Effects of Red Blood Cell Transfusions on Morbidity and Mortality in Non-Septic Critically Ill Patients; A Propensity Score Matched Study

RBC-transfusions can be lifesaving, but are also associated with harm. To further examine any effect of red blood cell (RBC)-transfusions given to critically ill patients that were not exposed to the risks of anemia or sepsis, we designed this retrospective propensity score matched study. The aim was to compare mortality and morbidity in non-septic critically ill patients that were given low-grade RBC-transfusions at hemoglobin level > 70 g/L with patients without RBC-transfusions any of the rst 5 days in intensive care.


Introduction
Anemia is common in critically ill patients and more than one fourth are transfused with allogenic red blood cell transfusions (RBC) [1,2]. RBC-transfusions can be lifesaving for many patients, but are also associated with harm such as transfusionassociated circulatory overload (TACO), transfusion-related immune modulation (TRIM), transfusion-related acute lung injury (TRALI) hemolytic reactions and infections [3]. However, also anemia is dangerous which makes risk-bene t assessment of RBC-transfusions important and necessary [4]. Many large randomized controlled trials (RCT) with high level of evidence demonstrate that a restrictive transfusion strategy (hemoglobin level > 70 g/L) is as safe as a liberal transfusion strategy (hemoglobin level > 90-100 g/L) [5][6][7][8][9][10]. In those RCTs, patients in both groups received RBC-transfusions and many patients may also have been exposed to the risk of anemia. Consequently, potentially adverse effects related to the low-grade RBCtransfusion itself, could be di cult to ascertain.
We have recently demonstrated that low grade RBC-transfusions given to septic patients were associated with increased mortality and morbidity in a liberal transfusion setting [11]. Given that RBC-transfusions may trigger TRIM, it is possible that harmful effects are more pronounced in septic patients than in other patient groups [11,12]. In an attempt to evaluate if the harmful effect of RBC transfusions in non-septic critically ill patients that were not exposed to the risks of anemia, we designed this retrospective propensity score matched study. The aim was to compare mortality and morbidity in critically ill patients without severe sepsis or septic shock that were given low-grade RBC-transfusions at hemoglobin level > 70 g/L with controls without RBC-transfusions any of the rst 5 days in intensive care. The hypothesis was that RBC-transfusions are harmful in non-septic critically ill patients without signi cant anemia.

Data collection and study population
The study was approved by Swedish Ethical Review Authority in Lund, Sweden (registration numbers 2014/916 and 2018/866) and the board waived the requirement for written informed consent. The manuscript was prepared according to the STROBE guidelines for observational studies [13].
All patients ≥ 18 years of age, admitted to the 9-bed general intensive care unit (ICU) at Skåne University Hospital, Lund, Sweden between 2007 and 2018 were eligible for inclusion. For patients with multiple admissions to the ICU during the time of the study, only the rst admission was included. To exclude patients with massive bleeding, patients who received high grade RBC-transfusion (de ned as > 670 ml or two units) any of the rst ve days in the ICU were excluded. All patients with severe sepsis or septic shock according to the Sepsis-2 de nition [14] were excluded. RBC-transfusions were given at the discretion of the treating physician. To exclude patients exposed to the risks of anemia, all patients with a pre-transfusion hemoglobin level <70 g/L were excluded.
Mortality data was collected from the Swedish intensive care quality register PASIVA (Otimo Data AB, Kalmar, Sweden). Physiological and laboratory data and pre-existing conditions (age, gender, chronic obstructive pulmonary disease (COPD), renal failure, diabetes), outcome variables (except mortality) and uid administration data were collected from raw data, i.e. from the electronic master chart system of the hospital (Melior, Cerner, N. Kansas City, MO, USA), or from the patient data management system at the ICU (Intellispace critical care and anaesthesia (ICCA), Philips, Amsterdam, the Netherlands).

Outcome variables
Mortality was assessed at 28, 90 and 180 days after ICU-admission and organ support was assessed by calculating days alive and free (DAF) of organ support for the rst 28 days after admission to the ICU. For patients who died in the ICU, we counted the days without the speci ed organ support before death as previously described [15]. Organ support measures were vasopressors for circulatory failure, invasive mechanical ventilation for respiratory failure and renal replacement therapy (RRT) for renal failure. Renal failure was also evaluated according to the acute kidney injury network (AKIN) scoring system.
The maximal AKIN score the rst 10 days after ICU admission was used for analysis. To obtain an overall measure of organ failure we also used the maximum sequential organ failure assessment (SOFA) score during the rst 28 days after admission.

Statistics
Patients receiving low grade RBC-transfusion (< 670 mL/day) during the rst 5 days of ICU admission were propensity score matched with non-transfused patients to adjust for differences in baseline variables associated with outcome. The propensity score was calculated with linear logistic regression using a one-to-many macro for SAS as previously described [16], with the covariates speci ed in Table 1. Physiological and laboratory variables used in the propensity score matching were collected within 90 min of admission to the ICU.
Sample size was based on the number of available patients during the study period. Variables were summarized using mean (standard deviation), median (interquartile range) or numbers (percentage). The propensity score matching was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) prior to any comparison between the groups. Kaplan-Meier survival analysis was performed and is presented in graphs with corresponding strati ed log-rank test. In accordance with previous recommendations comparisons between the groups after propensity score matching was performed with paired hypothesis testing [17]. The propensity score matching was performed by an independent statistician using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Other analyses were performed with SPSS Statistics version 26 (SPSS Inc., Chicago, Ill., USA). A two-sided P value of less than 0.05 was considered to indicate statistical signi cance.

Results
A consort diagram of all patients is presented in Figure 1. Out of 9 491 patients 5 240 patients remained after removing patients < 18 years of age, multiple admissions, high-grade RBC-transfusion (>670 ml/day), patients with pre-transfusion hemoglobin < 70 g/L and patients with severe sepsis or septic shock. After propensity score match 682 patients were included in the RBC-group and 682 patients in the control group. The annual inclusion rate in both groups was similar (Additional le 1). Baseline demographics, comorbidity, and clinical, physiologic, and laboratory data in both groups are summarized in Table 1 and 2. After the propensity score match the standardized difference between groups for included baseline variables were reduced to <10%. For the baseline variables that were not included in the matching, differences between the groups were eliminated after the matching for all variables except for "Reason for admission, central nervous system" ( Table 2).
All RBC-transfusions were leucoreduced. The median hemoglobin level before transfusion in the RBC-group was 98 g/L (91-106 g/L). The median hemoglobin level on day 0 was 108 (104-111 g/L) for the RBC group and 109 (106-111 g/L) for the control group (P=0.95). Daily median hemoglobin levels for the ve rst days for both groups are illustrated in Figure 2. The median volumes of RBC-transfusion in the RBC-group the rst ve days after admission are shown in Figure 3.

Outcomes
Detailed results are presented in Table 3. Mortality at 28, 90 and 180 days were higher in the RBC-group (Table 3 and Figure   4). The absolute risk increase for death at 180 days for patients in the RBC-group was 4.8% [95% CI: 2.5 to 7.2%]; (P<0.001).
RRT and AKINmax demonstrated an increased risk for acute renal failure in the RBC group. Low grade RBC-transfusion was also associated with circulatory and respiratory failure as well as higher SOFA-max score.

Fluids
There was no difference either in the median daily administration of colloids, crystalloids or total uid balance between the groups. The daily median total uid administration and urinary output was larger in the RBC-group compared to the controls, Table 4.

Discussion
In this propensity score matched study, low-grade leukoreduced RBC-transfusions given to non-septic critically ill patients without signi cant anemia, was associated with increased mortality, increased kidney-, circulatory-and respiratory-failure as well as with higher SOFA-max score. This con rms the hypothesis that RBC-transfusions given to non-septic critically ill patients without signi cant anemia are harmful.
We collected data from 2007, prior to many high quality RCTs recommending a transfusion thresh-hold of 70 g/L. As recommended in the beginning of the study period, RBC-transfusions were often given in an ambition to increase oxygen delivery [18]. Hence, the vast majority of patients in the RBC group were transfused at a "safe" hemoglobin level without being exposed to the risks of anemia, indicated by a median pre-transfusion hemoglobin level of 98 g/L (91-106). These data can therefore be used to evaluate the effect of RBC-transfusion itself on critically ill non-anemic, non-septic patients.
The propensity score-matching was performed to minimize the differences in baseline variables between the groups and to create the RBC-and the control-groups as similar as possible at ICU admission. Differences between the groups in variables not included in the matching, such as SAPS 3, disappeared after the matching with the exception of "Reason for admission, central nervous system", Table 2. This further underline the validity of the propensity score-matching.
Given that propensity score matching corrected for differences between the groups and that median hemoglobin level the rst day of ICU admission did not differ between groups (Figure 2), the results in the present study imply that any adverse effects of the RBC-transfusion itself are responsible for the worse outcomes in the RBC group. This has previously been suggested in several reports, studies and guidelines [1,3,4,11,[18][19][20][21]. In a retrospective registry study, similar to the present, Leal-Noval et al. included moderately anemic non-bleeding critically ill patients and matched patients that received RBCtransfusion with non-transfused patients [4]. Hospital mortality, ICU re-admissions, nosocomial infections and acute renal failure favored the non-transfused group. In contrast to the present study, pre-transfusion hemoglobin level was not reported and patients with nadir hemoglobin level > 95 g/L were excluded from that study. As the patients in the present study were transfused at a higher hemoglobin level, thus not exposed for the risk of anemia and as the results showed an even stronger correlation between RBC-transfusion and bad outcome, this further strengthens the evidence that RBC-transfusions should not be given to non-anemic critically ill patients.
The reasons RBC-transfusions are harmful for non-anemic non-septic critically ill patients remain elusive, but as mentioned above known adverse effects of RBC-transfusion include TACO, TRALI and TRIM. Given that the total uid balance between the groups did not differ (Table 4) TACO is a less likely explanation. Even if TRALI is the leading cause of direct transfusionrelated death, it is a rare event reported to occur in one case in 6 000 to 600 000 transfusions [22]. Also, TRALI is most common after plasma transfusion which makes this an unlikely cause of worse outcome after RBC-transfusion in the present study. RBC-transfusions contain many different immunomodulatory mediators that interact with and alter immune cell function in-vivo. The effect of these interactions may be both proin ammatory and immunosuppressive but are seldom obvious at the moment of the transfusion [23]. Nevertheless, these immunomodulatory properties of RBC-transfusions may be detrimental over time for critically ill septic and non-septic patients and may be responsible for the results in the present study [23].
The most obvious measure to avoid RBC-transfusions in critically ill would be to avoid anemia. As blood loss through diagnostic testing has been shown to be substantial and associated with RBC-transfusions it should be standard to minimize blood sampling and to use low-volume blood sampling tubes and in-line closed blood conservation devices on arterial lines for reinfusion of waste blood [24]. It would also be desirable to treat anemia without giving RBC-transfusion. A common type of anemia in critically ill patients is similar to the anemia described in chronic disease and the state of in ammation [1]. The pathophysiology of this anemia includes high levels of hepcidin leading to a state of functional iron de ciency [25] and a blunted response to erythropoietin [26]. Erythropoietin and iron supplementation have been studied in several RCTs but unfortunately without reduction in RBC-transfusions [27,28]. However, results of several ongoing trials on iron administration to critically ill patients are pending [21].
Finally, it is worth noting that our study has limitations and strengths. Limitations include that the study was retrospective and single center. Secondly baseline characteristics affecting outcomes were cautiously adjusted for, but it cannot be ruled out that undetected variables also were present. Strengths include that patients in neither group were exposed for the risk of anemia as patients with pre-transfusion hemoglobin level < 70g/L were excluded. This suggests that outcomes were less biased by any negative effect of anemia. Further, all physiological and laboratory variables and many pre-existing conditions were registered prospectively in electronic charts and collected as raw data directly from the electronic charts.

Conclusion
Low-grade leukoreduced RBC-transfusions given to non-septic critically ill patients without signi cant anemia correlated strongly with increased mortality, increased kidney-, circulatory-and respiratory-failure as well as with higher SOFA-max score. These ndings further strengthen the evidence supporting a restrictive use of RBC-transfusions in critically ill patients.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests Funding Department funding only.
Authors' contributions TK designed the study, built the database, wrote the rst version of the manuscript and performed the statistical analyses after the propensity score match that was performed by a statistician. PB contributed to study design and did the rst revision of the manuscript. All authors contributed to the interpretation of the data, revised the manuscript critically, and gave nal approval of the version to be published.