Quantification of IgM molecular response by droplet digital PCR as a potential tool for the early diagnosis of sepsis

Critical Care, Jun 2014

Eduardo Tamayo, Raquel Almansa, Elena Carrasco, Ana Ávila-Alonso, Ana Rodríguez-Fernández, John Wain, María Heredia, Esther Gomez-Sanchez, Susana Soria, Lucia Rico, et al.

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Quantification of IgM molecular response by droplet digital PCR as a potential tool for the early diagnosis of sepsis

Eduardo Tamayo 1 3 4 Raquel Almansa 0 1 2 3 Elena Carrasco 4 Ana vila-Alonso 8 Ana Rodrguez-Fernndez 8 John Wain 7 Mara Heredia 4 Esther Gomez-Sanchez 4 Susana Soria 4 Lucia Rico 0 1 2 3 Vernica Iglesias 0 1 2 3 ngel Martnez-Martnez 4 David Andaluz-Ojeda 1 3 6 Jose Ignacio Gmez Herreras 1 3 4 Jose Maria Eiros 5 Jess F Bermejo-Martin 0 1 2 3 0 Unidad de Apoyo a la Investigacion, Hospital Clinico Universitario de Valladolid, IECSCYL-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 1 Immunity, Risk of Infection and Sepsis group (IRIS), Hospital Clinico Universitario de Valladolid , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 2 Unidad de Apoyo a la Investigacion, Hospital Clinico Universitario de Valladolid, IECSCYL-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 3 Immunity, Risk of Infection and Sepsis group (IRIS), Hospital Clinico Universitario de Valladolid , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 4 Servicio de Anestesiologia y Reanimacion, Hospital Clinico Universitario-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 5 Hospital Clinico Universitario-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 6 Unidad de Cuidados Intensivos, Hospital Clinico Universitario-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain 7 Norwich Medical School, University of East Anglia , Norwich, Norfolk NR4 7TJ , UK 8 Servicio de Microbiologia, Hospital Clinico Universitario-SACYL , Avda Ramon y Cajal 3, 47005 Valladolid , Spain - Evaluation of host immune response to infection at the molecular level is a promising avenue to obtain diagnostic and prognostic tools for the clinical management of patients with sepsis. A recent report from Cajander and colleagues [1] has shown the potential of HLA-DR mRNA quantification by real-time PCR as a biomarker of immunosuppression in these patients. IgM is the first immunoglobulin produced in response to infection. In a pilot study, we have employed a next generation quantitative PCR method (nanoliter-sized droplet technology paired with digital PCR (ddPCR)) for detecting the early transcriptomic response of IgM in blood from patients with sepsis. Approval for the study protocol for both scientific and ethical aspects was obtained from the Committee for Clinical Research of Hospital Clnico Universitario, Valladolid, Spain. Written informed consent was obtained directly from each patient or a legal surrogate. The target gene transcript was IGHM, which encodes the constant region of the mu heavy chain, which defines the IgM isotype [2]. In blood, the cells producing IgM transcripts are B lymphocytes expressing CD20 [3], which was employed as housekeeping gene. Fifty-five patients with sepsis were recruited, 42 of them presenting criteria of septic shock (Additional file 1). Septic patients were predominantly older males (n = 40, 72.7%; mean age 72 years (standard deviation 9.3)). Mean Sepsis-related Organ Failure Assessment score was 8.4 (standard deviation 2.9). Overall ICU mortality was 34%. Emergency surgery was needed in 54% of cases, with cardiac and abdominal surgery the most frequent (45% and 40%, respectively). Respiratory infection was present in 34.5% of the cases. Frequency of abdominal infection was also 34.5%. Gram-negative bacteria were the most frequent isolated (56. 4% of cases). In parallel, we recruited 20 patients with post-surgical systemic inflammatory response syndrome (SIRS) and 15 healthy controls. Compared to real-time quantitative PCR, ddPCR offers greater precision and reproducibility [4]. ddPCR allowed us to identify the presence of an early molecular response of IgM in the blood of patients with sepsis compared with healthy controls and patients with SIRS. This response was more intense in the most severe patients (Figure 1). When accuracy and the predictive value of the IGHM/CD20 ratio for diagnosing sepsis were analyzed, the area under the receiver operating characteristic curve was 0.72 (95% confidence interval 0.60 to 0.85; P = 0.003; Figure 1). In conclusion, quantification of IgM response at the transcriptomic level by ddPCR represents a promising approach for the early detection of sepsis. 2014 Tamayo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Figure 1 IgM transcriptomic response in the presence/absence of sepsis. Left: comparison of immunoglobulin (Ig)M transcriptomic response between groups. HC, healthy control (n = 15); SIRS, systemic inflammatory response syndrome (n = 20); SEPSIS SOFA < =10, Sepsis with Sepsisrelated Organ Failure Assessment score 10 (n = 39); SEPS (...truncated)


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Eduardo Tamayo, Raquel Almansa, Elena Carrasco, Ana Ávila-Alonso, Ana Rodríguez-Fernández, John Wain, María Heredia, Esther Gomez-Sanchez, Susana Soria, Lucia Rico, Verónica Iglesias, Ángel Martínez-Martínez, David Andaluz-Ojeda, Jose Ignacio Herreras, Jose Eiros, Jesús F Bermejo-Martin. Quantification of IgM molecular response by droplet digital PCR as a potential tool for the early diagnosis of sepsis, Critical Care, 2014, pp. 433, 18, DOI: 10.1186/cc13910