purified fraction 1 showed more sensitive and specific reactions in the three tests, compared to CAWA or purified fraction 2 (P-F2) antigen. The other purified fractions (P-F3, P-F4 and P-F5) gave no positive reactions in any of the three tests. Using P-F1 antigen, ELISA was the most sensitive technique for diagnosis of both visceral and ocular toxocariasis followed by CIEP and then ID and the difference was statistically significant. However, CIEP was the most specific test followed by ELISA and lastly ID test. The ELISA test using Excretory-Secretory (E-S) larval antigen of Toxocara canis was less sensitive than the ELISA test using P-F1, although it was 100% specific. Thus, ELISA test using P-F1 is the test of choice for diagnosis of human toxocariasis, but when the specificity of a reaction is in doubt, CIEP test using the same antigen can be of value.

Comparison of counterimmunoelectrophoresis (CIE), latex agglutination (LA) and staphylococcal coagglutination (COAG) in pneumococcal antigen detection in vitro.

CIE was compared to agglutination assays employing commercial kits (Directigen, Phadebact), as well as our own LA and COAG reagents, in detection of pneumococcal capsular polysaccharide (PCP) antigens in vitro. Directigen provided the most sensitive assay. CIE was of comparable sensitivity except for PCP antigen types 7 and 14.

Anti-HBbrno: a new CIEP hepatitis B marker, more sensitive than HBsAg radioimmunoassay.

Evidence is presented for the new HBbrnoAg-Anti-HGbrno system association with the HBsAg. The association is displayed particularly in a prevalence of Ausria detectable HBsAg carriers among the anti-HBbrno carriers, suggesting the anti-HBbrno to be a marker of contact with hepatitis B or, possibly, an indicator of co-existing HBsAg antigenaemia, not detectable by radioimmunoassay. Identity of HBbrnoAg with HBsAg/a, d, y, w, is ruled out, the identity with the other HBV associated antigenic specificities remaining questionable.

The quantitation of rabies-specific antibodies. I. Modified counter immunoelectrophoresis. A rapid and sensitive method.

Modified counter immunoelectrophoresis was standardized with respect to dilution of tissue culture antigen and indicator serum, the incubation time for neutralization and the effect of an electric current. The technique was found to be sensitive enough to detect a minimum level of antibodies (0.5 IU/ml) by using a 16 mA current per slide for 2 h, indicator serum of 15 IU/ml and the use of an antigen at a concentration of 1:35. Above all, the incubation period did not affect the neutralization of the virus. The test was also applied to the detection of rabies-specific antibody levels in 73 human sera. The test was found to be simple, quick and economical for titration of rabies antibodies.

Sexual abuse of children. The detection of semen on skin.

OBJECTIVE: The detection of semen on the skin of children who present within 72 hours of an episode of sexual assault is critical to medical, forensic, and legal personnel. The Wood's Lamp, a UV light that causes semen to fluoresce, and four forensic laboratory techniques were compared to determine their sensitivity and decline in sensitivity over time. DESIGN: A descriptive study. PARTICIPANTS: Eleven adult female volunteers. MEASUREMENTS/MAIN RESULTS: Semen was placed on the skin of the volunteers. Samples of the dried semen were assessed during a 28-hour period with the Wood's Lamp, microscopy, the acid phosphatase assay, and two assays for the prostatic protein p30 (counterimmunoelectrophoresis and enzyme-linked immunosorbent assay). The intensity of the Wood's Lamp fluorescence of semen diminished dramatically by 28 hours; in contrast, the fluorescence of urine persisted up to 80 hours. Over time, the p30-enzyme-linked immunosorbent assay technique was more sensitive than microscopy, the acid phosphatase assay, and p30-counterimmunoelectrophoresis in detecting semen on skin. CONCLUSIONS: The Wood's Lamp is not a sensitive screening tool and should be used with caution. To improve the detection of sexual abuse in children, we recommend that the p30-enzyme-linked immunosorbent assay be used because of its potential as a more sensitive assay than those in current clinical use.

Evaluacion de la calidad de los reactivos que se utilizan en la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos / Evaluation of the quality of the reagents employed in the counterimmunoelectrophoresis technique for the determination of rabies antibodies

En este estudio se comprobo que el Instituto Butantan produce antigenos y sueros indicadores que se pueden utilizar con exito en la prueba de contrainmunoelectroforesis para titular anticuerpos antirrabicos en personas inmunizadas. No se pudieron demostrar diferencias estadisticamente significativas entre los resultados de las pruebas de estandarizacion realizadas en el Instituto Butantan y las pruebas de control de referencia llevadas a cabo en el Centro Panamericano de Zoonosis. Se propone que el Instituto Butantan produzca y distribuya a nivel nacional los reactivos para que los laboratorios de diagnostico apliquen la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos.