Background

Immune dysfunction plays a central role in a wide range of diseases including cancer, atherosclerosis, trauma, and infections [1,2,3,4]. In the context of sepsis, this dysfunction is demonstrated across innate and adaptive immunity, and is characterised by apoptosis of immune cells, dysfunction in cellular function of neutrophils and monocytes, and ‘T cell exhaustion’ [5,6,7,8,9]. The presence of these cellular dysfunctions is associated with poor clinical outcomes, including healthcare-associated infections, increased mortality, and prolonged hospital length of stay [10, 11]. Immune modulation in cancer through immune checkpoint blockade has revolutionised cancer treatment [12]. There is intense focus on investigating novel therapies to modulate the immune dysfunction in sepsis, in the hope of improving clinical outcomes [13].

There is no one test to identify patients with immune dysfunction and assays are highly specialised and not readily available in a hospital setting [14]. Lymphopenia is a window into the state of the immune system, available from routinely collected clinical data. Lymphopenia has been associated with increased mortality and healthcare-associated infection amongst patients with sepsis [15]. Adverse outcomes associated with lymphopenia have been recognised in a wider hospital setting, including in patients with pneumonia and following stroke [16, 17]. In the general population, lymphopenia is associated with an increased risk of hospitalisation secondary to infection, independent of clinical risk factors such as age and co-morbidities [18, 19].

The ability to identify and quantify this at-risk population is important for designing future studies to modulate the immune response and to investigate the longer-term impact of immune dysfunction in hospital. Since lymphopenia has been shown to lead to poor clinical outcomes in such a wide range of hospital populations, we sought to summarise the pooled prevalence of lymphopenia in hospitalised patients regardless of the cause of hospital admission. In addition, we aimed to determine the impact of lymphopenia on clinical outcomes including infection, mortality and length of hospital stay.

Methods

Protocol and registration

The systematic review was registered prospectively with PROSPERO (CRD42022327031) and was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [20, 21].

Study search strategy

PROSPERO and Cochrane Library searches confirmed that there were no previous systematic reviews of prevalence of lymphopenia in all-cause hospital admissions. Searches were performed on MEDLINE, Embase and CENTRAL databases. Studies that allowed extraction of prevalence data of lymphopenia were included. Records were not restricted by publication date. Records were extracted to Endnote (Thompson, Reuters, Philadelphia, PA, USA) to remove duplicate studies.

Rayyan was used for title and abstract screening [22]. A sample of 10% of results were reviewed by two reviewers to establish agreement (ZCE and TPH). Any disagreements were re-evaluated and resolved between the two reviewers. Data extraction was carried out by a single reviewer (ZCE).

Exclusion criteria

Narrative reviews, editorials, case reports, duplicate publications, qualitative studies, conference abstracts, and non-human studies were excluded. Studies were limited to adult populations and those published in English language. A protocol amendment, prior to formal screening of search results/data extraction, was published on PROSPERO to exclude studies where the primary focus were patients with pre-existing immunosuppression, HIV or COVID-19. This review aimed to summarise lymphopenia in the general hospital population, rather than in patients with immunosuppression (for example, secondary to chemotherapy), in whom lymphopenia and subsequent infection risk are well recognised. Studies of COVID-19 patients were excluded because lymphopenia in this context has been summarised in a recent systematic review [23]. This protocol update did not require a secondary amendment of the search strategy, as negative searching was not carried out.

Data collection process

Data were extracted from the selected papers onto a pre-formatted Excel worksheet (Microsoft, Redmond, WA, USA) containing the following characteristics: author and year of publication; country of origin and setting; study design; duration of study; demographics including age and sex; sequential organ failure assessment (SOFA) score; acute physiology and chronic health evaluation II (APACHE II) score; co-morbidities where available; and the definition of lymphopenia. Outcomes reported were extracted including healthcare-associated infection, all-cause mortality, and length of stay. The diagnostic criteria used for infection and healthcare-associated infection were also extracted.

The number of events was extracted for dichotomous outcomes. Means with standard deviation (SD) were extracted for continuous outcomes. Median values, interquartile ranges and sample size were used to estimate the sample mean and SD [24, 25].

Outcomes

The primary objective was to summarise the pooled prevalence of lymphopenia in all-cause hospital admissions. The primary clinical outcome was infection, including infection at admission and healthcare-associated infection. Secondary clinical outcomes included length of hospital stay, length of intensive care unit (ICU) stay, all-cause in-hospital mortality, 28/30-day mortality (defined as ‘early’) and 90-day/1-year mortality (defined as ‘late’).

The definition of lymphopenia was determined by the paper being analysed in the review. Absolute lymphocyte count (ALC) is expressed in units of 109/L. The normal range of ALC is often accepted to be between 1.5 to 4 x 109/L.

Risk of bias and quality of evidence assessment

The risk of bias was assessed by two reviewers (ZCE and TPH), using the Joanna Briggs Institute (JBI) critical appraisal checklist for observational studies [26].

An overall assessment of the evidence quality for outcome measures was reported according to the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) assessment [27]. The software program GRADEpro was used [28].

Statistical analysis

The meta-analysis was conducted using Review Manager and R Meta package [29,30,31]. A p value of less than 0.05 was accepted to be statistically significant. Dichotomous data were analysed using risk ratio (RR) with 95% confidence intervals (CIs). Continuous data were analysed using inverse variance (I-V) method to obtain mean difference (MD) and standard deviation (SD). Random-effects models for pooled analysis was used, independent of heterogeneity. Heterogeneity was assessed using the I2 statistic.

Results

Study selection

A total of 6006 studies were identified. After title and abstract screening, 236 potentially eligible studies underwent full-text review. The study flow diagram based on PRISMA guidelines demonstrates reasons for exclusion (Fig. 1). Following exclusion of 221 studies (Fig. 1), 15 studies were included in the analysis [15,16,17, 32,33,34,35,36,37,38,39,40,41,42,43].

Fig. 1
figure 1

PRISMA flow diagram showing literature search results. Fifteen studies were used for meta-analysis. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analysis [21]

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Study characteristics

A total of 72305 patients were included in the analysis (Table 1). Studies included a range of clinical conditions and settings, including elective admissions, critically ill patients, community-acquired pneumonia (CAP), ventilator-associated pneumonia (VAP), sepsis, spinal surgery, chest trauma, traumatic brain injury, and influenza A. Four studies were related to patients in ICUs only [34, 35, 40, 43].

Table 1 Characteristics of included papers. R* retrospective cohort study; P** prospective cohort study. p values*, where provided, are for comparisons between lymphopenic vs non-lymphopenic populations. HR, hazard ratio; OR, odds ratio; LOS, length of stay; IQR, interquartile range; RCT, randomised controlled trial; COPD, chronic obstructive pulmonary disease; UTI, urinary tract infection; CAP, community-acquired pneumonia; RT-PCR, reverse transcription polymerase chain reaction
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Nine studies were retrospective observational cohort studies and six were prospective cohort studies (Table 1). Lymphopenia was variably defined in all included papers, with a range of 0.5 to 1.2 x 109/L. Two studies did not report when ALC measures were collected [16, 42]. Eight studies reported ALC measures either on admission or within 24 hours of admission [17, 33, 34, 37,38,39, 41, 43]. Two studies measured ALC on specific days determined a priori; 4th day after a blood culture was taken in septic patients and postoperative day 7, respectively [15, 36]. Three studies measured ALC at multiple time points during hospital admission [32, 35, 40].

Risk of bias

Risk of bias was high amongst most included studies (Fig. 2). Areas of high risk of bias or uncertainty related to three main issues. Firstly, there were significant baseline differences between lymphopenic and non-lymphopenic groups in measures of demographics and disease severity. Secondly, completeness of data or strategies to deal with missing data were not reported. Thirdly, it was often unclear whether participants were free of the outcome at start of the study as these were not always measured in a reliable way.

Fig. 2
figure 2

Risk of bias using the JBI Critical Appraisal Checklist for Cohort Studies [26]. Plot created using robvis software [44]

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Funnel plots were visually inspected for identification of publication bias, where more than five publications reported a specific outcome. Visual inspection of funnel plots demonstrated high risk of publication bias.

Pooled prevalence of lymphopenia

The pooled prevalence of lymphopenia in all-cause hospitalisations was 38% (random effects model proportion 0.38; CI 0.34-0.42, I2 = 97%, p< 0.01) (Fig. 3a).

Fig. 3
figure 3

a Forest plot of pooled prevalence of lymphopenia (of any definition) across 15 studies. CI, confidence interval. b Subgroup analysis of ICU pooled prevalence of lymphopenia of any definition. c Subgroup analysis of septic shock pooled prevalence of lymphopenia of any definition. d Subgrouping of lymphopenia based on absolute lymphocyte count at time of admission (ALC, x 109/L)

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Given the significant study-related heterogeneity across all included studies, further subgroup analysis was carried out. Studies investigating an ICU population (n = 1020) demonstrated lymphopenia in 34% of admissions (random effects model proportion 0.34; 95% CI 0.26-0.44, I2 = 88%, p< 0.01) (Fig. 3b), although heterogeneity remained high [34, 35, 40, 43]. Subgroup analysis for septic shock populations (n = 1371) demonstrated higher prevalence of 54% with significant heterogeneity (random effects model proportion 0.54; 95% CI 0.30-0.76, I2 = 99%, p< 0.01) (Fig. 3c) [15, 16, 32, 34, 43].

Lymphopenia definition varied across studies, however eight studies defined lymphopenia based on ALC at hospital admission and/or within 24 hours of admission [17, 33, 34, 37,38,39, 41, 43]. Admission lymphopenia had a prevalence of 39% (random effects model proportion 0.39; 95% CI 0.31-0.47, I2 = 97%, p< 0.01) (Fig. 3d). Heterogeneity remained high.

Lymphopenia and infection

Two studies reported infection as the cause of admission [32, 39]. Lymphopenia was not associated with an infection diagnosis (RR 1.03; 95% CI 0.26-3.99, p=0.97, I2 = 55%) (Fig. 4). Subgroup analysis for this outcome was not possible due to the small number of studies. However, heterogeneity is noted between studies by Andreu-Ballester et al. and Rubio-Rivas et al.; lymphopenia definition (< 1 vs < 1.1 x 109/L), timing of lymphocyte measures (any point during hospital admission vs admission), and study population characteristics including age (> 14 vs ≥ 75 years), respectively (Table 1) [32, 39].

Fig. 4
figure 4

Forest plot of infection (as cause of admission) and lymphopenia (of any definition). CI, confidence interval; M-H, Mantel-Haenszel

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Seven studies reported the outcome of healthcare-associated infection and lymphopenia [15, 17, 33, 34, 36, 37, 42]. Lymphopenia was not associated with healthcare-associated infection (RR 1.31; 95% CI 0.78-2.20, p=0.31, I2 = 97%) (Fig. 5a). Sensitivity analysis was carried out based on lymphopenia definition, stratified as either less than 1.2 or less than 0.8 x 109/L (Fig. 5b). Heterogeneity was reduced in the analysis of ALC less than 1.2 x109/L but increased for studies of ALC greater than 0.8 x 109/L. The outcome remained non-significant.

Fig. 5
figure 5

a Forest plot of nosocomial infection and lymphopenia (of any definition). Funnel plot analysis demonstrates asymmetric shape. b Forest plot of nosocomial infection and lymphopenia, stratified by lymphopenia definition. Top panel ALC < 1.2 > 0.8 x 109L vs bottom panel ALC < 0.8 x 109L. CI, confidence interval; M-H, Mantel-Haenszel

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Four studies reported the outcome of septic shock and lymphopenia [16, 32, 34, 43]. Lymphopenia was associated with septic shock (RR 2.72; 95% CI 1.02–7.21, p = 0.04, I2 = 98%) (Fig. 6). Heterogeneity was high between the studies.

Fig. 6
figure 6

Forest plot of septic shock and lymphopenia (ALC stratified by cut-off, x 109/L). CI, confidence interval; M-H, Mantel-Haenszel

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Lymphopenia and mortality

In-hospital mortality was reported by seven studies (Fig. 7) [17, 32, 33, 37, 39, 40, 43]. Lymphopenia was associated with higher in-hospital mortality (RR 2.44; 95% CI 1.71-3.47, p < 0.00001, I2 =89%) (Fig. 7). Excluding Andreu-Ballester et al.’s study, which analysed data from 58260 hospital admissions, the significant heterogeneity is reduced to 41% (RR 2.13, 95% CI 1.72-2.65, p < 0.00001, I2 =41%) (Fig. 7b) [32].

Fig. 7
figure 7

a Forest plot of in-hospital mortality and lymphopenia (ALC stratified by 1.1 x 109/L as cut-off). Funnel plot demonstrates asymmetry. b Exclusion of Andreu-Ballester et al. changes to I2 = 41%. CI, confidence interval; M-H, Mantel-Haenszel

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‘Early’ (28/30-day) mortality was reported in six studies [15, 16, 34, 35, 38, 41]. Bermejo-Martin et al. studied two cohorts; ‘derivation’ multisite and ‘validation’ single-site cohorts annotated as [1] and [2], respectively (Fig. 8a) [16]. Lymphopenia was associated with higher early mortality (RR 2.05; 95% CI 1.64-2.56, p < 0.00001, I2 = 29%) (Fig. 8a). ‘Late’ (90-day/1-year) mortality was reported in two studies [15, 34]. Lymphopenia was associated with higher late mortality (RR 1.59; 1.33-1.90, p < 0.00001, I2 = 0%) (Fig. 8b).

Fig. 8
figure 8

a Forest plot of 28/30-day mortality (early) with lymphopenia of any definition. Bermejo-Martin et al. (1) and (2): data from derivation cohort and verification cohort, respectively. Funnel plot demonstrates asymmetry. b Forest plot of 90-day/1-year mortality (late) with lymphopenia of any definition. CI, confidence interval; M-H, Mantel-Haenszel

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Lymphopenia and length of stay

Hospital length of stay (LOS) was reported in five studies (Fig. 9) [17, 33, 37, 38, 41]. Mendez et al. reported hospital LOS of a population of patients with CAP based on two lymphopenia definitions of ALC ≤ 1 x109/L and ALC ≤ 0.724 x 109/L [38]. LOS has been reported separately in the analysis for these two lymphopenia thresholds (Fig. 9) [38]. The overall mean difference in hospital LOS is 1.25 days (95% CI 0.32-2.18, p = 0.008, I2 = 89%) longer for patients with lymphopenia (Fig. 7).

Fig. 9
figure 9

Forest plot of hospital Length of Stay (LOS) and lymphopenia. Mendez et al. (1) and (2): absolute lymphocyte count cut off less than 1.0 x109/L vs. less than 0.724 x109/L, respectively. a Lymphopenia defined ALC less than 1 x 109/L. b Lymphopenia defined as ALC < 0.724 x109/L. CI, confidence interval; I-V, Inverse Variance method

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Sensitivity analysis was performed based on lymphopenia definition. Four studies, defining lymphopenia as less than 1 x 109/L, demonstrated that lymphopenic populations stayed in hospital 1.85 days (95% CI 1.03-2.66, p < 0.0001, I2 = 74%) longer than non-lymphopenic populations [17, 33, 37, 38]. Two studies, defining lymphopenia as less than 0.724 x 109/L, demonstrated that lymphopenic patients stayed in hospital for 0.35 days longer than the non-lymphopenic population, however this was not statistically significant (95% CI -0.10-0.81, p = 0.13, I2 = 0%) [38, 41].

ICU LOS was reported by three studies (Fig. 10) [15, 33, 37]. Mean difference was 0.17 days longer for non-lymphopenic subgroup, however this was not statistically significant (95% CI -0.34-0.68, p= 0.50, I2 = 60%) (Fig. 10).

Fig. 10
figure 10

Forest plot of ICU LOS and lymphopenia (of any definition). CI, confidence interval; I-V, Inverse Variance method

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Discussion

This systematic review adds to the growing evidence that lymphopenia is associated with adverse clinical outcomes. We demonstrate that lymphopenia is common in hospitalised patients, occurring in 38% of patients. We demonstrate that lymphopenia is associated with increased early and late mortality. In addition, there is prolonged hospital stay. The analysis did not demonstrate a significant difference in risk of admission with an infection or acquiring a hospital-acquired infection if lymphopenic. However, there was an increased risk of septic shock in lymphopenic patients. The fifteen studies included demonstrate that lymphopenia is seen across a wide range of pathologies including infection, trauma, and intracranial haemorrhagic conditions.

Lymphopenia has been associated with increased mortality and infection risk in a wide range of settings including community populations, perioperative, and sepsis [15, 18, 19, 45]. The studies have broadly shown that lymphopenia is associated with an increased risk of infections and mortality. Given the range of clinical settings in which lymphopenia has been shown to result in adverse clinical outcomes, we summarised for the first time, the prevalence of lymphopenia in all-cause hospitalised patients. Our findings are broadly in line with other studies. In a meta-analysis of peri-operative patients, lymphopenia was associated with a three-fold increase in mortality and a higher rate of postoperative complications and infections [45]. While we demonstrated increased mortality in both ‘early’ and ‘late’ deaths, we did not demonstrate an increase in risk of infection. An increased risk of infection seems intuitively associated with lymphopenia. Lymphopenia is a hallmark of immune dysfunction in sepsis and is associated with healthcare-associated infections [46]. In a single centre observational study, it was persistent lymphopenia lasting beyond the fourth day of sepsis admission, that was associated with a significant increase in secondary infections [15]. Furthermore, in a large population study of 98, 344 individuals, lymphopenia was associated with an increased risk of acquiring infections, including sepsis [18]. When summarising the risk of infection across a broad range of conditions, we did not find a significant association between an infectious cause of hospital admission or healthcare-associated infection. We did, however, show a 3-fold increased risk of septic shock with lymphopenia (RR 2.72; 95% CI 1.02–7.21, p = 0.04, I2 = 98%).

Our review suggests there is a ‘dose-response’ between severity of lymphopenia and adverse clinical outcome. In a retrospective study, Bermejo-Martin et al., identified a subgroup of patients with CAP who were lymphopenic (ALC less than 0.724 x 109/L) that accounted for a significant portion of individuals who developed septic shock and demonstrated a significantly higher risk of 30-day mortality [16]. Consistent with this finding, in a large cohort study, Andreu-Ballester et al. demonstrated that the lowest absolute values were demonstrated in sepsis and septic shock, with severe low absolute counts of lymphocytes associated with higher risk of mortality [32]. Drewry et al. stratified lymphopenia definitions as moderate and severe persistent lymphopenia. This stratification demonstrated a higher incidence of nosocomial infections alongside higher 28-day and 1-year mortality rates in the severe cohort compared to the moderate cohort [15]. These findings suggest a relationship between severity of lymphopenia and outcome, specifically in subgroups of septic shock.

Given the spectrum of conditions that lymphopenia is present in, there is uncertainty whether lymphopenia is an epiphenomenon of an unwell patient or whether it plays a central role in morbidity and mortality. The significance of lymphopenia in different clinical settings and populations is uncertain. Studies to date indicate that lymphopenia reflects a wider dysfunctional immune system. This is certainly shown in studies in sepsis, where immune dysfunction is characterised not only by lymphopenia, but also low monocyte HLA-DR, increased PD-1 and increased regulatory T cells [5,6,7,8,9]. Other routinely measured biomarkers reflect immune dysfunction and have been shown to be associated with increased mortality. In a large population of 31,178 outpatients, in addition to lymphopenia, high levels of C-reactive protein (CRP) were also associated with reduced survival [19]. A follow-up study of sepsis survivors identified a hyperinflammation/immunosuppression phenotype with a significantly higher 1-year mortality risk, demonstrated by CRP as a marker of ongoing inflammation and additional markers of immunosuppression including soluble PD-L1 [10]. Although our review cannot conclude that lymphopenia in the included studies is due to immune dysfunction, our findings are consistent with current understanding of immune perturbations in acute illness.

There are limitations to this study. We aimed to determine the prevalence of lymphopenia in a ‘general’ hospital population. For this reason, we excluded studies that specifically focused on immunosuppressed populations in which the prevalence of lymphopenia and the associated infection risk would be much higher. These populations are immunosuppressed secondary to medical treatments for cancer or inflammatory diseases, and so represent a different population to those with immune dysfunction because of an acute disease. However, it is possible that some patients within the included studies of ‘general’ populations would be on immunosuppressive medications and contribute to the lymphopenic population. In addition, it can be argued that patients with COVID-19 should be represented in the general in-patient population. We excluded these studies because lymphopenia is a well-recognised characteristic and systematic review of lymphopenia in COVID-19 patients has been recently published [23].

This review is further limited by the range of lymphopenia definitions used in the studies, resulting in high levels of heterogeneity in the meta-analysis. Definition of lymphopenia ranged from 0.5 to 1.2 x 109/L. The lack of a unified definition of lymphopenia demonstrates the need for further research in causality, and in clarifying whether there is a potential count-dependent relationship between severity of lymphopenia and outcome.

Lastly, the conclusions made by this meta-analysis are limited by the quality of studies included. Most of the studies had a high risk of bias or uncertainty regarding risk of bias. Since the included studies were observational studies, the GRADE quality of evidence was often downgraded (Fig. 11). However, large sample sizes in studies such as Andreu-Ballester et al., Bermejo-Martin et al., and Campbell et al. allowed upgrading of quality due to large effect size demonstrated across multiple outcomes (Fig. 11) [16, 32, 33].

Fig. 11
figure 11

Summary of findings table and GRADE assessment of outcomes. a Wide confidence intervals for overall effect estimate. b Lack of confounding variables identification [17, 33, 36]. Follow-up time not reported/unclear [15, 36]. Significant baseline differences between lymphopenic and non-lymphopenic subpopulations [37]. c Statistically significant high heterogeneity. d Asymmetrical funnel plot. e Ceccato et al. determined lymphopenia cut-off based on previous analysis [34]. f Lack of generalisability to wider hospitalised population; Ceccato et al.'s study population was related to intensive care-related nosocomial infection while Zhou et al.'s study concentrated on patients with severe influenza A patients [34, 42]. g Significant differences between lymphopenic and non-lymphopenic subpopulations [37, 43]. Unclear reporting of characteristics across groups in Andreu-Ballester et al. and Vulliamy et al. [32, 40] h Significant differences between lymphopenic and non-lymphopenic subpopulations [16, 38, 41]. Potential confounding factors not identified [34]

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In conclusion, this meta-analysis shows that lymphopenia is common across all-cause hospitalisations and associated with increased risk of mortality and length of stay. Moreover, given the consistent findings across several types of pathology, the data suggest a link between lymphopenia at any point during a hospital stay and poor outcome. This meta-analysis highlights the paucity of available high-quality evidence. By summarising the prevalence of lymphopenia in hospitalised patients, this review may inform the design of future studies investigating outcomes and novel treatments for immune dysfunction in hospitalised patients. In particular, prospective studies of lymphocyte count and its potential correlation with detailed immunophenotyping and longer term patient outcomes may provide further insight into the value of lymphopenia as a marker of immune dysfunction and prediction of illness trajectory after hospitalisation.