Ortho Diagnostic Systems Research Articles

Lymphocytes in peripheral blood and thyroid tissue in children with Graves’ disease

This study was undertaken to analyze subsets of lymphocytes in peripheral blood in the early phase and in the thyroid tissue in the late phase of Graves' disease (GD) in children. The study included 30 children with GD and 30 healthy children. Monoclonal antibodies were used to define peripheral blood lymphocyte subsets and they were analyzed using the flow cytometer Ortho Diagnostic System. After thyroidectomy, T cells were detected by CD3, CD4, CD8 antibodies, B cells by CD79alpha antibodies, and the antigen presenting dendritic cells (APCs) by CD1alpha antibodies (DakoCytomation) in the thyroid tissue. Before the treatment, an increased percentage of CD4+ T helper cells and B cells and decreased CD8+ T suppressor/cytotoxic cells were observed in peripheral blood in all the GD children. The number of lymphocytes and dendritic cells in the thyroid tissue increased in the children with GD in comparison to the control group, especially T cells subsets CD4+ and CD8+ and CD79alpha+ B cells. The percentage of T cells in the thyroid tissue was lower and that of B cells was higher than in peripheral blood. In their structure, thyrocytes can have components similar to alpha-chains connected with beta-microglobulins, which were characteristic for APCs. The primary defect of immunoregulation in children with GD is probably dependent on a large number and the activity of T helper cells and on a small number and hypofunction of T suppressor cells. The amount of lymphocytes decreased proportionally to the duration of methimazole treatment. The thyrocytes probably can present antigens.

LYMPHOCYTES IN PERIPHERAL BLOOD AND THYROID TISSUE IN CHILDREN WITH GRAVES' DISEASE

OBJECTIVE: Our goal was to analyze interactions of lymphocytes in peripheral blood and thyroid tissue in children with Graves' disease (GD). METHODS: The prospective study concerned 15 children affected with GD and 15 healthy children. The levels of autoantibodies against thyrotropin receptor, thyroid peroxidase, and thyroglobulin were assayed. Monoclonal antibodies (Ortho Diagnostic Systems, Raritan, NJ) were used to define peripheral blood lymphocyte subsets and analyzed by using a flow cytometer. After thyroidectomy, thyroid specimens were stained with hematoxylin/eosin. T cells were detected by CD3+, CD4+, and CD8+ antibodies and the antigen-presenting dendritic cells with CD1a+ and CD35+ antibodies (DakoCytomation, Glostrup, Denmark). RESULTS: Before treatment, all children with GD had increased thyroid autoantibody levels, an increased percentage of CD4+ helper cells, and decreased levels of CD8+ suppressor/cytotoxic T cells, which resulted in an elevated CD4/CD8 ratio. The percentage of CD19+Cd5+ B cells was increased, although the total population of CD19+ B cells did not differ from that of the control group. The number of lymphocytes in the thyroid was decreased in 10 patients after long-term thiamazole treatment. In 5 patients with short-term therapy (<6 months after relapse of GD), the lymphocytes had formed lymphatic follicles: antigen-presenting dendritic cells CD1a+CD35+ in the germinal center and T-helper CD4+, T-suppressor CD8+, and B cells CD79+ on the edges. CONCLUSIONS: The primary defect of immunoregulation in GD consists of an increase of T-helper lymphocytes with a simultaneous decrease in the number of T-cytotoxic/suppressor cells. Thiamazole therapy probably leads to reduction of the lymphocyte amount in the thyroid.

Lower CD4+ T-Cell Number Expressing CD7+ Characterizes HIV/Hepatitis C Virus Coinfection Compared With HIV and Hepatitis C Virus Monoinfection

Lower CD4+ T-Cell Number Expressing CD7+ Characterizes HIV/Hepatitis C Virus Coinfection Compared With HIV and Hepatitis C Virus Monoinfection

Hepatitis C Infection and Risk of Chronic Liver Disease in Lagos

Objectives: This case-control study sets out to investigate the prevalence of antibodies to hepatitis C virus (anti-HCV) and its association with the presence of hepatitis B surface antigen (HBsAg) in Nigerians with chronic liver disease. Method: Seventy-four (74) biopsy proven cases of chronic liver disease and 74 age and sex matched controls without liver disease were evaluated. Questionnaire interview for risk factors was administered to all subjects. Anti-HCV was determined using a highly sensitive third generation enzyme immuno-assay (ELISA 3, Ortho Diagnostics Systems Gmbh, Germany). Hepatitis B surface antigen was evaluated using an ELISA test system (Murex Diagnostics Limited, UK). Results: The prevalence of anti-HCV was significantly higher in patients with chronic liver disease than in the controls (12.2% vs 1.4%, p=

Routine HCV PCR screening of blood donations to identify early HCV infection in blood donors lacking antibodies to HCV.

Detection of early hepatitis C infection of blood donors is still a major problem for blood transfusion. Common anti-HCV screening assays show differences in sensitivity and specificity. The often mild symptoms of acute hepatitis C also cause difficulties in the identification of early HCV infection. The feasibility and efficacy of routine screening of blood donations for HCV RNA were investigated. Blood donations (n = 251,737) were screened for HCV RNA over 4 years. RNA extraction, amplification, and detection were done by two commercial HCV PCR kits (HCV Cobas Amplicor and HCV Cobas Amplicor 2.0, Roche Diagnostics). Screening was done by pool testing with a maximum pool size of 40 serum samples. Three donations out of 251,737 were HCV RNA positive and anti-HCV negative. ALT levels of these donations were 271, 32, and 10 U per L. The HCV infection of a fourth HCV RNA-positive donor could not be identified by routine, second-generation HCV EIA (Abbott Diagnostika). In this case, two previous donations were also HCV RNA positive, and three second-generation test systems (Abbott) could not detect anti-HCV, whereas third-generation anti-HCV screening assays detected antibody with different sensitivity. The first HCV RNA-positive donation was identified only by the HCV ELISA 3.0 (Ortho Diagnostic Systems). The results of confirmatory assays like RIBA HCV 3.0 (Ortho) and Matrix (Abbott) indicate a restricted immune response to NS3 only. HCV RNA detection by PCR can be carried out routinely in blood donor screening without significant delay of release of the components. The residual risk of transmission can be reduced by identification of early infection, which can lead to an improved safety of blood components. RNA screening can also be advantageous in cases of incomplete or lack of antibody response to HCV.

Prevalence of hepatitis C virus (HCV) infection and its vertical transmission in Egyptian pregnant women and their newborns.

We studied the prevalence of hepatitis C virus (HCV) antibody seropositivity using ELISA (Ortho Diagnostic system, 3rd generation test) polymerase chain reaction testing of HCV-RNA (PCR, Promega) and serum alanine transferase (ALT) level in 100 healthy, HIV-negative, pregnant women who delivered spontaneously at the Alexandria University Hospital, and their newborns. Some risk factors were studied using Fisher's exact test. Nineteen per cent of pregnant women were HCV seropositive and 14 of them (14/19) had circulating HCV-RNA, detected by PCR. Nine of the babies born to the 19 HCV seropositive females had circulating antibodies, whereas HCV-RNA was detected in five of them. This gives a vertical transmission risk of 5/14 (36 per cent) for mothers carrying the HCV-RNA and 5/19 (26 per cent) for those having circulating HCV antibodies. History of previous blood transfusion, elevated serum ALT level, and history of infection with schistosomiasis were significant risk factors for HCV infection in mothers. In addition to the previous factors, maternal history of jaundice, stillbirth and hepatomegaly were significant risk factors for neonatal infection. The occurrence of early jaundice and the presence of congenital anomalies in the newborns were non-significant risk factors. In conclusion, our data indicate a high prevalence of HCV seropositivity in Egyptian HIV-negative pregnant women with a significant high rate of vertical transmission of HCV.

Bcl-2 expression by eosinophils in a patient with hypereosinophilia

The hypereosinophilic syndrome (HES) represents a heterogenous group of disorders characterized by hypereosinophilia in the blood and bone marrow, as well as eosinophilic tissue infiltration. The mechanisms of eosinophilic accumulation in HES are largely unknown. It has been recently suggested that the tissue eosinophilia observed in allergic disorders is, at least partially, caused by inhibition of eosinophil apoptosis by T cell‐, mast cell‐, and eosinophil-derived cytokines.1 Therefore we hypothesized that abnormalities in the process of eosinophil apoptosis may play a part in the etiology of HES. Members of the B-cell lymphoma-2 (Bcl-2) family of proteins are important regulators of apoptosis in many cellular systems.2 The first member of the family, Bcl-2, was originally cloned from the breakpoint of a t(14;18) translocation present in many human B-cell lymphomas. Increased production of Bcl-2 protein as a result of t(14;18) translocations contributes to neoplastic B-cell expansion by preventing B-cell death. 2 In this article we describe an unusual case of HES with blood and tissue eosinophils that expressed, in contrast to normal eosinophils, the Bcl-2 gene. Because it was impossible to induce the Bcl-2 gene by IL-5 or GM-CSF in normal eosinophils, it is unlikely that the observed eosinophil Bcl-2 expression in this subject was the result of stimulation with eosinophil hematopoietins in vivo. Moreover, overexpression of Bcl-2 was associated with delayed death of blood eosinophils in vitro, suggesting that Bcl-2 did act as an apoptosis repressor in this cellular system.

Effect of superimposed infections on viral replication in human immunodeficiency virus type 1-infected children.

Effect of superimposed infections on viral replication in human immunodeficiency virus type 1-infected children.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

The gel test: sensitivity and specificity for unexpected antibodies to blood group antigens

The recently FDA-licensed anti-IgG gel test for pretransfusion antibody detection requires crossover validation before implementation. Six hundred coded samples sent for routine pretransfusion tests were used to compare GEL (ID-MTS, Ortho Diagnostic Systems Inc., Raritan, NJ) with Löw and Messeter's low-ionic-strength saline (LISS). There were 456 GEL-LISS-, 97 GEL+LISS+, 45 GEL-LISS+, and 2 GEL+LISS- tests. The 144 positive tests involved 157 antibodies; 67 of these (cold auto, anti-M, -Le, etc.) were considered harmless with respect to transfusion management. GEL-LISS+ tests included seven samples containing potentially significant antibodies (assumed from specificity): anti-K(4), -Jka, -Fyb, and -S. Two potentially significant antibodies (anti- C and -D) were GEL+LISS-. Sensitivity and specificity for potentially significant antibodies were 92% and 96% for GEL, and 98% and 90% for LISS, respectively. The seven GEL-LISS+ samples associated with potentially significant antibodies were from six patients. Five of these antibodies, all detected in immune-suppressed patients, reacted predominantly as agglutinins in LISS. None of these seven antibodies were detected reliably by polyethylene glycol and LISS-additive tube methods. In light of the immune status of the patients with GEL-LISS+ agglutinins with specificity normally considered potentially significant, and because other valid methods did not detect these antibodies, their clinical importance is questionable. Excluding these questionable antibodies, GEL has the same sensitivity and better specificity than LISS. GEL is a valid method for pretransfusion antibody detection.

5597688 Cell fixative and method of analyzing virally infected cells : Connelly Mark; Chakraborty Utpal; Brooks Houston G Doylestown, PA, United States Assigned to Ortho Diagnostic Systems Inc

5597688 Cell fixative and method of analyzing virally infected cells : Connelly Mark; Chakraborty Utpal; Brooks Houston G Doylestown, PA, United States Assigned to Ortho Diagnostic Systems Inc

5491067 Agglutination reaction and separation vessel : Setcavage Thomas; Reis Kathleen J; Davies Donald M; Mazur Edward J Milford, NJ, United States assigned to Ortho Diagnostic Systems Inc

5491067 Agglutination reaction and separation vessel : Setcavage Thomas; Reis Kathleen J; Davies Donald M; Mazur Edward J Milford, NJ, United States assigned to Ortho Diagnostic Systems Inc

5486454 Nucleic acid probe for the detection of Salmonella human pathogens : Madonna M Jan; Woods Derek Middlesex, NJ, United States assigned to Ortho Diagnostic Systems Inc

5486454 Nucleic acid probe for the detection of Salmonella human pathogens : Madonna M Jan; Woods Derek Middlesex, NJ, United States assigned to Ortho Diagnostic Systems Inc

HTLV-I/II seroprevalence in 55 Brazilian patients with idiopathic uveitis.

Human T-cell lymphotropic virus type I has been associated with adult T-cell leukemia and lymphoma, neurological disease (tropical spastic paraparesis or HTLV-I associated myelopathy) and, more recently, uveitis. This virus is highly endemic in some regions of the world, especially in southwest Japan, Caribbean islands, South A m erica and parts o f Central A frica1. The presence of HTLV-I/II in Brazil varies according to the geographical area, with seroprevalence in healthy blood donors ranging from 0.08 to 1.35% in studies conducted by the Ministry of Health5. In Belo Horizonte, the capital of Minas Gerais State, a seroprevalence of 0.32%4 has been observed in eligible blood donors. In Ja p a n , sero p rev alen ce for HTLV-I antibodies among patients with idiopathic uveitis ranges from 35.4 to 44.8%2. In Brazil, to our knowledge, there is no data on HTLV-I seroprevalence among individuals with this diagnosis. From March to September of 1994, we tested 55 patients with idiopathic uveitis for the presence of antibodies to HTLV-I/II by using an ELISA (Ortho Diagnostic Systems, N ew J e r s e y , U SA ). P o s itiv e te s ts w e re confirmed by Western blot (Cambridge Biotech, Maryland, USA). None of the patients presented clinical or laboratorial evidence of systemic disease related to other uveitis types such as toxoplasmosis, syphilis, tuberculosis, ankylosing spondylitis, sarcoidosis, Behcet’s disease or Vogt-Koyanagi-Harada’s disease. The patients’age ranged from 9 to 78 years (mean, 36.2, SD, 17.52). Thirty-six (65.4%) were females and 19 (34.6%) were males. The distribution of the anatomical types of uveitis

5422277 Cell fixative composition and method of staining cells without destroying the cell surface : Connelly Mark; Chakraborty Utpal R; Brooks Houston Doylestown, PA, United States Assigned to Ortho Diagnostic Systems Inc

5422277 Cell fixative composition and method of staining cells without destroying the cell surface : Connelly Mark; Chakraborty Utpal R; Brooks Houston Doylestown, PA, United States Assigned to Ortho Diagnostic Systems Inc

Immunological events during the incubation period of hepatitis C virus infection in immunocompromised individuals: [formula omitted]. ∗Athens University Medical School, ∗∗Ortho Diagnostic Systems Inc. Raritan, NJ

Immunological events during the incubation period of hepatitis C virus infection in immunocompromised individuals: [formula omitted]. ∗Athens University Medical School, ∗∗Ortho Diagnostic Systems Inc. Raritan, NJ

Evaluation of Performance of Reused HIVCHEK 1 + 2 Test Blocks which have Shown Negative Result: A Reliable Method for Rural Hospital?

The performance of the reusing of test membranes which have been used previously for negative tests for the detection of antibody to HIV (HIVCHEK 1 + 2 of Ortho Diagnostic Systems, Paris, France) was evaluated under field conditions. The sensitivity and specificity of the reusing strategy compared with a HIV determination obtained by using new HIVCHECK 1 + 2 tests were 89.1% and 100%, respectively. The positive predictive value was 100% and the negative predictive value was 91.5%. The authors conclude that the reduction in sensitivity of the reusing strategy in comparison with the use of new tests makes this strategy ethically unacceptable for the detection of HIV infection in blood donors. On the other hand, the reusing strategy could be very useful for diagnostic purpose and for epidemiological HIV surveillance in resource-poor countries.

Prevalence, severity, and risk factors of liver disease in blood donors positive in a second-generation anti—hepatitis c virus screening test

In a cohort of 483 blood donors positive for antibody to hepatitis C virus on second-generation enzyme-linked immunosorbent, the confirmatory second-generation recombinant immunoblot assay (Ortho Diagnostic Systems) was positive in 172 cases (36%), indeterminate in 113 (23%), and negative in 198 (41%). We further studied 94 of the donors (recombinant immunoblot assay positive in 85, indeterminate in 6, and negative in 3). Alanine transaminase (ALT) activity, assayed on three occasions, was elevated in at least one assay in 85% of the 85 recombinant immunoblot assay-positive donors. Liver disease was present in 95% of these patients (chronic persistent hepatitis, 35%; chronic active hepatitis, 53%; cirrhosis, 7%). Ten of the 13 recombinant immunoblot assay-positive donors with normal ALT activity had liver disease; polymerase chain reaction testing for viral RNA was predictive of liver disease in most cases. Donors with cirrhosis differed significantly from cirrhosis-free donors in terms of age, sex ratio, ALT activity, and excessive alcohol consumption. Three of the 6 recombinant immunoblot assay-indeterminate donors (isolated C 22) who underwent histological examination had elevated ALT activity and liver disease. The 3 recombinant immunoblot assay-negative donors evaluated were free of liver disease. This study shows that anti-HCV second-generation enzyme-linked immunosorbent positivity is confirmed in fewer than 40% of blood donors by the second-generation recombinant immunoblot assay, and that liver disease is present in 95% of recombinant immunoblot assay-positive donors. Recombinant immunoblot assay positivity combined with viremia is frequently associated with the existence of liver disease, regardless of transaminase activity. Excessive alcohol consumption may be an important factor in the onset of cirrhosis in anti-HCV-positive blood donors.

Prevalence, severity, and risk factors of liver disease in blood donors positive in a second-generation anti—hepatitis C virus screening test†

In a cohort of 483 blood donors positive for antibody to hepatitis C virus on second-generation enzyme-linked immunosorbent, the confirmatory second-generation recombinant immunoblot assay (Ortho Diagnostic Systems) was positive in 172 cases (36%), indeterminate in 113 (23%), and negative in 198 (41%). We further studied 94 of the donors (recombinant immunoblot assay positive in 85, indeterminate in 6, and negative in 3). Alanine transaminase (ALT) activity, assayed on three occasions, was elevated in at least one assay in 85% of the 85 recombinant immunoblot assay-positive donors. Liver disease was present in 95% of these patients (chronic persistent hepatitis, 35%; chronic active hepatitis, 53%; cirrhosis, 7%). Ten of the 13 recombinant immunoblot assay-positive donors with normal ALT activity had liver disease; polymerase chain reaction testing for viral RNA was predictive of liver disease in most cases. Donors with cirrhosis differed significantly from cirrhosis-free donors in terms of age, sex ratio, ALT activity, and excessive alcohol consumption. Three of the 6 recombinant immunoblot assay-indeterminate donors (isolated C 22) who underwent histological examination had elevated ALT activity and liver disease. The 3 recombinant immunoblot assay-negative donors evaluated were free of liver disease. This study shows that anti-HCV second-generation enzyme-linked immunosorbent positivity is confirmed in fewer than 40% of blood donors by the second-generation recombinant immunoblot assay, and that liver disease is present in 95% of recombinant immunoblot assay-positive donors. Recombinant immunoblot assay positivity combined with viremia is frequently associated with the existence of liver disease, regardless of transaminase activity. Excessive alcohol consumption may be an important factor in the onset of cirrhosis in anti-HCV-positive blood donors.

Beware of commercial human thrombins used to stimulate cultured endothelial cells

In the field of in vitro biocompatibility testing, the investigation of cell response at the interface with a biomaterial is of great importance; there is a need for standard conditions and thus of well-defined and reliable sources of materials for an objective evaluation of cellular function. Thrombin is often used in vitro as a stimulating agent to check the specific functions of cultured endothelial cells. In the present work, and in order to select a thrombin of commercial origin, two criteria were borne in mind: purity towards the presence of the von Willebrand factor (vWF) and effectiveness towards vWF release by human umbilical venous endothelial cells (HUVEC) that have been submitted to four human commercial thrombins. We detected the presence of vWF in some thrombin solutions that have not yet been in contact with HUVEC. The different thrombins contained vWF antigen ranging from less than 0.1 mUnit per NIH unit of thrombin (from Diagnostica Stago and Sigma Chemical Co.) to 10–20 mUnit per NIH unit of Fibrindex ® thrombin (from Ortho Diagnostic Systems). Thus, if vWF is present in commercial thrombins, it contributes to and overestimates the vWF appearance in the media resulting from cell stimulation. Consequently, we fixed on thrombin from Diagnostica Stago for further studies involving HUVEC on biomaterials.