Paper microfluidic device for early diagnosis and prognosis of acute myocardial infarction via quantitative multiplex cardiac biomarker detection.

The early detection of acute myocardial infarction (AMI) upon the onset of chest pain symptoms is crucial for patient survival. However, this detection is challenging, particularly without a persistent elevation of ST-segment reflected in an electrocardiogram or in blood tests. A majority of the available point-of-care testing devices allow accurate and rapid diagnosis of AMI. However, AMI diagnosis is reliable only at intermediate and later stages, with myocardial injury (> 6 h) and MI, based on the expression of specific cardiac biomarkers including troponin I or T (cTnI or cTnT), creatine kinase-MB (CK-MB), and myoglobin. Diagnosis at the early myocardial ischemia stage is not possible. To overcome this limitation, a sensitive and rapid microfluidic paper-based device (µPAD) was developed for the simultaneous detection of multiple cardiac biomarkers for the early and late diagnosis of AMI. The glycogen phosphorylase isoenzyme BB (GPBB) was detected during early (within first 4 h) ischemic myocardial injury. On the same µPAD platform, detection of prolonged elevation of levels of cTnT and CK-MB, which are only produced 6 h after the onset of chest pain in human serum, was possible. Sandwich immunoassay performed on the µPAD achieved reproducibility (RSD approximately 10% and intra-and inter-day precision (CV 10-20%, 99th percentile), as well as consistently stable test results for 28 days, with strong correlation (r2= 0.962), using the standard Siemens Centaur XPT Immunoassay system. The present findings indicate the potential of the µPAD platform as a point-of-care device for the early diagnosis and prognosis of AMI.

Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.

Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Limitaciones de la determinación de los niveles de los biomarcadores séricos durante el aislamiento de venas pulmonares / Limitations of biomarkers serum levels during pulmonary vein isolation

Introducción y objetivos. Se han usado varios biomarcadores para la evaluación y la cuantificación de la lesión miocárdica tras ablación. Estudiamos las posibles diferencias en la estabilidad térmica y las posibilidades de uso de las proteínas liberadas por las células cardiacas lesionadas mediante diferentes fuentes de energía. Métodos. En primer lugar, estudiamos la estabilidad térmica in vitro de la creatincinasa (CK), la isoenzima miocárdica de la creatincinasa (CK-MB), las troponinas I (cTnI) y las troponinas T (cTnT) en muestras de sangre obtenidas de 15 pacientes con infarto agudo de miocardio con elevación del segmento ST (IAMCEST) confirmado. En segundo lugar, se obtuvieron y se analizaron los biomarcadores en 82 pacientes tratados mediante ablación con radiofrecuencia (ARF) y en 79 pacientes tratados mediante crioablación con balón (CAB). Resultados. Los experimentos in vitro mostraron que todos los biomarcadores eran estables a temperaturas bajas (–30°C). Las troponinas se mostraron estables al analizarlas a altas temperaturas. En cambio, se observó un descenso importante en los valores de CK y CK-MB a 50 y 40°C, respectivamente. El estudio in vivo mostró que el aumento de las cifras de CK-MB fue significativamente elevado en pacientes sometidos a CAB exclusivamente. Se observaron valores patológicos de CK-MB en el 24% de los pacientes con ARF y en el 98% de los pacientes sometidos a CAB. Se observaron valores patológicos de cTnI en todos los pacientes y el aumento de la concentración de cTnI fue muy significativo en ambos grupos tras la ablación. Conclusiones. Tanto los resultados in vitro como los obtenidos in vivo muestran que la CK-MB no puede usarse para la determinación cuantitativa de las lesiones miocárdicas producidas por la energía de radiofrecuencia. Sólo las troponinas reflejan las lesiones miocárdicas independientemente de la fuente de energía, y se debería utilizarlas para comparar los efectos en los biomarcadores de la crioablación frente a la ablación con radiofrecuencia (AU)

Introduction and objectives: Several biomarkers have been used for evaluation and quantification of myocardial injury after effective ablation. Westudied possible different thermal stability and usability of the proteins released by cardiac cells injured by different energy sources. Methods: Firstly, we tested in vitro thermal stability of creatinine kinase (CK), myocardial bound creatinine kinase (CKMB), cardiac troponins I (cTnI) and cardiac troponins T (cTnT) in collected blood samples from 15 patients (pts) with confirmed ST-segment elevated myocardial infarction (STEMI). Secondly, the biomarkers were collected and analyzed in 82 pts treated with radiofrequency ablation (RFA) and in 79 pts treated with cryo-balloon ablation (CBA). Results: In vitro experiment showed that all biomarkers were stable in low temperature of -30oC. Troponins were stable in the high temperatures analyzed. A substantial drop in CK and CKMB levels were measured at 50 8C and 408 C, respectively. In vivo study showed that the increase in CKMB levels was highly significant in CBA pts only. Pathological CKMB values were observed in 24% of RFA pts and 98% of CBA pts. Pathological cTnI values were observed in all pts and the rise in cTnI levels was highly significant in both groups after ablation. onclusions: Both in vitro and in vivo results show that CKMB cannot be used for quantitative determination of myocardial injury produced by radiofrequency energy. Only cardiac troponins reflect myocardial injury, regardless of energy source, and may be considered in future studies for comparison of biomarkers effects of cryo versus radiofrequency ablation (AU)