RESUMO
CONTEXTO: A criptococose é uma micose sistêmica causada por duas espécies do basidiomiceto encapsulado do gênero Cryptococcus. A infecção acomete principalmente o sistema nervoso central (SNC) e o trato respiratório e sua apresentação mais comum é a meningite criptocócica (MC) ou meningoencefalite. Mundialmente, a MC é resultado de cerca de 15% de todas as mortes relacionadas ao HIV. Estima-se que os 223.100 casos de MC, em 2014, resultaram em 181.100 óbitos, sendo 75% (135.900) na África Subsaariana, seguida pelo Sudeste Asiático e América Latina. Após o surgimento da terapia antirretroviral (TARV) para o HIV, a incidência da criptococose diminuiu significativamente em países desenvolvidos (39%), entretanto, a incidência e a mortalidade pela infecção criptocócica ainda é extremamente alta em países com epidemia de HIV incontrolada e acesso limitado aos medicamentos e aos cuidados de saúde. TECNOLOGIA: Teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA). PERGUNTAS DE PESQUISA: 1) O teste CRAG-LFA é sensível e específico na detecção da infecção por Cryptococcus em PVHIV com células CD4+ ≤ 200 cél/mm³ comparado ao teste de aglutinação do látex? 2) O teste CRAG-LFA é sensível e específico no diagnóstico da meningite criptocócica em PVHIV sintomáticos, independente da contagem de células CD4+, se comparado ao teste de aglutinação do látex e tinta da China, em amostra de líquor? EVIDÊNCIAS CLÍNICAS: Para a Pergunta 1 de pesquisa, a estimativa de sensibilidade de CRAG-LFA na detecção da infecção criptocócica foi de 100% e as estimativas de especificidade variaram de 99% a 100%, em relação ao teste de aglutinação por látex (CRAG-LA). Já a sensibilidade combinada dos dois estudos primários incluídos foi 100% (IC95%; 96 - 100) e a especificidade combinada de 99% (IC95%; 99 - 100). Para a Pergunta 2 de pesquisa, a estimativa de sensibilidade de CRAGLFA no diagnóstico de meningite criptocócica em PVHIV foi de 100% e as estimativas de especificidade variaram de 98% a 99%. A sensibilidade combinada dos três estudos primários meta-analisados foi de 100% (IC95%; 92 - 100) e a especificidade combinada de 99% (IC95%; 97 - 100), em relação ao teste de tinta da China. Como evidências analisadas para responder à Pergunta 2 de pesquisa, também foram incluídas duas revisões sistemáticas com meta-análise, a primeira reportou que os valores de sensibilidade e especificidade combinados de CRAG-LFA no líquor foram 98,9% (IC 95%, 97,9% a 99,5%) e 98,9% (IC 95%, 98,0% a 99,5%), respectivamente, já a segunda revisão descreveu que, no líquor, CRAG-LA (10 coortes diagnósticas, 1810 participantes) teve uma sensibilidade resumida de 97,1% (91,9 - 99) e uma especificidade de 99,1% (93,8 - 99,9) enquanto o teste CRAG-LFA (6 coortes diagnósticas, 3.099 participantes) teve uma sensibilidade resumida de 99,5% (97,2 - 99,9) e especificidade de 99,5% (94,2 - 99,9). Embora houvesse alguma evidência estatística fraca de que o CRAG-LFA pode ter melhor sensibilidade no líquor (p = 0,07) do que CRAG-LA, suas especificidades foram comparáveis (p = 0,54). EVIDÊNCIAS ECONÔMICAS: Foi conduzida avaliação econômica do tipo Microssimulação de Monte Carlo, com horizonte temporal de um ano. Considerou-se dois desfechos primários de efetividade: anos de vida e anos de vida ajustados por qualidade (QALY). A análise de custo-efetividade mostrou que os dois testes, CRAG-LA e CRAG-LFA, são custo-efetivos, dominando a alternativa tinta da China e o cenário de não realização da detecção. Para o diagnóstico de meningite criptocócica em PVHIV sintomáticas o uso do teste diagnóstico CRAG-LFA revelou-se custo-efetivo comparado ao teste CRAG-LA, e ao teste tinta da China. A análise de sensibilidade determinística e probabilística indica um favorecimento, na maioria das vezes, do teste CRAG-LFA. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: A análise de impacto orçamentário (AIO) foi realizada para um horizonte temporal de cinco anos, assumindo-se um market share inicial de 20% para o LFA, com incrementos anuais no mesmo valor, chegando a 100% no quinto ano. Para a pergunta 1 de pesquisa a incorporação de LFA teria um custo adicional de aproximadamente 52,7 milhões de reais. Estimou-se também o impacto orçamentário de 100% de adoção da detecção em PVHIV CD4≤ 200 cél/mm³ assintomáticos. Nesse contexto, ocorreria uma economia da ordem de 55 milhões de reais em cinco anos, considerando detecção precoce e tratamento preemptivo, em relação ao custo de tratamento da meningite. Para a pergunta 2, para cada 1.000 PVHIV por ano, em cinco anos teríamos 809 indivíduos tratados a um custo adicional de R$ 10.697.431,27. RECOMENDAÇÕES INTERNACIONAIS: Foi realizada busca por recomendações de uso do teste CRAG-LFA por outras instituições e agências de ATS, a recomendação identificada é proveniente da Organização Mundial da Saúde (OMS). De acordo com a OMS, o diagnóstico e o tratamento precoce da meningite criptocócica são essenciais para reduzir a mortalidade por doença criptocócica em pacientes com infecção pelo vírus da imunodeficiência humana (HIV). A recomendação do órgão é para que os países deem prioridade a ensaios de antígeno criptocócico de diagnóstico rápido, de preferência ensaios de fluxo lateral como o CRAG-LFA, para uso no líquor, soro, plasma ou sangue total. Para adultos, adolescentes e crianças vivendo com HIV com suspeita de um primeiro episódio de meningite criptocócica, a punção lombar imediata com medição da pressão de abertura do líquor e aplicação de um teste diagnóstico rápido para o antígeno criptocócico é recomendado como o preferido abordagem diagnóstica. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Foram identificados dois outros testes registrados no segundo semestre de 2020 na Anvisa: CRAG Lateral Flow Assay e Biosynex® CryptoPS como testes rápidos CRAG-LFA. Na técnica de LA foram localizados 4 (quatro) registros sanitários na ANVISA. Já no FDA foram encontrados 3 (três) registros sanitários, sendo 1 (um) utilizando a técnica de sonda de DNA. Também foram consultadas as bases de dados de Propriedade Intelectual da WIPO e Espacenet Patent para buscar depósitos de testes de diagnósticos para meningite criptocócica. A consulta buscou os dados recentes de depósitos concedidos entre 2017 e 2020. Foram localizadas 05 patentes concedidas, sendo 04 (quatro) para metodologias em biologia molecular e um (1) para espectroscopia de Raman. CONSIDERAÇÕES FINAIS: Dadas as evidências apresentadas, o teste CRAG-LFA tem potencial custo-efetivo para a detecção da infecção criptocócica e prevenção da mortalidade relacionada à meningite criptocócica em PVHIV. RECOMENDAÇÃO PRELIMINAR: Diante do exposto, a Conitec, em sua 95ª reunião ordinária, realizada no dia 03 de março de 2021, deliberou que a matéria fosse disponibilizada em consulta pública com recomendação preliminar favorável à incorporação no SUS do teste point of care de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção de infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com CD4+ ≥200 células/mm³ e diagnóstico de meningite criptocócica em PVHIV independente da contagem de células CD4+. Os membros do plenário concordaram, a partir das evidências apresentadas que o teste diagnóstico é custo-efetivo, tem baixo impacto orçamentário e, além disso, foi considerado um teste de fácil aplicação na prática clínica, superando os testes já disponíveis, utilizados como comparadores. A matéria foi disponibilizada em consulta pública. CONSULTA PÚBLICA: A Consulta Pública nº 18/2021 foi realizada entre os dias 18/03/2021 e 06/04/2021. Foram recebidas 82 contribuições, sendo 45 pelo formulário para contribuições técnico-científicas e 38 pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. Foram consideradas apenas as contribuições encaminhadas no período estipulado e por meio do site da Conitec, em formulário próprio. As contribuições recebidas destacaram as vantagens clínicas do uso do teste CRAG-LFA, considerando economia de recursos, na identificação precoce da condição e tratamento preemptivo. Vantagens relacionadas ao manuseio do teste, sua aplicabilidade e facilidade na interpretação também foram citadas. Não FORAM ADICIONADAS REFERÊNCIAS QUE ALTERASSEM A ANÁLISE DA EVIDÊNCIA APRESENTADA NO RELATÓRIO. RECOMENDAÇÃO FINAL: Os membros do plenário presentes na 97ª reunião ordinária da Conitec, no dia 05 de maio de 2021, deliberaram, por unanimidade, recomendar a incorporação, no SUS, do teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção da infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com células CD4+ ≤ 200 cél/mm³ e diagnóstico de meningite criptocócica em PVHIV sintomáticos, independente da contagem de células CD4+, conforme estabelecido pelo Ministério da Saúde. Não foram adicionadas referências que alterassem a análise da evidência apresentada. Foi assinado o Registro de Deliberação nº 610/2021. DECISÃO: Incorporar o teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção de infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com CD4+ ³200 células/mm³ e diagnóstico de meningite criptocócica em PVHIV independente da contagem de células CD4+, no âmbito do Sistema Único de Saúde SUS, conforme Portaria nº 28, publicada no Diário Oficial da União nº 108, Seção 1, página 177, em 11 de junho de 2021.(AU)
Assuntos
Testes de Aglutinação/instrumentação , HIV , Meningite Criptocócica/diagnóstico , Criptococose/diagnóstico , Testes Imediatos , Avaliação da Tecnologia Biomédica , Análise Custo-Eficiência/métodos , Sistema Único de SaúdeRESUMO
OBJECTIVE: Commercial kits of column tests for pre-transfusion testing have progressively replaced conventional tube tests in most laboratories. Aim of this study was to compare three commercial test cell panels for the identification of irregular red blood cell (RBC) alloantibodies. Overall, 44 samples with a positive indirect antiglobulin test (IAT) by routine testing were used for comparison of following panels: Ortho RESOLVE® panelC (Ortho Clinical Diagnostics (OCD), Milan, Italy), ID-DiaPanel(-P) (Bio-Rad Laboratories, CA, USA) and Identisera Diana(P) (Grifols, Barcelona, Spain). Column agglutination techniques were used, with microtubes containing either microgel (Bio-Rad/Grifols) or glass bead microparticles (Ortho). RESULTS: Alloantibody identification was possible in 38 samples, of which identical identification was shown in 33 samples by all methods. The remaining samples showed differences between certain methods, with the gel card system being superior to the glass card system for analyzing stored samples Considering that not all samples were evaluated in all three methods, the concordance rate reached 100% between Bio-Rad and Grifols, 90.5% between Bio-Rad and OCD, 86.5% between OCD and Grifols and 90.5% between all methods. Although differences in sensitivities were seen for specific antibodies, the three methods showed comparable performance for the identification of RBC alloantibodies.
Assuntos
Testes de Aglutinação/normas , Tipagem e Reações Cruzadas Sanguíneas/normas , Eritrócitos/imunologia , Isoanticorpos/sangue , Testes de Aglutinação/instrumentação , Testes de Aglutinação/métodos , Tipagem e Reações Cruzadas Sanguíneas/instrumentação , Tipagem e Reações Cruzadas Sanguíneas/métodos , Eritrócitos/citologia , Humanos , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade , Medicina Transfusional/métodosRESUMO
This paper presents a novel design of a capillary stop valve with a chamfered side that can be used as a flow regulator to hold an injected microfluid in the valve position in a capillary force-driven microfluidic device. Biochemical analysis can be conducted if the chamfer-type valves are placed at strategic positions according to the test protocol. Hence, the stored reagent can be dragged out of the valve for further reaction when the specimen passes through. However, countercurrent phenomena were observed in the commonly used T-type capillary stop valve (without the chamfered side). In blood typing tests, the countercurrent led to incomplete dragging and the fluid stopped flowing at the complicated mixing channel; thus, the blood typing reaction was attenuated. On the contrary, the chamfer-type valve reduced the countercurrent phenomena and ameliorated the blood typing reaction. Consequently, agglutination results can be easily discriminated from nonagglutination cases.
Assuntos
Tipagem e Reações Cruzadas Sanguíneas/instrumentação , Tipagem e Reações Cruzadas Sanguíneas/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Testes de Aglutinação/instrumentação , Testes de Aglutinação/métodos , HumanosRESUMO
The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses.
Assuntos
Bioprótese , Imunoensaio/instrumentação , Rim/imunologia , Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência/instrumentação , Smartphone , Testes de Aglutinação/instrumentação , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Imunoensaio/métodos , Rim/efeitos dos fármacos , Rins Artificiais , Microscopia de Fluorescência/métodos , Aplicativos Móveis , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Testes de Toxicidade/instrumentação , Testes de Toxicidade/métodos , Interface Usuário-ComputadorRESUMO
Antibody titration is traditionally performed using a conventional test tube (CTT) method, which is subjected to interlaboratory variations because of a lack of standardization and reproducibility. The aim of this study is to compare newer methods such as get column technology (GCT) and erythrocyte magnetized technology (EMT) for antibody titration in terms of accuracy and precision. Patient serum samples that contained immunoglobin G (IgG) red blood cell (RBC) alloantibodies of a single specificity for Rh or K anitgens were identified during routine transfusion service testing and stored. Titration and scoring were performed separately by and stored. Titration and scoring were performed separately by different laboratory personnel on CTT, GCT, and EMT. Testing was performed a total of three times on each sample. Results were analyzed for accuracy and precision. A total of 50 samples were tested. Only 20 percent of samples tested with GCT shoed titers identical to CTT, whereas 48 percent of samples tested with EMT showed titers identical to CTT. Overall, the mean of th titer difference from CTT was higher using GCT (+0.31) compared with that using EMT (+0.13). Precision shown by CTT was 30 percent, EMT was 76 percent, and GCT was 92 percent on repeat testing. GCT showed higher titer values in comparison with CTT but was found to be the most precise. EMT titers were comparable to CTT, and its precision was intermediate. Further studies to validate this method are required.
Assuntos
Testes de Aglutinação/instrumentação , Testes de Aglutinação/métodos , Eritrócitos/imunologia , Isoanticorpos/análise , Campos Magnéticos , Feminino , Humanos , Isoanticorpos/imunologia , MasculinoRESUMO
Aimed to explore an agglutination test which can simultaneously detect two pathogenic bacteria, an agglutination test based on colored silica nanoparticles (colored-SiNps) was established in this work. Monodisperse colored-SiNps were used as agglutination test carriers; red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red 136 and C.I. Reactive Blue 14. Then the red-SiNps were sensitized with antibodies against E. sakazaki and denoted as IgG-red-SiNps; The blue-SiNps were coated with antibodies against S. pullorum and S. Gallinarum and denoted as IgGblue- SiNps. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could simultaneously agglutinate with E. sakazakii and S. pullorum and S. gallinarum on glass slide. The E. sakazakii and S. pullorum and S. gallinarum could be simultaneously detected by agglutination test with obvious agglutination phenomena. The E. sakazakii and S. pullorum and S. gallinarum could both be detected in a range from 4×10(3) to 4×10(9) CFU/mL. The pullorum and S. gallinarum and E. sakazakii in the infected food sample were detected by mixture solution of IgG-red-SiNps and IgG-blue-SiNps too. This agglutination test was easy and rapid, it might be useful for in situ rapid detection method for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.
Assuntos
Testes de Aglutinação/métodos , Enterobacter/isolamento & purificação , Nanopartículas , Salmonella/isolamento & purificação , Dióxido de Silício/química , Testes de Aglutinação/instrumentação , Microscopia Eletroquímica de Varredura , Nanopartículas/química , Nanopartículas/ultraestruturaAssuntos
Proteínas ADAM/sangue , Testes de Aglutinação/métodos , Ensaios Enzimáticos Clínicos/métodos , Microangiopatias Trombóticas/enzimologia , Proteína ADAMTS13 , Testes de Aglutinação/instrumentação , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Reações Antígeno-Anticorpo , Automação , Catálise , Compostos Cromogênicos/análise , Ensaios Enzimáticos Clínicos/instrumentação , Ensaio de Imunoadsorção Enzimática , Glutationa Transferase/análise , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Coloide de Ouro , Humanos , Nefelometria e Turbidimetria , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade , Fator de von Willebrand/análise , Fator de von Willebrand/genéticaRESUMO
BACKGROUND AND OBJECTIVES: ABO-incompatible red blood cell transfusions still represent an important hazard in transfusion medicine. Therefore, some countries have introduced a systematic bedside ABO agglutination test checking that the right blood is given to the right patient. However, this strategy requires an extremely time-consuming learning programme and relies on a subjective interpretation of ABO test cards agglutination. We developed a prototype of a fully automated device performing the bedside agglutination test that could be completed by reading of a barcoded wristband. This POCT checks the ABO compatibility between the patient and the blood bag. MATERIALS AND METHODS: Proof of concept and analytical validation of the prototype has been completed on 451 blood samples: 238 donor packed red blood cells, 137 consecutive unselected patients for whom a blood group determination had been ordered and on 76 patient samples selected with pathology that could possibly interfere with or impair performances of the assay. RESULTS: We observed 100% concordance for ABO blood groups between the POCT and the laboratory instrument. CONCLUSION: These preliminary results demonstrate the feasibility of ABO determination with a simple POCT device eliminating manipulation and subjective interpretation responsible for transfusion errors. This device should be linked to the blood bank system allowing all cross-check of the results.
Assuntos
Sistema ABO de Grupos Sanguíneos/análise , Testes de Aglutinação/métodos , Tipagem e Reações Cruzadas Sanguíneas/métodos , Testes Imediatos , Testes de Aglutinação/instrumentação , Automação , Bancos de Sangue , Incompatibilidade de Grupos Sanguíneos , Tipagem e Reações Cruzadas Sanguíneas/instrumentação , Feminino , Humanos , Masculino , Kit de Reagentes para DiagnósticoRESUMO
Today, when monoclonal antibodies (mAbs) have become one of the most important classes of therapeutic drugs, it is easy to forget how much they have transformed our healthcare in other ways. One of the first clinical areas, as this paper shows, where mAbs made their mark was in the field of blood typing. The adoption of mAbs for this purpose was done with little public fanfare or funding. Nonetheless, it radically transformed the accuracy and cost of blood typing and shifted the procedure away from a dependence on reagents made from human blood donated by volunteers. This paper argues that the development of mAbs as reagents for blood typing laid the foundation for the first large-scale production of mAbs thereby paving the way to the advent of mAb diagnostics and therapeutics.
Assuntos
Testes de Aglutinação/métodos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Sistema ABO de Grupos Sanguíneos , Testes de Aglutinação/instrumentação , Testes de Aglutinação/tendências , Tipagem e Reações Cruzadas Sanguíneas/economia , Tipagem e Reações Cruzadas Sanguíneas/tendências , Humanos , Sistema do Grupo Sanguíneo MNSs , Reprodutibilidade dos Testes , Sistema do Grupo Sanguíneo Rh-HrRESUMO
BACKGROUND: Detection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers. METHODS: For ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs. RESULTS: Regardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O. CONCLUSIONS: There were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in interpreting ABO Ab titer results, taking into consideration the detection method used and the blood group.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Testes de Aglutinação/normas , Anticorpos/análise , Citometria de Fluxo , Adulto , Idoso , Testes de Aglutinação/instrumentação , Anticorpos Anti-Idiotípicos/análise , Eritrócitos/química , Eritrócitos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , TemperaturaRESUMO
BACKGROUND: Most immune reactions related to transfusion and transplantation are caused by IgM ABO antibodies. However, IgG also plays an important role in these reactions. Therefore, a method to measure antibodies, including IgG, is necessary. We investigated ABO antibody titers of healthy individuals using a column agglutination technique (CAT) with or without dithiothreitol (DTT) and compared them with titers obtained using a conventional tube method. METHODS: Among healthy adults who underwent a medical examination, 180 individuals (60 with blood group A, 60 with group B, and 60 with group O) were selected. Antibody titrations were performed using the immediate spin (IS) tube, anti-human globulin (AHG) tube, and CAT with or without DTT methods. RESULTS: Higher median values of anti-B and anti-A titers in groups A and B individuals, respectively, were obtained using the IS method than using the AHG method. Higher values for group O individuals were obtained using the AHG method. Higher median titers of anti-B and anti-A in group O individuals were obtained using CAT without DTT than using the AHG method. Median titers of anti-B and anti-A in all blood groups were higher in CAT without DTT than in CAT with DTT, especially for group O individuals. CONCLUSIONS: We recommend CAT with and without DTT for titration of anti-A and anti-B, especially in group O individuals, to provide more sensitive results that include IgG data. Adjustment of insurance coverage of fees associated with antibody titration might be necessary, considering the actual cost of reagents and personnel.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Testes de Aglutinação , Anticorpos/análise , Adulto , Testes de Aglutinação/instrumentação , Anticorpos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Influenza A H1N1/2009 is a highly infectious, rapidly spreading airborne disease that needs to be monitored in near real time, preferably in a microfluidic format. However, such demonstration is difficult to find as H1N1 concentration in aerosol samples is extremely low, with interference from dust particles. In this work, we measured Mie scatter intensities from a microfluidic device with optical waveguide channels, where the antibody-conjugated latex beads immunoagglutinated with the target H1N1 antigens. Through careful optimizations of optical parameters, we were able to maximize the Mie scatter increase from the latex immunoagglutinations while minimizing the background scatter from the dust particles. The aerosol samples were collected from a 1:10 mock classroom using a button air sampler, where a nebulizer generated aerosols, simulating human coughing. The detection limits with real aerosol samples were 1 and 10 pg/mL, using a spectrometer or a cell phone camera as an optical detector, respectively. These are several orders of magnitudes more sensitive than the other methods. The microfluidic immunosensor readings are in concordance with the results of reverse transcription polymerase chain reaction. The assay time was 30 s for sampling and 5 min for the microfluidic assay.
Assuntos
Testes de Aglutinação/instrumentação , Anticorpos Imobilizados/metabolismo , Anticorpos Antivirais/metabolismo , Tosse/virologia , Monitoramento Ambiental/instrumentação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Aerossóis/química , Automação Laboratorial , Telefone Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Limite de Detecção , Microfluídica/instrumentação , Microesferas , Miniaturização , Reprodutibilidade dos Testes , Espectrofotometria/instrumentação , VentilaçãoRESUMO
Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection.
Assuntos
Testes de Aglutinação/métodos , Blastomyces/isolamento & purificação , Blastomicose/diagnóstico , Testes de Aglutinação/instrumentação , Animais , Anticorpos Antifúngicos/imunologia , Blastomyces/imunologia , Blastomicose/imunologia , Blastomicose/microbiologia , Humanos , Coelhos , Sensibilidade e EspecificidadeRESUMO
Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.
Assuntos
Testes de Aglutinação/métodos , Imunoensaio/métodos , Magnetismo , Testes de Aglutinação/instrumentação , Reações Antígeno-Anticorpo , Imunoensaio/instrumentação , Microfluídica , Óleos/química , Estreptavidina/química , Água/químicaRESUMO
The measurement of time and distance can be used for determining agglutination in small (nL) samples of liquid. We demonstrate the use of this new scheme of detection in typing and subtyping blood in a simple microfluidic system that monitors the speed of flow of microdroplets. The system (i) accepts small samples of liquids deposited directly onto the chip, (ii) forms droplets on demand from these samples, (iii) merges the droplets, and (iv) measures their speed in a microchannel. A sequence of measurements on different combinations of blood and antibodies can thus be used to determine blood type with the estimated probability of mistyping being less than 1 in a million tests. In addition, in the agglutinated samples, red blood cells concentrate at the rear of the droplets yielding an additional vista for detection and suggesting a possible mechanism for separations.
Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Aglutinação , Testes de Aglutinação/instrumentação , Testes de Aglutinação/métodos , Tipagem e Reações Cruzadas Sanguíneas/instrumentação , Movimento Celular , Desenho de Equipamento , Eritrócitos/citologia , Eritrócitos/imunologia , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To determine if column agglutination technology (CAT) for titration of anti-D and anti-c concentrations produces comparable results to those obtained by continuous flow analyser (CFA). BACKGROUND: Anti-D and anti-c are the two commonest antibodies that contribute to serious haemolytic disease of the foetus and neonate (HDFN). Current practice in the UK is to monitor these antibodies by CFA quantification, which is considered more reproducible and less subjective than manual titration by tube IAT (indirect antiglobulin test). CAT is widely used in transfusion laboratory practice and provides a more objective endpoint than tube technique. MATERIALS AND METHODS: Antenatal samples were (i) quantified using CFA and (ii) titrated using CAT with the reaction strength recorded by a card reader and expressed as a titre score (TS). RESULTS: The TS rose in accordance with levels measured by quantification and was able to distinguish antibody levels above and below the threshold of clinical significance. CONCLUSION: CAT titre scores provided a simple and reproducible method to monitor anti-D and anti-c levels. The method was sensitive to a wide range of antibody levels as determined by quantification. This technique may have the potential to replace CFA quantification by identifying those cases that require closer monitoring for potential HDFN.
Assuntos
Testes de Aglutinação/instrumentação , Isoanticorpos/sangue , Cuidado Pré-Natal/métodos , Adulto , Testes de Aglutinação/métodos , Automação , Teste de Coombs , Eritroblastose Fetal/prevenção & controle , Feminino , Humanos , Recém-Nascido , Projetos Piloto , Gravidez , Reprodutibilidade dos Testes , Imunoglobulina rho(D)RESUMO
Reliability of the Widal tube agglutination test has been the subject of many controversies over the years. This study was performed to assess the effect of certain modifications on the performance of Widal test in a novel microplate assay. Sera from 37 patients (21 males; 16 females) (mean age 28 +/- 7 years) were tested in the Immunology Unit at King Khalid University Hospital, Riyadh. Among them were 26 patients with suspected typhoid fever and 11 had bacteriologically confirmed diagnosis of Salmonella infection. The modifications included either the use of 0.5% bovine serum albumin (BSA), absorption of sera with sheep red blood cells (SRBC) or heat inactivation of sera. Compared with Widal tube agglutination test, microplate assay with SRBC absorption of the sera from patients with suspected typhoid fever was not only associated with enhancement of detection titers for both H (p < or = 0.001) and O (p < or = 0.005) Salmonella agglutinins but also the percentage of reactivity. The presence of BSA augmented detection titers for Salmonella H agglutinins (p < or = 0.02) only. Heat inactivation of sera however was found to be associated with reduction in the detectable titers for both H (p < or = 0.03) and O (p < or = 0.01) agglutinins. Increased titers of Salmonella agglutinins were also evident in 11 patients with confirmed diagnosis of Salmonella infection. The novel microplate agglutination assay using the SRBC absorption was associated with enhancement in Widal test reactivity and appears to be a useful alternative for the diagnosis of Salmonella infection.
Assuntos
Testes de Aglutinação/métodos , Febre Tifoide/diagnóstico , Adulto , Testes de Aglutinação/instrumentação , Aglutininas/análise , Aglutininas/imunologia , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Feminino , Humanos , Masculino , Salmonella typhi/imunologia , Salmonella typhi/isolamento & purificação , Febre Tifoide/microbiologia , Adulto JovemRESUMO
Fibrinogen can transform fibrin through an agglutination reaction, finally forming fibrin polymer with grid structure. The density and viscosity of the reaction system changes drastically during the course of agglutination. In this research, we apply an independently-developed piezoelectric agglutination sensor to detect the fibrinogen agglutination reaction in patients with coronary heart diseases. The terminal judgment method of determining plasma agglutination reaction through piezoelectric agglutination sensor was established. In addition, the standard curve between plasma agglutination time and fibrinogen concentration was established to determinate fibrinogen content quantitatively. The results indicate the close correlation between the STAGO paramagnetic particle method and the method of piezoelectric agglutination sensor for the detection of Fibrinogen. The correlation coefficient was 0.91 (γ = 0.91). The determination can be completed within 10 minutes. The fibrinogen concentration in the coronary heart disease group was significantly higher than that of the healthy control group (P < 0.05). The results reveal that high fibrinogen concentration is closely correlated to the incurrence, development and prognosis of coronary heart diseases. Compared with other traditional methods, the method of piezoelectric agglutination sensor has some merits such as operation convenience, small size, low cost, quick detecting, good precision and the common reacting agents with paramagnetic particle method.
Assuntos
Testes de Aglutinação/instrumentação , Técnicas Biossensoriais/instrumentação , Doença das Coronárias/sangue , Fibrinogênio/análise , Testes de Aglutinação/métodos , Técnicas Biossensoriais/métodos , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , QuartzoRESUMO
Red blood cell (RBC) aggregation in the blood stream is prevented by the zeta potential created by its negatively charged membrane. There are techniques, however, to decrease the zeta potential and allow cell agglutination, which are the basis of most of antigen-antibody tests used in immunohematology. We propose the use of optical tweezers to measure membrane viscosity, adhesion, zeta potential, and the double layer thickness of charges (DLT) formed around the cell in an electrolytic solution. For the membrane viscosity experiment, we trap a bead attached to RBCs and measure the force to slide one RBC over the other as a function of the velocity. Adhesion is quantified by displacing two RBCs apart until disagglutination. The DLT is measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta potential is obtained by measuring the terminal velocity after releasing the RBC from the trap at the last applied voltage. We believe that the methodology proposed here can provide information about agglutination, help to improve the tests usually performed in transfusion services, and be applied for zeta potential measurements in other samples.
Assuntos
Testes de Aglutinação/instrumentação , Testes de Aglutinação/métodos , Separação Celular/instrumentação , Membrana Eritrocítica/fisiologia , Adesões Focais/fisiologia , Pinças Ópticas , Adesividade , Separação Celular/métodos , Células Cultivadas , Elasticidade , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Potenciais da Membrana , Estresse MecânicoRESUMO
A simple rapid piezoelectric immunoagglutination assay (PEIA) technique with antibody-modified liposome has been developed for direct quantitative detection of human immunoglobulin G (hIgG). This technique is based on specific agglutination of antibody-coated liposome particles in the presence of the corresponding antigen, which can be monitored by the frequency shift of a piezoelectric device. Compared with conventional piezoelectric assays, this liposome-based PEIA does not require the immobilization of antigen or antibody on the quartz crystal surface, making the developed technique especially useful for rapid and renewable immunochemical determination. To alleviate non-specific adsorption of serum proteins, modification of the quartz crystal surface by different protocols and the composition of the assay medium have been investigated. The results indicate that the background interference can be substantially minimized through modifying the quartz crystal surface with a bovine serum albumin (BSA) layer and introducing an appropriate amount of BSA in the assay medium. The effects of the liposome composition, the liposome concentration and the concentration of poly(ethylene glycol) (PEG) in the assay medium, have also been investigated. The frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05-6 microg mL(-1) with a detection limit of 50 ng mL(-1).