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)