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Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis
Örebro University, School of Health Sciences. Department of Infectious Diseases.
Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory.
Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory.
Clinical Epidemiology and Biostatistics, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 5, e0154690Article in journal (Refereed) Published
Abstract [en]

Introduction: A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).

Aims: The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA-mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis.

Methods and Findings: Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1-28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1-2, HLA-DRA 0.40 (0.28-0.59) p<0.001, CIITA 0.48 (0.32-0.72) p = 0.005, mHLA-DR 0.63 (0.45-1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46-0.77) p<0.001, CIITA 0.56 (0.41-0.76) p<0.001, mHLA-DR 0.81 (0.66-1.00) p = 0.28.

Conclusion: We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library of Science , 2016. Vol. 11, no 5, e0154690
National Category
Infectious Medicine
Identifiers
URN: urn:nbn:se:oru:diva-50323DOI: 10.1371/journal.pone.0154690ISI: 000375676400061PubMedID: 27144640OAI: oai:DiVA.org:oru-50323DiVA: diva2:931460
Note

Funding Agencies:

Nyckelfonden (Örebro, Sweden)

Research committee of Örebro County Council

Available from: 2016-05-27 Created: 2016-05-16 Last updated: 2017-04-25Bibliographically approved
In thesis
1. Dynamics of Human Leukocyte Antigen-D Related expression in bacteremic sepsis
Open this publication in new window or tab >>Dynamics of Human Leukocyte Antigen-D Related expression in bacteremic sepsis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monocytic human leukocyte antigen-D related (mHLA-DR) expression determined by flow cytometry has been suggested as a biomarker of sepsisinduced immunosuppression.

In order to facilitate use of HLA-DR in clinical practice, a quantitative real-time PCR technique measuring HLA-DR at the transcription level was developed and evalutated. Levels of HLA-DR mRNA correlated to mHLADR expression and were robustly measured, with high reproducibility, during the course of infection. Dynamics of mHLA-DR expression was studied during the first weeks of bloodstream infection (BSI) and was found to be dependent on the bacterial etiology of BSI. Moreover, mHLA-DR was shown to be inversely related to markers of inflammation. In patients with unfavourable outcome, sustained high C-reactive protein level and high neutrophil count were demonstrated along with low mHLA-DR expression and low lymphocyte count. This supports the theory of sustained inflammation in sepsis-induced immunosuppression. The association between mHLA-DR and bacterial etiology may be linked to the clinical trajectory via differences in ability to cause intractable infection. Staphylococcus aureus was the dominating etiology among cases with unfavourable outcome. With focus on patients with S. aureus BSI, those with complicated S. aureus BSI were found to have lower HLA-DR mRNA expression during the first week than those with uncomplicated S. aureus BSI. If these results can be confirmed in a larger cohort, HLA-DR measurement could possibly become an additional tool for early identification of patients who require further investigation to clear infectious foci and achieve source control.

In conclusion, PCR-based measurement of HLA-DR is a promising method for measurements of the immune state in BSI, but needs further evaluation in the intensive care unit setting to define the predictive and prognostic value for deleterious immunosuppression. The etiology of infection should be taken into consideration in future studies of translational immunology in sepsis.

Place, publisher, year, edition, pages
Örebro: Örebro University, 2017. 97 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 161
Keyword
monocyte HLA-DR, sepsis, immunosuppression, bloodstream infection, HLA-DRA, CIITA, qRT-PCR
National Category
Family Medicine
Identifiers
urn:nbn:se:oru:diva-56125 (URN)978-91-7529-191-8 (ISBN)
Public defence
2017-05-19, Örebro universitet, Campus USÖ, hörsal C3, Södra Grev Rosengatan 32, Örebro, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2017-05-24Bibliographically approved

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