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

Abstract

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.

Fig 1

(A) Monocyte HLA-DR (mHLA-DR), (B) HLA-DRA mRNA and (C) CIITA mRNA expression in bacteraemic sepsis categorized by sepsis severity on admission. Interaction tests calculated by mixed model demonstrated significantly different linear associations over time between the severity groups in Fig 1A and 1B. Boxplots are defined by medians (line within the boxes), quartiles (box range), min-max (whiskers) if no outliers were present otherwise circle markers if outliers more than 1.5 box lengths from the box and asterisks (*) if outliers more than 3 box lengths from the box.

Fig 2

Monocyte HLA-DR (A) and HLA-DRA mRNA expression (B) in 20 patients with bacteraemic severe sepsis categorized by the Sequential [Sepsis-Related] Organ Failure Assessment Score on admission. On assessment 1–2 days after admission, the median levels of mHLA-DR (AB/c) and HLA-DRA (ratio) in patients with SOFA scores of ≥ 5 and < 5 were; 12400/14100 (p = 0.167) and 0.93/1.52 (p = 0.009) respectively. On day 3, median levels of mHLA-DR (AB/c) were 10800/15600 (p = 0.024) and HLA-DRA (ratio) 0.87/1.9 (p = 0.014) in the two SOFA score groups. No significant differences between groups were demonstrated on day 7, 14 or 28.