RESUMO
The evaluation of a prolonged aPTT often includes Lupus Anticoagulant, Antiphospholipid Antibodies, and Factor VIII (FVIII) inhibitors. We have noticed that patient samples positive for lupus antibody (LA) are frequently also positive for FVIII IgG antibodies in an enzyme-linked immunosorbent assay (ELISA), indicating the need for follow-up testing with a more labour-intensive functional assay for FVIII inhibition. This study evaluates the potential for a FVIII IgG ELISA to yield false-positive results in patient samples positive for LA or other antiphospholipid antibodies. A total of 289 residual de-identified patient samples positive for LA (n = 143), anti-cardiolipin IgG (n = 84), or beta2-glycoprotein antibody (n = 62) were tested for FVIII IgG using a commercial ELISA. Samples with positive FVIII IgG ELISA results were further tested for FVIII activity using a clot-based FVIII inhibitor assay. The FVIII IgG ELISA yielded positive results in 39 (13%) of the samples tested, including 13/143 (13%) LA-positive, 15/85 (18%) aCL IgG-positive and 6/62 (10%) ß2-glycoprotein IgG-positive samples. The clot-based FVIII inhibitor assay yielded negative results in all 39 FVIII IgG-positive specimens tested, indicating discrepancy with the FVIII IgG ELISA results. Patient specimens positive for LA, aCL IgG, or ß2-glycoprotein IgG may yield false-positive results for FVIII antibodies. Caution is warranted in interpreting FVIII antibody results in these cases.
Assuntos
Anticorpos Antifosfolipídeos/sangue , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Fator VIII/antagonistas & inibidores , Fator VIII/imunologia , Inibidor de Coagulação do Lúpus/sangue , Anticorpos Anticardiolipina/sangue , Anticorpos Neutralizantes/sangue , Testes de Coagulação Sanguínea , Reações Falso-Positivas , Hemofilia A/sangue , Hemofilia A/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , beta 2-Glicoproteína I/antagonistas & inibidores , beta 2-Glicoproteína I/imunologiaRESUMO
Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive 'standardized' laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-ß(2)glycoprotein I [anti-ß(2)GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these 'criteria' aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-ß(2)GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to 'criteria' aPL tests in an evidence-based manner, during the 13(th) International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-ß(2)GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-ß(2)GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of 'criteria' aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.
Assuntos
Comitês Consultivos , Anticorpos Antifosfolipídeos/análise , Síndrome Antifosfolipídica/diagnóstico , Congressos como Assunto , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/classificação , Síndrome Antifosfolipídica/imunologia , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Feminino , Guias como Assunto , Humanos , Gravidez , Inquéritos e Questionários , TexasRESUMO
Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73-1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44-0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.
Assuntos
Testes de Coagulação Sanguínea/métodos , Doenças de von Willebrand/metabolismo , Fator de von Willebrand/metabolismo , Técnicas de Laboratório Clínico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Mutação/genética , Ligação Proteica , Reprodutibilidade dos Testes , Doenças de von Willebrand/genética , Fator de von Willebrand/análise , Fator de von Willebrand/genéticaRESUMO
Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable. SUMMARY: Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time [APTT] and dilute Russell viper venom [dRVV]). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.
Assuntos
Síndrome Antifosfolipídica/sangue , Testes de Coagulação Sanguínea/métodos , Inibidor de Coagulação do Lúpus/sangue , Tempo de Tromboplastina Parcial , Adolescente , Adulto , Idoso , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Distribuição Normal , Plasma/química , Tempo de Protrombina/métodos , Valores de Referência , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Six members of a family with hypofibrinogenaemia had fibrinogen concentrations ranging from 0.5 to 1.1 mg/ml and, after sequencing the entire coding region and the intron exon boundaries of all three fibrinogen genes, a single heterozygous ACT-->ATT mutation was identified in the gamma gene. This novel mutation was not detected in normal family members or unrelated controls. The gamma371 Thr-->Ile substitution occurs at a conserved threonine in the gammaD domain, but molecules containing the new isoleucine were not present in circulating fibrinogen. The evidence for this was that purified gamma chains had a normal mass of 48375 Da compared to a control of 48374 Da, and tryptic peptide maps were entirely normal. The mutation predicts a mass increase of 12 Da in peptide T-36, but on mass mapping only the normal [M+2H] ion was detected, at 948 m/z. There was no new signal at 954 m/z that would indicate expression of variant chains. Also the normal 948 m/z signal was at the same intensity in digests from the proposita and controls. Crystal structures show a hydrogen bond from the threonine hydroxyl to the main chain and this case suggests this bond is critical in maintaining the structure of the gammaD domain.
Assuntos
Fibrinogênio/genética , Fibrinogênios Anormais/genética , Mutação , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Éxons , Feminino , Fibrinogênio/química , Fibrinogênios Anormais/química , Heterozigoto , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , TripsinaRESUMO
The dilute Russell's viper venom time (dRVVT) and the kaolin clotting time (KCT) are two among the most commonly used coagulation tests for the detection of lupus anticoagulants. The dRVVT seems superior to the KCT in identifying LA-positive patients at risk of thrombosis. However, this relationship is greatly influenced by both the source of reagents and the instrumentation employed to carry out the assays. Therefore, 4 dRVVTs ("home-made" dRVVT, DVV test, Bioclot LA, LA Screen), and one KCT (Kaoclot) were performed in two centers and compared for their retrospective correlation with the thrombotic complications of 72 patients with a previously established diagnosis of lupus anticoagulants. Two other assays ("home-made" KCT, and Colloidal Silica Clotting Time, CSCT) were performed in one of the two centers, and compared with Kaoclot for their clinical correlations in the same population of patients, 44 of whom (61%) had suffered from arterial and/or venous thrombosis. A rather good degree of inter-laboratory and inter-assay correlations of the different tests was found. However, a statistically significant association with thrombosis was found only with the coagulation profile generated using the "home-made" dRVVT. When the commercially available dRVVTs were used, none of the coagulation profiles remained associated with thrombosis. When the assays were analyzed separately, the association with thrombosis was statistically significant for LA screen (p = 0.0019), DVV test (p = 0.0043), and Bioclot (p = 0.0255), and of borderline significance for the "home-made" dRVVT (p = 0.0503) in one center. This last assay was also significantly associated with thrombosis in the other center (p = 0.0139). When venous and arterial thrombosis were considered separately, DVV test was statistically associated with venous thrombosis in both centers (p = 0.0076 and p = 0.0187, respectively), and LA screen in one center (p = 0.0303). No dRVVT was found to correlate with arterial thrombosis. Kaoclot, Colloidal Silica Clotting Time, and the "home-made" KCT did not correlate with thrombosis. The prevalence of IgG and/or IgM antibodies to cardiolipin, beta2-glycoprotein I and prothrombin were 74%, 86% and 85%, respectively. Increased titers of IgG anticardiolipin antibodies were associated with arterial thrombosis (p = 0.0375), whereas IgM anti-beta2-glycoprotein I antibodies were associated with venous thrombosis (p = 0.0433). In conclusion, these retrospective data support the notion that the dRVVT, rather than other coagulation or ELISA tests, are able to identify lupus anticoagulant-positive patients at risk of thrombosis. This property appears common to several commercially available dRVVT kits, making this type of assay the ideal target of future efforts of laboratory standardization.
Assuntos
Inibidor de Coagulação do Lúpus/efeitos adversos , Kit de Reagentes para Diagnóstico/normas , Trombose/etiologia , Adulto , Idoso , Autoanticorpos/sangue , Testes de Coagulação Sanguínea/normas , Cardiolipinas/imunologia , Feminino , Glicoproteínas/imunologia , Humanos , Testes Imunológicos , Inibidor de Coagulação do Lúpus/sangue , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Protrombina/imunologia , Tempo de Protrombina , Estudos Retrospectivos , Trombose/sangue , beta 2-Glicoproteína IRESUMO
Many of the autoantibodies in antiphospholipid syndrome (APS) are directed against beta2-glycoprotein I (beta2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of beta2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed > or = threefold selectivity for beta2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti-beta2 antibodies are localized to the amino terminal domain of beta2-GPI.
Assuntos
Anticorpos Anticardiolipina/imunologia , Glicoproteínas/imunologia , Adulto , Anticorpos Anticardiolipina/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/análise , Autoanticorpos/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Epitopos/análise , Epitopos/química , Epitopos/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , beta 2-Glicoproteína IRESUMO
To clarify the involvement of annexin V (ANX) in antiphospholipid antibody (APA) specificities, we studied antiANX antibodies (aANX) using 2 kinds of enzyme-linked immunosorbent assay plates (plain and gamma-irradiated) and anti-beta 2-glycoprotein I antibodies (a-beta 2GPI) in 53 patients with antiphospholipid syndrome (APS). The incidence of aANX IgG-positive results in the autoimmune APS group was significantly higher than that of healthy control subjects. However, we could not demonstrate a significantly higher incidence in the infection- or drug-induced group. Nor could we find an increased incidence of IgM isotype. When the 2 plates were compared, the discrepancies of positivity were demonstrated in both isotypes. We speculated that these discrepancies between the plate surfaces were attributed to the altered antigenicity of ANX. Although positivity of a-beta 2GPI was associated significantly with clinical manifestations, no significant associations were demonstrated between the incidence of aANX-positive results and clinical manifestations. We inferred that the involvement of aANX in the pathogenic mechanism of APS is unlikely.
Assuntos
Anexina A5/imunologia , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Trombose/imunologia , Adulto , Anexina A5/sangue , Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/sangue , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Glicoproteínas/imunologia , Humanos , Imunoglobulina G/classificação , Imunoglobulina M/classificação , Masculino , Pessoa de Meia-Idade , Tempo de Tromboplastina Parcial , Gravidez , Tempo de Protrombina , Proteínas Recombinantes , Dodecilsulfato de Sódio , beta 2-Glicoproteína IRESUMO
The prevalence and clinical significance of plasma oxidized low-density lipoprotein (oxLDL) and antibodies against oxLDL (anti-oxLDL) were evaluated in patients with antiphospholipid syndrome (APS). OxLDL and IgG anti-oxLDL were determined by enzyme-linked immunosorbent assay in plasma samples from 80 patients with APS. Positive values (mean + 3 SD) for oxLDL and anti-oxLDL were found in 21 (26%) and 19 (24%) of 80 patients with APS, respectively These values were significantly higher than those in healthy subjects. Levels of oxLDL and anti-oxLDL antibodies in subgroupings of patients with APS who had experienced thrombotic events were compared. There were significant differences among the groups for the levels of both oxLDL and anti-oxLDL antibodies. Pairwise comparisons between the groups yielded similar but not identical results. There was a significant, positive correlation between levels of plasma oxLDL and anti-oxLDL. These results suggest that elevated levels of plasma oxLDL and anti-oxLDL may be risk factors and potential markers for thrombosis, especially for arterial thrombotic events, in patients with APS.
Assuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/sangue , Lipoproteínas LDL/imunologia , Trombose/imunologia , Adulto , Idoso , Síndrome Antifosfolipídica/complicações , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/análise , Masculino , Pessoa de Meia-Idade , Trombose/etiologiaRESUMO
BACKGROUND: Chemokines effect their proinflammatory and growth regulatory roles through interaction with serpentine receptors. One such receptor, CXCR2, binds multiple CXC chemokines, including interleukin 8, GRO-alpha, GRO-beta, GRO-gamma, and NAP-2. We have previously identified CXCR2 expression on myeloid cells, notably mature granulocytes, and projection neurons. OBJECTIVE: To determine the expression of CXCR2 by cells of the neuroendocrine system. DESIGN: Archival specimens from normal neuroendocrine tissues and their malignant counterparts were analyzed by immunohistochemistry with monoclonal antibodies specific for CXCR1 and CXCR2. RESULTS: Immunohistochemical analysis revealed high-level expression of CXCR2 by cells in the pituitary, adrenal medulla, pancreatic islets, thyroid C cells, scattered Kulchitsky cells in the bronchi, and counterpart neuroendocrine cells in the stomach, small bowel, colon, and appendix. Neuroendocrine neoplasms that demonstrated high-level CXCR2 expression included (1) primary carcinoids localized to the stomach, small bowel, colon, appendix, fallopian tube, ovary, and lung; (2) atypical carcinoids of the lung; (3) metastatic carcinoids; (4) pituitary adenomas; (5) pheochromocytomas; and (6) medullary carcinomas of the thyroid. Small cell lung carcinomas, large cell neuroendocrine carcinomas of the lung, small cell carcinoma of the cervix, Merkel cell carcinomas, neuroblastomas, and malignant melanomas lacked evidence of CXCR2 expression. CONCLUSIONS: The expression of CXCR2 by normal neuroendocrine cells and neoplastic counterparts that have retained phenotypic features of this differentiation program suggests that chemokines may play an important role in functions that are characteristic of this cell type. In addition, this raises the possibility that chemokines may modulate secretion of biologically active products of these cells and their neoplastic counterparts.
Assuntos
Neoplasias/imunologia , Neoplasias/patologia , Tumores Neuroendócrinos/patologia , Sistemas Neurossecretores/imunologia , Receptores de Quimiocinas/análise , Receptores de Interleucina/análise , Anticorpos Monoclonais , Antígenos CD/análise , Feminino , Neoplasias Gastrointestinais/patologia , Neoplasias dos Genitais Femininos/patologia , Humanos , Imuno-Histoquímica/métodos , Interleucina-8/imunologia , Tumores Neuroendócrinos/imunologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/patologia , Especificidade de Órgãos , Receptores de Interleucina-8A , Receptores de Interleucina-8B , Valores de ReferênciaAssuntos
Síndrome Antifosfolipídica/diagnóstico , Síndrome Antifosfolipídica/imunologia , Programas de Rastreamento/métodos , Adulto , Algoritmos , Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Inibidor de Coagulação do Lúpus/sangue , Masculino , Tempo de Tromboplastina ParcialRESUMO
OBJECTIVE: The presence of endocervical metaplasias and their association with cervical squamous and glandular neoplasias were studied. MATERIALS AND METHODS: Cervical specimens were examined microscopically to identify the presence of glandular metaplasias. The type of cervical neoplastic abnormality, when present, also was recorded. Statistical analyses of the microscopical findings included frequency observation counts and contingency analysis (Fisher's exact test). RESULTS: One hundred eighty-seven cervices were examined. Diagnoses included adenocarcinoma (8), adenocarcinoma in situ (14), glandular dysplasia (4), squamous carcinoma (2) and cervical intraepithelial neoplasia (78); 4 cervices had combined squamous and glandular abnormalities. Eighty-seven had no evidence of cervical neoplasias. The glandular metaplasias were tubal (79 ciliated cell, 3 eosinophilic cell) and intestinal (22); 14 cases had combined tubal and intestinal metaplastic change. Ninety-seven cervices had no evidence of glandular metaplasia. Intestinal metaplasia was intimately associated (p = .0002) with neoplastic changes, particularly glandular abnormalities. Ciliated cell metaplasia also was associated with glandular or squamous abnormalities (p < .04), but more than one-third of specimens with this metaplasia had no associated abnormality. CONCLUSIONS: A high degree of association exists between cervical intestinal glandular metaplasia and cervical glandular and squamous neoplasias. Patients with this metaplastic change should be followed up closely if no associated lesion is found initially.