Study design

This is a retrospective, single-centre, observational study with an analytical scope nested in a Frisian ICU survivors cohort, as established by Beumeler et al., [11]. Data were collected in a tertiary teaching hospital with a mixed ICU, located in Leeuwarden, the Netherlands. The participants were adult patients admitted to the ICU between May and November 2019 with a length of stay (LOS) of ≥ 48 h. Only patients for whom plasma mitochondrial measurements were successfully assessed were included in the analysis.

Quantification of mitochondrial DNA levels and mitochondrial DNA damage using real-time qPCR

We employed real-time quantitative polymerase chain reaction (qPCR) to assess the mtDNA extracted from plasma samples collected from venous blood, as described previously by van der Slikke et al., [10], at baseline (< 72 h after admission) and 12 months post-ICU. To estimate mtDNA levels, cycle threshold (CT) values of NADH dehydrogenase 1 (ND1) and beta-2 microglobulin (β2M) were evaluated, with the ND1/β2M ratio reflecting the relative abundance of the mitochondrial genome. For amplification of the DNA the following protocol was used: 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 61 °C for 60 s. All reactions were carried out in duplicate and CT values were averaged. A standard curve was used to determine the efficiency, linear range, and reproducibility of the qPCR assay. The difference in the ct value between ND1 and B2M was used to quantify the relative abundance of the mitochondrial genome. MtDNA damage was assessed by quantifying long-range PCR, where the ratio between a long and short mtDNA fragment was calculated [10]. TaKaRa LA Taq DNA polymerase kit (Takarabio, Kusatsu, Japan) was used for the long-range PCR. A 10 kb mtDNA template, stretching from ND5 to ND1, representing more than two-third of the mitochondrial genome, was amplified by long-range PCR (Biorad, California, USA), while the reference consisted of a short mtDNA fragment of approximately 200bp (D-loop; a regulative region). 5 μl of DNA was used. For the amplification of the long 10 kb mtDNA part, the following thermal profile was used: To amplify the long-range DNA, 94 °C for 1 min, followed by 18 cycles of 15 s at 94 °C and 12 min at 64 °C was used, followed by 10 min at 72 °C. The short fragment was amplified using: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 61 °C for one min. Both PCR products were run on a 1% agarose gel (45 min, 100 V). The intensity of the bands on the gel was analysed using Image Lab (Biorad, California, USA). The ratio of the intensity of the short stable fragment to the long unstable fragment was calculated to quantify mtDNA damage. A higher ratio reflects more mtDNA damage.

Physical functioning and group allocation

Evaluation of the PF domain of health-related quality of life (HRQoL) was performed by the use of a validated Dutch equivalent of the 36-item Short Form Health Survey, the Research and Development-36 (RAND-36) questionnaire[12]; administered at baseline (to assess pre-ICU physical functioning) and 12 months after discharge from ICU. In cases where patients were unable to fill in the questionnaires at baseline, proxies were asked to perform this task[11]. Patients were then categorized into recovery (R) and non-recovery (NR) groups based on their 12-month PF score. To establish appropriate cut-off values, we used age-specific thresholds in RAND-36 PF domain score obtained from Table 5 in the manual by van der Zee et al., [12].

Demographic factors and clinical characteristics

The following demographic factors and clinical characteristics were collected: age on admission, gender, LOS ICU, LOS hospitalization (LOS hos), body mass index (BMI), type of admission, sepsis, need for cardiopulmonary resuscitation (CRP) and Clinical Frailty Scale (CFS) score at admission. The medical comorbidities such as the presence of malignancy, diabetes, chronic obstructive pulmonary disease (COPD), history of cerebrovascular accident (CVA) and chronic kidney disease (CKD) were included following the data dictionary of the National Intensive Care Evaluation [13]. Additionally, information on indices of severity of illness during ICU stay was recorded, including the need for renal replacement therapy (RRT), the need for intubation, the number of days on mechanical ventilation and the Acute Physiology and Chronic Health Evaluation (APACHE III) score.

Statistical analysis

The descriptive statistics were applied to characterize the population, combined with a Shapiro–Wilk test was applied to numerical variables to assess the data for normality. Consequently, scale variables were summarized as median and interquartile range, while categorical variables were described as proportions. A p-value of less than 0.05 was considered the threshold for statistical significance.

Differences between the recovery (R) and non-recovery (NR) groups were evaluated using a Wilcoxon Rank Sum Test for scale variables. Fisher’s Exact Test was employed to evaluate contingency tables for categorical variables such as gender, admission type, and comorbidities. Correlation coefficients were calculated for the relationship between mitochondrial markers and the scores from PF evaluated at baseline and 12 months, reported as Spearman’s Rho (rs). Additionally, the correlation between clinical variables and mitochondrial markers and PF was evaluated. While the sample size may limit statistical power, the primary goal was identifying trends and generating hypotheses.

Due to the exploratory nature of this study, a visual overview was created using boxplots to identify potential patterns, for which PF scores were categorized into quartiles and mitochondrial markers were evaluated in the PF quartiles.

All statistical analyses were performed using the statistical software package R version 4.3.1 (Beagle Scouts). Tables for the final report were made using Microsoft Excel (Microsoft Office Professional Plus 2019). The results of this study were reported using the Strengthening the Reporting of Observational Studies (STROBE) checklist [14].