RESUMO
Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.
Assuntos
Doadores de Sangue , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Western Blotting , Humanos , RNA Viral/sangue , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04 percent) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.
O uso de testes de ácidos nucleicos (NAT) na rotina de triagem de doadores de sangue tornou-se uma realidade ao final da década de 1990. Descreve-se aqui uma metodologia de RT-PCR multiplex "in-house" que permite a detecção simultânea dos RNAs dos vírus HIV e HCV além de uma molécula artificial de RNA usada como controle externo. O método detecta todos os subtipos de HIV do grupo M e também do grupo N e O, com uma sensibilidade de 500 UI/mL. Após validação, este teste substituiu o do antígeno p24, até então na rotina de triagem em nosso laboratório, desde 1996. De julho de 2001 a fevereiro de 2006 foram testadas 102.469 doações e 41 (0.04 por cento) foram NAT reativas. Uma doação NAT isoladamente reativa (anticorpo não-reativa) foi detectada com soroconversão subseqüente do doador, portanto, o rendimento do NAT nesta população até o presente momento é de 1:102.469. Este número contrasta com a experiência obtida internacionalmente, onde taxas de 1:600.000 - 1:3.100.000 foram descritas.
Assuntos
Humanos , Doadores de Sangue , HIV , /sangue , Infecções por HIV/diagnóstico , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Western Blotting , HIV , Reprodutibilidade dos Testes , RNA Viral/sangue , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To evaluate, in vitro, the effects of collecting and cryopreserving fresh dry platelet concentrates (PCs). MATERIAL AND METHODS: Standard and dry PCs were collected in the same apheresis procedure. PCs were evaluated by mean platelet volume (MPV), pH, glucose and LDH levels. Activation was examined by flow cytometry using anti-CD41, anti-CD42 and anti-CD62p monoclonal antibodies and annexin binding assay. Platelet function was assessed by aggregation using ADP, collagen and arachidonic acid as agonists. Dry PCs were compared to standard PCs and to cryopreserved dry PCs. We also compared the use of ThromboSol to 5% DMSO as cryoprotectives. RESULTS: Dry PCs presented a significantly reduced pH and glucose (p<0.001), increased LDH levels and CD62p expression (p<0.001) and diminished aggregation response to ADP (p<0.001). Platelet cryopreservation was associated with platelet lysis, activation and loss of function. Dry PCs cryopreserved with TS were associated with statistically higher LDH levels (p<0.001) and a higher percentage of annexin binding (p=0.005), in addition to a lower number of CD42 positive platelets (p=0.01). CONCLUSION: Dry PCs should be rapidly frozen after collection to avoid a fall in pH and platelet activation. 5% DMSO performed better than TS to cryopreserve dry PCs.
