RESUMEN
BACKGROUND AND OBJECTIVES: Enumeration of residual white blood cells in leucoreduced blood components is essential part of quality control. Digital PCR has substantially facilitated quantitative PCR and was thus evaluated for measurements of leucocytes. MATERIALS AND METHODS: Target for quantification of leucocytes by digital droplet PCR was the blood group gene RHCE. The SPEF1 gene was added as internal control for the entire assay starting with automated DNA extraction. The sensitivity of the method was determined by serial dilutions of standard samples. Quality control samples were analysed within 24 h, 7 days and 6 months after collection. Routine samples from leucodepleted red blood cell concentrates (n = 150) were evaluated in parallel by flow-cytometry (LeucoCount) and by digital PCR. RESULTS: Digital PCR reliably detected at least 0·4 leucocytes per assay. The mean difference between PCR and flow-cytometric results from 150 units was -0·01 (±1·0). DNA samples were stable for up to at least six months. PCR measurement of leucocytes in samples from plasma and platelet concentrates also provided valid results in a pilot study. CONCLUSION: Droplet digital PCR to enumerate leucocytes offers an alternative for quality control of leucoreduced blood products. Sensitivity, specificity and reproducibility are comparable to flow-cytometry. The option to collect samples over an extended period of time and the automatization introduce attractive features for routine quality control.
Asunto(s)
Seguridad de la Sangre , ADN/sangre , ADN/genética , Transfusión de Eritrocitos , Eritrocitos/citología , Citometría de Flujo , Humanos , Recuento de Leucocitos/métodos , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Reacción a la Transfusión/prevención & controlRESUMEN
Antibodies against human leukocyte antigens (HLAs) have long been associated with transfusion-related acute lung injury (TRALI). In contrast to febrile transfusion reactions and refractoriness to platelet transfusions in immunized patients, the causative antibodies in TRALI are present in the transfused blood component, i.e. they are formed by the blood donor and not by the recipient. Consequently, blood components with high plasma volume are particularly associated with TRALI. In addition to antibodies against HLAs, antibodies directed against human neutrophil antigens (HNAs) present in the plasma of predominantly multiparous female blood donors can induce severe TRALI reactions. Especially, antibodies to HLA class II and HNA-3a antigens can induce severe or even fatal ALI in critically ill patients. Over the last decade, the clinical importance of TRALI as major cause for severe transfusion-related morbidities has led to the establishment of new guidelines aimed at preventing this condition, including routine testing for HLA and -HNA antibodies for plasma donors with a history of allogeneic sensitization. This, in turn, poses new challenges for close collaboration between blood transfusion centers and histocompatibility and immunogenetics laboratories, for sensitive and specific detection of the relevant antibodies.
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Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/prevención & control , Prueba de Histocompatibilidad/tendencias , Histocompatibilidad/fisiología , Inmunogenética/tendencias , Reacción a la Transfusión , Transfusión Sanguínea/métodos , Transfusión Sanguínea/normas , Transfusión Sanguínea/tendencias , Femenino , Antígenos HLA/inmunología , Prueba de Histocompatibilidad/métodos , Humanos , Inmunogenética/métodos , Modelos Biológicos , Medicina Regenerativa/métodos , Medicina Regenerativa/normas , Medicina Regenerativa/tendenciasRESUMEN
Patients with refractory or relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) rarely have prolonged responses to salvage therapy, including imatinib, resulting in a short opportunity for potentially curative stem cell transplantation. To identify minimal residual disease (MRD) parameters predictive of imminent relapse, we quantitated Bcr-Abl expression by real-time PCR in peripheral blood (PB) and bone marrow (BM) of 24 Ph+ALL patients after achieving a complete response and MRD minimum. The ratio of Bcr-Abl and glyceraldehyde-3-phosphate dehydrogenase copies, magnitude of increase and velocity of increase were evaluated regarding subsequent time intervals to relapse, death or censoring. High Bcr-Abl levels >/=5 x 10(-4) in PB (n=23) and >/=10(-4) in BM (n=18) were significantly associated with short time periods to relapse. Bcr-Abl increases >2 logarithmic units (log) in PB, but not in BM preceded short-term relapse. The velocity of Bcr-Abl increases predicted response duration in PB (cutoff: 1.25 log/30 days) and BM (0.6). Bcr-Abl level and velocity of increase in BM as well as magnitude of increase in PB correlated with remaining periods of survival and predicted relapse within 2 months in nine of 10, 10 of 11 and four of four patients, respectively. Thus, these MRD parameters may guide timing and intensity of therapeutic modifications.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasia Residual/diagnóstico , Piperazinas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirimidinas/uso terapéutico , ARN Mensajero/análisis , Benzamidas , Médula Ósea/metabolismo , Médula Ósea/patología , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Mesilato de Imatinib , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Neoplasia Residual/tratamiento farmacológico , Neoplasia Residual/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Neoplásico/genética , Inducción de Remisión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Terapia Recuperativa , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND AND OBJECTIVES: DNA typing of the human Rh blood groups generally shows good agreement with serologically defined phenotypes. However, in the present report we describe four individuals who were declared Rh e negative by genotyping although they express the Rh e antigen. MATERIALS AND METHODS: Serotyping was performed using mono- and polyclonal Rh antisera. Fluorescent multiplex sequence-specific polymerase chain reactions (PCR-SSPs) identified RHD exons and the polymorphisms usually associated with the Rh E/e or Rh C/c/C(W) antigens. Additional PCR amplification reactions, which were carried out to reveal RHCE-D-CE hybrid genes, analysed exon 5 of the RH genes, the location of the polymorphism (676C-->G) coding for the Rh E and Rh e antigens. RESULTS: Four individuals were identified who expressed Rh e antigens but were negative by PCR-SSP typing for common Rhe-coding sequences. In one family analysed in detail, an RHCE-D5-CE hybrid gene associated with Rh e antigen expression was identified. A concomitant RHcE allele accounted for a seemingly regular typing pattern by conventional RH PCR. CONCLUSIONS: The presence of RHCE-D5-CE hybrid alleles may cause false-negative DNA-typing results for the Rh e antigen that are easily overlooked unless appropriate RH hybrid PCR-SSPs are incorporated into conventional DNA-typing protocols. These and previous data strongly caution against an uncritical interpretation of RH DNA-typing results.
Asunto(s)
Reordenamiento Génico , Glicoproteínas/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Tipificación y Pruebas Cruzadas Sanguíneas , Epítopos/análisis , Reacciones Falso Negativas , Genotipo , Humanos , LinajeRESUMEN
While blood products become more safe in terms of viral contamination, the risk of transfusion-related bacterial infection has re-emerged to one of the major hazards in transfusion medicine. In recent prospective studies the rate of contaminated platelets ranged from 0.04 to 0.5%, and a rate of transfusion reactions between 0.007% and 0.046%. It is generally agreed that most of the organisms isolated from donated blood originate from the normal skin flora or from the environment. As it is unlikely that antiseptic methods can achieve absolute sterilization of the skin before venepuncture, blood banks have to rely on laboratory tests to detect contaminated blood products before release. But most of the currently available methods detecting bacterial contaminations do not have the potential to be sensitive and fast enough for a routine contamination screening in transfusion services. Here we present two alternative strategies based on molecular genetic techniques (Real-Time-PCR and Haystack processing) that detect or semi-quantify bacterial rRNA gene sequences for the majority of bacterial species. In addition we discuss some aspects on target selection, routine preparation and residual 16S-rDNA-contamination of enzymes.