Open Access

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

  • Eduardo Tamayo1, 2,
  • Raquel Almansa2, 3,
  • Elena Carrasco1,
  • Ana Ávila-Alonso4,
  • Ana Rodríguez-Fernández4,
  • John Wain5,
  • María Heredia1,
  • Esther Gomez-Sanchez1,
  • Susana Soria1,
  • Lucia Rico2, 3,
  • Verónica Iglesias2, 3,
  • Ángel Martínez-Martínez1,
  • David Andaluz-Ojeda2, 6,
  • Jose Ignacio Gómez Herreras1, 2,
  • Jose Maria Eiros7 and
  • Jesús F Bermejo-Martin2, 3Email author
Contributed equally
Critical Care201418:433

https://doi.org/10.1186/cc13910

Published: 6 June 2014

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 Clínico 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.
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 Sepsis-related Organ Failure Assessment score ≤10 (n = 39); SEPSIS SOFA >10, sepsis with SOFA score >10 (n = 16). Results for the ratio are expressed as (Copies IGHM/Copies CD20). Right: receiver operating characteristic curve of IGHM/CD20 predicting presence of sepsis in the comparison (sepsis versus SIRS). For this comparison septic patients were considered as a single group.

Notes

Abbreviations

ddPCR: 

droplet digital PCR

Ig: 

Immunoglobulin

PCR: 

Polymerase chain reaction

SIRS: 

Systemic inflammatory response syndrome.

Declarations

Acknowledgements

The authors deeply thank Eva Obregón, Angel Burgaleta from BioRad Spain and Yann Jouvenot from BioRad USA for their technical support with the ddPCR assay. This work was supported by 'Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III', Spain, grant number PI13/02110.

Authors’ Affiliations

(1)
Servicio de Anestesiología y Reanimación, Hospital Clínico Universitario-SACYL
(2)
Immunity, Risk of Infection and Sepsis group (IRIS), Hospital Clínico Universitario de Valladolid
(3)
Unidad de Apoyo a la Investigación, Hospital Clínico Universitario de Valladolid
(4)
Servicio de Microbiología, Hospital Clínico Universitario-SACYL
(5)
Norwich Medical School, University of East Anglia
(6)
Unidad de Cuidados Intensivos, Hospital Clínico Universitario-SACYL
(7)
Hospital Clínico Universitario-SACYL

References

  1. Cajander S, Bäckman A, Tina E, Strålin K, Söderquist B, Källman J: Preliminary results in quantitation of HLA-DRA by real-time PCR: a promising approach to identify immunosuppression in sepsis. Crit Care. 2013, 17: R223-10.1186/cc13046.PubMedPubMed CentralView ArticleGoogle Scholar
  2. Lefranc M-P, Giudicelli V, Ginestoux C, Jabado-Michaloud J, Folch G, Bellahcene F, Wu Y, Gemrot E, Brochet X, Lane J, Regnier L, Ehrenmann F, Lefranc G, Duroux P: IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 2009, 37: D1006-D1012. 10.1093/nar/gkn838.PubMedPubMed CentralView ArticleGoogle Scholar
  3. Bemark M, Holmqvist J, Abrahamsson J, Mellgren K: Reconstitution after haematopoietic stem cell transplantation - revelation of B cell developmental pathways and lineage phenotypes. Clin Exp Immunol. 2012, 167: 15-25. 10.1111/j.1365-2249.2011.04469.x.PubMedPubMed CentralView ArticleGoogle Scholar
  4. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL, Tewari M: Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods. 2013, 10: 1003-1005. 10.1038/nmeth.2633.PubMedPubMed CentralView ArticleGoogle Scholar

Copyright

© Tamayo et al.; licensee BioMed Central Ltd. 2014

This article is published under license to 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.

Advertisement