[About Minimization of Expenses on Allergy Diagnosis in Children: Analysis of Consistency of in Vitro- and in Vivo-Allergic Examinations Results].

UNLABELLED: High morbidity rate of atopic diseases among children, including high importance of grass pollen as a sensitizing agent, determine the relevance ofstudies on diagnostic examination systems for appointment of adequate therapy. The research of the most relevant allergens for patients to excludeof duplicating and uninformative tests became urgent after development of a new type of diagnostic tests that does not require expensive equipment. The objective of this research was to evaluate the results of in vitro- and in vivo-diagnostic examinations of children with various forms of atopic disease caused by pollen of meadow grasses, and to choose the most significant prognostic parameters for the diagnosis. METHODS: 277 children aged 4-16 years with various forms of atopic disease were included in the study. There were performed skin prick tests and determination of IgE-antibodies levels to allergen extracts of cocksfoot (g3), meadow fescue (g4), timothy grass (g6). RESULTS: In the studied group of patients 32-50% of children have antibodies to grass allergens. There was a close correlation of antibody response on the investigated allergens, quantitative coincidence of IgE-antibodies to g3 andg4 allergens levels. IgE (g6) concentration was close to the IgE(g3) and IgE(g4) levels (85.0 ± 21.6%). Analysis of the skin tests results showed that 44% of patients have a positive response to grass allergens, and in vivo-tests results coincide with serologicaltests results, mostly in a qualitative sense. The most significant relationship was noted between in vivo and in vitro-tests in the results of testing the response to meadow fescue pollen. CONCLUSION: Based on these data IgE concentration index to meadow fescue allergens can be used as a prognostic marker to determine the sensitization of patients with different nosology forms of allergy and can help to improve allergic diagnostics.

Diagnostic accuracy of immunological methods in patients with tuberculous pleural effusion from Venezuela.

In recent years, better diagnostics for tuberculosis (TB) has received increasing attention, especially the diagnosis of tuberculous pleural effusion, which is difficult and at present the main tool in TPE diagnostic is pleural effusion smear and culture, but unfortunately, sensitivities are low, therefore better TPE diagnostic tools are needed. The aim of this study was to find a diagnostic algorithm to assess the progress in TPE diagnostic at the Hospital Vargas de Caracas, that permits identification of the majority of patients, at a satisfactory cost-benefit ratio, evaluating the levels of IFN-gamma and IL-12p40 in pleural effusion and serum, as well as the antibody reactivity in order to compare it with microbiological tests. A total of 60 individuals with pleural effusion were studied; 20 patients with tuberculous pleural effusion (TPE) formed the patient group and 40 patients with non-tuberculous pleural effusion (NTPE) formed the control group. The levels of IFN-gamma and IL-12p40 in effusion and serum and class and subclasses of IgG reactivity to Mycobacterium tuberculosis antigens were measured by ELISA. The utility of these methods for diagnosis of TPE was evaluated using receiver operating characteristic (ROC) curve analysis. The results of the 11 immunological methods evaluated showed that the anti-PPD IgG2 method was able to reach the highest specificity of 95% (CI: 88.3-101.8), positive predictive value (PPV) = 75 (at 30% sensitivity); while that the overall sensitivity of methods was between 95% and 30%, of these, two methods reached higher sensitivities; increased levels of pleural IFN-gamma, with a sensitivity of 95% (CI: 85.5-104.5) with the highest negative predictive value (NPV) = 97, (at 82.5% specificity), followed by decreased levels of serum IL-12p40 with a sensitivity of 95% (CI: 85.5-104.5), NPV = 95.2 (at 50% specificity). In contrast, microbiological methods showed that smear had a sensitivity of only 20%, while smear plus culture had, a sensitivity of 70%. Considering that TPE represents approximately 15 percent of all the TB clinically diagnosed at the Hospital Vargas de Caracas, in those patients with preliminary microbiology negativity in the effusion, the combined analysis of pleural IFN-gamma and anti-PPD IgG2 could represent a fast and effective diagnostic algorithm for improving the diagnosis previous to obtain culture results. In this way treatment against TB could be initiated or the need to cytological and pleural biopsy could be considered.

Efficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells.

BACKGROUND: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step, using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes, whereas single cell suspensions from tonsils contain relatively few red blood cells. RESULTS: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however, the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells, but not sheep red blood cells, reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. CONCLUSION: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods.

Testing practices and volume of non-Lyme tickborne diseases in the United States.

Large commercial laboratories in the United States were surveyed regarding the number of specimens tested for eight tickborne diseases in 2008. Seven large commercial laboratories reported testing a total of 2,927,881 specimens nationally (including Lyme disease). Of these, 495,585 specimens (17%) were tested for tickborne diseases other than Lyme disease. In addition to large commercial laboratories, another 1051 smaller commercial, hospital, and government laboratories in four states (CT, MD, MN, and NY) were surveyed regarding tickborne disease testing frequency, practices, and results. Ninety-two of these reported testing a total of 10,091 specimens for four tickborne diseases other than Lyme disease. We estimate the cost of laboratory diagnostic testing for non-Lyme disease tickborne diseases in 2008 to be $9.6 million. These data provide a baseline to evaluate trends in tickborne disease test utilization and insight into the burden of these diseases.

Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.

BACKGROUND: The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents. RESULTS: In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements. CONCLUSIONS: These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur.

A cost-effective analysis of the optimum number of stool specimens collected for immunochemical occult blood screening for colorectal cancer.

This study was carried out to assess, from the viewpoint of cost-effectiveness, the optimum number of faecal specimens to collect for use in immunochemical occult blood testing as a means of screening for colorectal cancer. 3300 asymptomatic individuals were subjects of this study. They gave samples for an immunochemical faecal occult blood test, monohaem and colonoscopy was carried out during a medical check-up. For evaluation of the optimum number of sampling specimens, the results of the first day of sampling, those of the first and second days, and those of samples taken for 3 consecutive days were considered as the single-day method, the 2-day method and the 3-day method respectively. The average cost to detect 1 patient with colorectal cancer, the detection rate and the false-positive rate of these three faecal sample collection methods were evaluated. The average costs for one cancer case detected were calculated as $3,630.68 for the single-day method, $3,350.65 for the 2-day method and $4,136.36 for the 3-day method, respectively. The detection rate and the false-positive rate were calculated as 47 and 3.5% for the single-day method, 82 and 4.7% for the 2-day method and 88 and 5.3% for the 3-day method, respectively. This detection rate was significantly different between the single- and the 2-day methods, as well as between the single- and the 3-day methods (P<0. 05). No significant differences in the false-positive rate amongst the three testing methods were observed. This analysis suggests that a 2-day faecal collection method is recommended for immunochemical occult blood screening by Monohaem from the aspects of cost-effectiveness and diagnostic accuracy.

Two-graph receiver operating characteristic (TG-ROC): update version supports optimisation of cut-off values that minimise overall misclassification costs.

TG-ROC was recently introduced as a novel PC-assisted device for cut-off optimisation and evaluation of quantitative serodiagnostic tests (Greiner, 1995, J. Immunol. Methods 185, 145; Greiner et al., 1995, J. Immunol. Methods 185, 123). In this notice a recent extension of the programme is described which facilitates the cut-off selection when estimates of costs associated with false-positive and false-negative results are available.

A freezing method for cell fusions to distribute and reduce labor and permit more thorough early evaluation of hybridomas.

A method is described whereby cell fusions can be bulk-frozen shortly after the hybridization step. Recoveries are shown to be comparable to those obtained for control hybridomas cultured without freezing. Advantages are discussed in terms of labor distribution and antibody assay and evaluation strategies. It is further shown that peritoneal feeder cell preparations can be conveniently frozen as a means of workload reduction.

A novel antibody immobilization and its application in immunoaffinity chromatography.

A novel antibody immobilization and its application in immunoaffinity chromatography (IAC) were presented. Using acrylamide (AM) as monomer, ethylene glycoldimethacrylate (EGDMA) as cross-linker and bulk polymerization as synthetic method, we prepared a polymer in which the Cu(II) was embedded. The Cu(II)-embedded polymer was tested for its binding with protein. It was found that Cu(II)-embedded polymer displayed a strong binding with bovine serum albumin (BSA). At 80% of methanol, no BSA was released from Cu(II)-embedded polymer. The Cu(II)-embedded polymer was then used as a novel solid support for antibody immobilization. IAC column was prepared by immobilizing polyclonal antibody (pAb) against clenbuterol (CL) on Cu(II)-embedded polymer and packing the Cu(II)-embedded polymer-pAb into a common solid phase extraction (SPE) cartridge. Under optimal extraction conditions, the IAC column was characterized in terms of maximum binding capacity for target analyte, extraction efficiency and reusability. It was revealed that, for IAC column packed with 0.1 g of solid support immobilized with antibody, the maximum capacity for CL was 616 ng; the extraction recoveries of the column for CL from three spiked food samples were 84.4-95.2% with relative standard deviation (RSD) of 9.3-15.5%; after more than 30 times repeated usage, there was not significant loss of specific recognition. The results demonstrated the feasibility of the prepared IAC column for CL extraction. The proposed antibody immobilization method exhibiting the properties of simplicity, low cost, strong binding for target analyte, no leaching of antibody, etc., would be a very useful tool applied in the field of IAC.

Diagnostic accuracy of immunological methods in patients with tuberculous pleural effusion from Venezuela / Precisión diagnóstica de métodos inmunológicos en pacientes con tuberculosis pleural de Venezuela

In recent years, better diagnostics for tuberculosis (TB) has received increasing attention, especially the diagnosis of tuberculous pleural effusion, which is difficult and at present the main tool in TPE diagnostic is pleural effusion smear and culture, but unfortunately, sensitivities are low, therefore better TPE diagnostic tools are needed. The aim of this study was to find a diagnostic algorithm to assess the progress in TPE diagnostic at the Hospital Vargas de Caracas, that permits identification of the majority of patients, at a satisfactory cost-benefit ratio, evaluating the levels of IFN-g and IL-12p40 in pleural effusion and serum, as well as the antibody reactivity in order to compare it with microbiological tests. A total of 60 individuals with pleural effusion were studied; 20 patients with tuberculous pleural effusion (TPE) formed the patient group and 40 patients with non-tuberculous pleural effusion (NTPE) formed the control group. The levels of IFN-g and IL-12p40 in effusion and serum and class and subclasses of IgG reactivity to Mycobacterium tuberculosis antigens were measured by ELISA. The utility of these methods for diagnosis of TPE was evaluated using receiver operating characteristic (ROC) curve analysis. The results of the 11 immunological methods evaluated showed that the anti-PPD IgG2 method was able to reach the highest specificity of 95% (CI: 88.3-101.8), positive predictive value (PPV)=75 (at 30% sensitivity); while that the overall sensitivity of methods was between 95% and 30%, of these, two methods reached higher sensitivities; increased levels of pleural IFN-g, with a sensitivity of 95% (CI: 85.5-104.5) with the highest negative predictive value (NPV)=97, (at 82.5% specificity), followed by decreased levels of serum IL-12p40 with a sensitivity of 95% (CI: 85.5-104.5), NPV=95.2 (at 50% specificity). In contrast, microbiological methods showed that smear had a sensitivity of only 20%, while smear plus ...

Recientemente existe un gran interés hacia un mejor y más rápido diagnóstico de tuberculosis (TB), especialmente de tuberculosis pleural, el cual es difícil. Al presente las principales herramientas diagnósticas son la baciloscopia y el cultivo de líquido pleural; desafortunadamente, las sensibilidades de estos métodos son bajas, por lo que el desarrollo de nuevas herramientas diagnósticas es necesario. El objetivo del presente estudio consistió en encontrar un algoritmo que permita la rápida identificación de la mayoría de los pacientes con TB pleural que ingresan en el Hospital Vargas de Caracas a un buen costo-beneficio. Para esto se evaluaron los niveles de las citocinas Interferón-gamma (IFN-g) y la Interleucina 12p40 (IL-12p40) en líquido pleural y suero, así como la reactividad de anticuerpos contra antígenos de Mycobacterium tuberculosis. Se estudiaron 60 individuos con derrame pleural; 20 individuos con líquido pleural tuberculoso (LPT) conformaron el grupo de pacientes y 40 individuos con líquido pleural no tuberculoso (LPNT) el grupo de controles. La técnica de inmunoensayo de ELISA fue utilizada para medir los niveles de IFN-g y IL-12p40; así como las reactividades de los diversos isotipos y subclases de inmunoglobulina G (IgG) frente a antígenos del bacilo. La utilidad de los métodos fue evaluada utilizando el análisis de las curvas ROC (receiver operating characteristic). Los resultados de los 11 métodos inmunológicos evaluados mostraron que el método IgG2 anti-PPD alcanzó la mayor especificidad de 95%, (CI: 88,3-101,8) con un valor predictivo positivo (VPP) de 75. La sensibilidad de los métodos estuvo entre 30% y 95%; dos métodos alcanzaron altas sensibilidades: los altos niveles de IFN-g en líquido pleural, con sensibilidad de 95% (CI: 85,5-104,5), con un valor predictivo negativo (VPN) de 97, seguido de los bajos niveles de IL-12p40 en suero, con una sensibilidad de 95% (CI: 85,5-104,5) con un VPN de 95,2. En contraste, ...

Hemagglutination treponemal test for syphilis.

Sera from 290 hospital patients were tested to compare the sensitivity, specificity, and reproducibility of the hemagglutination treponemal test for syphilis (HATTS) with the fluorescent treponemal antibody absorption test (FTA-ABS). Complete agreement was obtained between the methods when 142 syphilitic sera from patients with various stages of syphilis were tested. By using clinical histories, the specificity with 148 nonsyphilitic sera was determined to be 100% for the HATTS and 96.6% (143 of 148) for the FTA-ABS. Satisfactory reproducibility was obtained with both methods. Compared with the FTA-ABS, the HATTS was more specific, easier, and more economical to perform. We therefore recommend the HATTS as a suitable alternative to the FTA-ABS.

The effect of calf serum on human lymphoblastoid cell-derived interferon production.

Substitution of low-cost calf serum for fetal bovine serum was evaluated in Namalva cell production of human alpha-lymphoblastoid interferon [HuIFN-alpha(Ly)] in 50-liter fermentors. Namalva cell growth and IFN yields were greatly reduced and protein precipitate weight increased from calf serum-supplemented cell cultures. The overall reduction in IFN production was greater than threefold, making the substitution impractical.

Evaluation of five immunological methods for the assay of serum netilmicin.

Three specific immunological methods for the determination of netilmicin and two enzyme immunoassays for gentamicin were compared for assaying serum netilmicin. The commercial kits we used were Radioimmunoassay (RIA), Substrate Labelled Fluorescence Immunoassay (gentamicin) ( SLFIA ), Homogeneous Enzyme Immunoassay (gentamicin and netilmicin) (EMIT) and Fluorescence Polarization Immunoassay ( FPIA ). After analysing spiked calf serum samples in the range of 0-15.8 mg X 1(-1) netilmicin, extensive statistical analysis was performed. The Performance Index was used to qualify the different methods. Results show that the SLFIA (gentamicin) kit is a very acceptable method for the determination of serum netilmicin levels, well comparable with the EMIT (gentamicin) kit and the EMIT (netilmicin) kit. FPIA and RIA require expensive specialized apparatus so it is only economically feasible when other drugs or substances are also determined on the same apparatus. The abilities and specific needs of each laboratory will play an important role in the choice of a method, because the tested kits are all within the range we need from a clinical point of view.

Rapid and inexpensive microdetermination of serum fructosamine results in diabetics, uraemics, diabetics with uraemia and healthy subjects.

This paper describes a simple, reliable and inexpensive method for rapid determination of serum fructosamine. The assay is based on commercially available reagents and utilizes equipment accessible in most laboratories (i.e. an automated ELISA-reader interfaced with an IBM computer). In contrast to HbA1c determination, the fructosamine method presented can be used in diabetes complicated by uraemia. In the clinically relevant measuring range, fructosamine is uninfluenced by serum albumin concentration in diabetics with or without uraemia. Eighty microlitres of non-haemolysed capillary serum suffices for a duplicate determination. One year's storage of normal serum induced no change in serum fructosamine estimates. s-Fructosamine in 18 healthy subjects was 2.1 +/- 0.3 mmol/l (mean +/- SD) in venous blood and 2.2 +/- 0.4 mmol/l in capillary blood. In diabetics 3.4 +/- 0.6 mmol/l and 3.3 +/- 0.6 mmol/l (n = 38) were found. The method is well-suited for routine use in the diabetic out-patient clinic.

The epidemiology of mumps in southern Alberta 1980-1982.

The full spectrum of mumps in Southern Alberta was studied over the years 1980-1982. In the community, a random sample of 3,497 children was tested for prevalence of immunity to mumps. The 1,816 who were not immune were retested after one year. The incidence of new immunity was 16.5%. Most seroconverters had no illness suggestive of mumps. History of previous mumps or immunization were found to be unreliable predictors of serologic immune status. In the family study, information was obtained on 342 cases diagnosed as mumps by family physician recorders. The overall complication rate was 10.8% and the secondary attack rate within families was 11.7%. Hospital records of all 25 patients admitted in Calgary with mumps during the same period were analyzed. Estimated costs of unopposed mumps over 20 years compared with the estimated costs of vaccination showed that a vaccination program could give a benefit-cost ratio between 6.4 and 247. These studies show that mumps is a mild disease with relatively few serious complications or sequelae. Even so, there would be definite medical and economic benefit with immunization. A low-cost addition of mumps vaccine to the immunization program would be justified.

Determination of CD4+ and CD8+ lymphocytes with the cytosphere assay: a comparative study with flow cytometry and the immunoalkaline phosphatase method.

The proportion and absolute numbers of CD4+ and CD8+ lymphocytes in peripheral blood were determined using a new manual method, the cytosphere assay (CA). This method uses small latex beads coated with monoclonal antibodies directed against the CD4 and CD8 receptors, respectively. The CA was compared with two other methods for determination of T lymphocyte subsets, flow cytometry (FC) and the immunoalkaline phosphatase (IA) method, by testing HIV-seropositive and HIV-seronegative samples from Denmark (44) and Ivory Coast (79). For HIV-seropositive samples, both the proportion and the absolute number of CD4+ lymphocytes determined by CA showed a good correlation with results obtained by FC (correlation coefficients were 0.92 and 0.74 in Denmark and The Ivory Coast, respectively) and IA (correlation coefficients were 0.94 and 0.66 in Denmark and The Ivory Coast, respectively). However, for HIV-seronegative samples the corresponding correlation coefficients were low. CD4% determinations deviated more from FC counts at higher CD4 counts than at lower levels for both seronegative and seropositive individuals. In conclusion, the CA performed best for samples from HIV-infected individuals. Before a more general utilization of the method, it is necessary to improve its repeatability and standardize its performance at all levels of CD4+ T cells.

Quantitation of serum immunoglobulins.

This article reviews critically the current "state of the art" in the quantitation of immunoglobulins in serum and other body fluids. The methodologies reviewed include (a) those occurring in solution, for example, automated immunoprecipitin and laser nephelometric techniques, rate analysis techniques and radioimmunoassays; (b) techniques involving radial diffusion in agarose gels, with and without secondary development steps; and (c) those techniques involving solid supports, such as fluoroimmunometric assays and solid phase radioimmunoassays. The theory of each technique is presented with an analysis of the strengths and weaknesses, particularly sources of error, and the techniques are compared in terms of accuracy, precision, sensitivity, test cost, equipment cost, feasibility for use in different laboratory settings, and ease of handling. Problems associated with the antisera used, with standards, and with quality controls are discussed and solutions suggested.

[National study of screening practices for hepatitis C among hemodialysis patients. Hepatitis Group]. / Etude nationale des pratiques de dépistage de l'hépatite C chez les patients hémodialysés. Hepatitis Group.

French and American consensus conferences on hepatitis C confirmed the burden of that disease, especially in high risk populations. In France, the seroprevalence of HCV is about 20% among haemodialysed patients. This study aimed at describing the French screening practices in haemodialysed patients. In 1995, 1213 self-administered questionnaires were sent to nephrologists working in 715 dialysis units. The response rate was 48% (585/1213) and 485 questionnaires were analysed. In 98% of questionnaires nephrologists answered that they prescribed screening test. Routine screening with alanine amino-transferase (ALT) was reported in 98% of questionnaires, usually once a month (57%) or four times a year (23%). Routine anti-HCV serology was reported by 96%, usually once (28%) or twice (46%) a year. The two main annual strategies combining ALT and anti-HCV serology were 12 ALT and 2 serologies (21%), or 12 ALT and 1 serology (14%) per year. HCV RNA detection was reported mainly in the case of positive anti-HCV serology (70%). The study suggested heterogeneity in screening practices and revealed the need to determine the cost-effectiveness ratios of the various strategies.