The discriminatory ability of day 1 and day 4 absolute lymphocyte counts to predict 28-day mortality is demonstrated in Figure 4 using ROC plots. Day 4 absolute lymphocyte count, but not day 1 absolute lymphocyte count, demonstrated a significantly higher AUC compared with a reference of 0.5. The performance of specific day 4 absolute lymphocyte count thresholds to predict 28-day mortality is indicated on the graph. The most accurate discriminatory threshold was calculated to be 1.0 × 103 cells/μL, which demonstrated a sensitivity of 76% and a specificity of 56%.
Information regarding immune phenotype in patients with sepsis is vital before any consideration of immunomodulatory therapeutic interventions can occur. The current study provides new findings in this domain and offers potential insight into areas for further research. Our findings have several implications.
After initial predominance of a proinflammatory cytokine-driven response, many patients with sepsis develop persistent and profound immunosuppression prior to succumbing to the syndrome (3, 7, 8, 15). The results of this study demonstrate that a persistently low level of circulating lymphocytes on the fourth day following the diagnosis of sepsis independently predicts short- and long-term survival and may serve as a biomarker for sepsis-induced immunosuppression. The immune response to sepsis is extremely variable and can change dramatically as the syndrome progresses. Patients who die early do so as a result of overwhelming hyperinflammation marked by cardiovascular collapse and multiple organ failure (4, 29). Many of the patients who survive this phase, however, go on to develop a compensatory anti-inflammatory response characterized by increased inhibitory receptors on T cells and antigen-presenting cells, decreased production of proinflammatory cytokines, expansion of myeloid-derived suppressor cells, and apoptosis-related loss of lymphocytes and dendritic cells (7, 9, 30–32). In the current study, only 42 patients (who otherwise would have met inclusion criteria) died prior to the fourth day after the diagnosis of sepsis. This accounted for only 35.6% of the total deaths due to sepsis, signifying that the majority of patients who ultimately died were at risk for sepsis-induced immunosuppression.
Previous studies have demonstrated persistently decreased levels of specific subpopulations of B and T cells in adult septic nonsurvivors compared with survivors but have not shown significant differences in the overall absolute lymphocyte count (11–13, 16). Our study, which included a large number of septic nonsurvivors, demonstrated that whereas absolute lymphocyte counts decrease to similarly low levels in survivors and nonsurvivors at the onset of sepsis, nonsurvivors’ absolute lymphocyte counts remain persistently low, while survivors experience lymphocyte recovery. We theorize that the initial fall in circulating lymphocytes at the onset of infection in patients with sepsis reflects two separate processes. First, lymphocytes are recruited out of the peripheral circulation to areas of infection and inflammation, and second, sepsis induces a number of stimuli that trigger lymphocyte apoptosis (18). Furthermore, we postulate that the persistent lymphopenia seen in nonsurvivors is most likely due to ongoing sepsis-induced lymphocyte apoptosis secondary to continued release of proapoptotic stimuli during the immunosuppressive phase of sepsis (12, 32–34). These findings are especially striking because the optimal host response to infection should be to increase lymphocyte proliferation and thereby augment the number of lymphocyte effector cells.
Another significant finding of our study is that patients with severe persistent lymphopenia had a significantly higher incidence of secondary infections compared with patients whose absolute lymphocyte counts had recovered to normal by day 4, and there was also a strong trend toward increased secondary infections in the group with moderate persistent lymphopenia. These results suggest that increased susceptibility to new infections may be contributing to the higher mortality seen in persistently lymphopenic patients.
For this study, we chose to focus on day 4 absolute lymphocyte counts based on previously published evidence that indicated that lymphocyte counts on this day would discriminate between survivors and nonsurvivors (11–13, 24). The time point at which patients with sepsis transition from a predominantly proinflammatory state to one of hypoinflammation is unknown and is likely extremely variable because of host-, pathogen-, and disease-related factors. Therefore, we also analyzed day 3 lymphocyte counts in the subset of patients who had complete blood counts measured on that day to determine whether persistent lymphopenia at an earlier time point could also predict death. We found that day 3 absolute lymphocyte counts were not associated with short- or long-term mortality after accounting for other covariates. Despite this, determining the time at which continued lymphopenia becomes most clinically relevant is difficult to ascertain, and there are likely subsets of patients (e.g., elderly patients) in whom the lymphocyte pattern diverges between survivors and nonsurvivors at an earlier or later time point than that seen in this study. Similarly, lymphopenia was defined a priori as an absolute lymphocyte count less than 1.2 × 103 cells/μL for this study because this was the lower limit of normal specified by the laboratory at our institution. Based on our ROC analysis, however, a day 4 absolute lymphocyte count less than 1.0 × 103 cells/μL may be a more accurate discriminatory threshold to optimally predict 28-day mortality. In the future, larger studies that could stratify patients by age or disease severity may clarify whether different absolute lymphocyte cutoff values would be more useful in specific subpopulations of patients with sepsis.
This study has several limitations. As a retrospective study, it was prone to limitations inherent in this study design, such as imbalance between the study groups. In contrast to previous studies of lymphopenia in patients with sepsis, though, we attempted to account for major care-related and patient-specific determinants of survival by adjusting for these factors in a multivariable model. In fact, an important aspect of this study was that we included a sufficient number of nonsurvivors to show that persistent lymphopenia increased the risk of death at 28 days and 1 year even after accounting for other known predictors of mortality such as age, APACHE II score, time until appropriate antibiotic coverage, and comorbidities. As a retrospective study, data collection was limited to variables that were obtained during the usual clinical care of the patients, so we were unable to correlate persistent lymphopenia with other known biomarkers of sepsis-induced immunosuppression such as decreased monocyte HLA-DR expression, overexpression of programmed cell death 1 receptor on T cells, or decreased ex vivo lipopolysaccharide-induced tumor necrosis factor α levels. Instead, we used the development of secondary infections as a clinically relevant surrogate marker of immunosuppression. Going forward, future clinical trials in this area should measure not only clinical outcomes, but also biomarkers of immunosuppression to further establish causation.
Another limitation of this study is that the exact time of sepsis onset could not be precisely determined. For the purpose of this study, we defined the onset of sepsis to be the time that the first positive culture was ordered by the treating physician. The majority of included patients, however, had blood cultures drawn in the emergency department, so potential delays in presentation to the hospital further complicate interpretation of timing in this study. Furthermore, we excluded patients who died or were discharged prior to day 4. Although this may limit some of the generalizability of our results, it allowed us to focus on sepsis-induced immunosuppression by eliminating patients experiencing early death (which is typically due to cardiovascular collapse and not immunosuppression) or early clinical recovery.
Finally, the main limitation of this study is that the results do not allow us to conclude whether persistent lymphopenia directly contributes to mortality in patients with sepsis or whether it is simply a marker of disease severity. We are also not able to presume that immune-stimulatory therapy aimed at reversing lymphopenia would alter patient outcomes. We postulate that ongoing lymphocyte apoptosis caused by the continued release of proapoptotic stimuli contributes to morbidity and mortality in patients with sepsis by inhibiting clearance of primary infections and increasing susceptibility to secondary infections. This hypothesis is supported by multiple animal studies, which have suggested that lymphocyte apoptosis is a key pathogenic mechanism in sepsis and that prevention of lymphocyte apoptosis improves survival (40–43). Still, prospective studies are needed to establish lymphopenia as a causative factor in late mortality in sepsis.
The clinical value of persistent lymphopenia is yet to be elucidated. Because of the enormous overlap in lymphocyte counts among survivors and nonsurvivors, day 4 absolute lymphocyte count would most likely be useful as a predictor of mortality in groups of patients rather than an accurate predictor of death in any individual patient. More important, perhaps, is the potential value of day 4 absolute lymphocyte count in predicting an increased risk of secondary infection and sepsis-induced immunosuppression. Currently, in the clinical setting, a persistently low absolute lymphocyte count in patients with sepsis should prompt clinicians to reevaluate their patients’ response to therapy and assess for the presence of new or untreated infections. In the future, sepsis therapies could be tailored to individuals’ immunological phenotypes. Potential immunotherapeutic agents, such as interleukin 7 and anti–programmed cell death 1 receptor antibody, act to increase CD4 and CD8 T-cell production, block lymphocyte apoptosis, and prevent T-cell exhaustion. These therapies may be most effective if administered selectively to patients with evidence of lymphocyte dysfunction or loss. Identification of patients with persistent lymphopenia will select not only for those at high risk for short- and long-term mortality, but also for those with the specific immunological derangements that these therapies aim to improve.
The authors thank Karen Steger-May, with the Division of Biostatistics at Washington University in St Louis, for her assistance with statistical analysis.
1. Lagu T, Rothberg MB, Shieh MS, Pekow PS, Steingrub JS, Lindenauer PK: Hospitalizations, costs, and outcomes of severe sepsis in the United States 2003 to 2007. Crit Care Med 40 (3): 754–761, 2012.
2. Munford RS, Pugin J: Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med 163 (2): 316–321, 2001.
3. Hotchkiss RS, Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med 348 (2): 138–150, 2003.
4. Angus DC, van der Poll T: Severe sepsis and septic shock. N Engl J Med 369 (9): 840–851, 2013.
5. Ward PA: Immunosuppression
in sepsis. JAMA 306 (23): 2618–2619, 2011.
6. Torgersen C, Moser P, Luckner G, Mayr V, Jochberger S, Hasibeder WR, Dunser MW: Macroscopic postmortem findings in 235 surgical intensive care patients with sepsis. Anest Analg 108 (6): 1841–1847, 2009.
7. Boomer JS, To K, Chang KC, Takasu O, Osborne DF, Walton AH, Bricker TL, Jarman SD 2nd, Kreisel D, Krupnick AS, et al.: Immunosuppression
in patients who die of sepsis and multiple organ failure. JAMA 306 (23): 2594–2605, 2011.
8. Schefold JC, Hasper D, Volk HD, Reinke P: Sepsis: time has come to focus on the later stages. Med Hypotheses 71 (2): 203–208, 2008.
9. Hotchkiss RS, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Matuschak GM, Buchman TG, Karl IE: Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 27 (7): 1230–1251, 2009.
10. Venet F, Davin F, Guignant C, Larue A, Cazalis MA, Darbon R, Allombert C, Mougin B, Malcus C, Poitevin-Later F, et al.: Early assessment of leukocyte alterations at diagnosis of septic shock. Shock 34 (4): 358–363, 2010.
11. Monserrat J, de Pablo R, Reyes E, Diaz D, Barcenilla H, Zapata MR, de la Hera A, Prieto A, Alvarez-Mon M: Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients. Crit Care 13 (1): R26, 2009.
12. Hein F, Massin F, Cravoisy-Popovic A, Barraud D, Levy B, Bollsert PE, Gibot S: The relationship between CD4+
regulatory T cells and inflammatory response and outcome during shock states. Crit Care 14 (1): R19, 2010.
13. Monserrat J, de Pablo R, Diaz-Martin D, Rodriguez-Zapata M, de la Hera A, Prieto A, Alvaraez-Mon M: Early alterations of B cells in patients with septic shock. Crit Care 17 (3): R105, 2013.
14. Inoue S, Suzuki-Utsunomiya K, Okada Y, Taira T, Iida Y, Miura N, Tsuji T, Yamagiwa T, Morita S, Chiba T, et al.: Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly. Crit Care 41 (3): 810–819, 2013.
15. Felmet KA, Hall MW, Clark RS, Jaffe R, Carcillo JA: Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure. J Immunol 174 (6): 3765–3772, 2005.
16. Cheadle WG, Pemberton RM, Robinson D, Livingston DH, Rodriguez JL, Polk HC Jr: Lymphocyte subset responses to trauma and sepsis. J Trauma 35 (6): 844–849, 1993.
17. Oberholzer C, Oberholzer A, Bahjat FR, Minter RM, Tannahill CL, Abouhamze A, LaFace D, Hutchins B, Clare-Salzler MJ, Moldawer LL: Target adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival
in murine sepsis. Proc Natl Acad Sci U S A 96 (20): 14541–14546, 2001.
18. Hotchkiss RS, Tinsley KW, Swanson PE, Change KC, Cobb JP, Buchman TG, Korsmeyer SJ, Karl IE: Prevention of lymphocyte cell death in sepsis improves survival
in mice. Proc Natl Acad Sci U S A 96 (25): 14541–14546, 1999.
19. Meisel C, Shefold JC, Pschowski R, Baumann T, Hetzger K, Gregor J, Weber-Carstens S, Hasper D, Keh D, Zuckermann H, et al.: Granulocyte-macrophage colony-stimulating factor to reverse sepsis-associated immunosuppression
: a double-blind, randomized, placebo-controlled multicenter trial. Am J Respir Crit Care Med 180 (7): 640–648, 2009.
20. Hall MW, Knatz NL, Vetterly C, Tomarello S, Wewers MD, Vold HD, Carcillo JA: Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome. Intensive Care Med 37 (3): 525–532, 2011.
21. Angus DC: The search for effective therapy for sepsis: back to the drawing board? JAMA 306 (23): 2614–2615, 2011.
22. Lewis RT, Klein H: Risk factors in postoperative sepsis: significance of preoperative lymphocytopenia. J Surg Res 26 (4): 365–371, 1979.
23. de Jager C, van Wijk P, Mathoera RB, de Jongh-Leuvenink J, van der Poll T, Wever PC: Lymphocytopenia and neutrophil-lymphocyte count ratio predict bacteremia
better than conventional infection markers in an emergency care unit. Critical Care 14 (5): R192, 2010.
24. Heffernan DS, Monaghan SF, Thakkar RK, Machan JT, Cioffi WG, Ayala A: Failure to normalize lymphopenia following trauma is associated with increased mortality, independent of the leukocytosis pattern. Crit Care 16 (1): R12, 2012.
25. Von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP; for the STROBE Initiative: The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Int Med 147 (8): 573–577, 2007.
26. Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, Cohen J, Opal SM, Vincent JL, Ramsay G: International Sepsis Definitions Conference: 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med 29 (4): 530–538, 2003.
27. Vincent JL, de Mendonca A, Cantraine F, Moreno R, Takala J, Suter PM, Sprung CL, Colardyn F, Blecher S: Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Crit Care Med 26 (11): 1793–1800, 1998.
28. Vincent JL, Moreno R, Takala J, Willatts S, de Mendonça A, Bruining H, Reinhart CK, Suter PM, Thijs LG: The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. Intensive Care Med 22 (7): 707–710, 1996.
29. for the Early Goal-Directed Therapy Collaborative Group: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med 345 (19): 1368–1377, 2001.
30. Venet F, Chung CS, Monneret G, Huang X, Garber M, Ayala A: Regulatory T cell populations in sepsis and trauma. J Leukoc Biol 83 (3): 523–535, 2008.
31. Ertel W, Kremer JP, Kenney J, Steckholzer U, Jarrar D, Trentz O, Schildberg FW: Downregulation of proinflammatory cytokine release from whole blood from septic patients. Blood 85 (5): 1341–1347, 1995.
32. Weighardt H, Heidecke CD, Emmanuilidis K, Maier S, Bartels H, Siewert JR, Holzmann B: Sepsis after major visceral surgery is associated with sustained and interferon-gamma–resistant defects of monocyte cytokine production. Surgery 127 (3): 309–315, 2000.
33. Rigato O, Salomao R: Impaired production of interferon-gamma and tumor necrosis factor-alpha but not of interleukin 10 in whole blood of patients with sepsis. Shock 19 (2): 113–116, 2003.
34. Sinistro A, Almerighi C, Ciaprini, Natoli S, Sussarello E, Di Fino S, Calò-Carducci F, Rocchi G, Bergamini A: Downregulation of CD40 ligand response in monocytes from sepsis patients. Clin Vaccine Immunol 15 (12): 1851–1858, 2008.
35. Munoz C, Carlet J, Fitting C, Misset B, Blériot JP, Cavaillon JM: Dysregulation of in vitro
cytokine production by monocytes during sepsis. J Clin Invest 88 (5): 1747–1754, 1991.
36. Monneret G, Lepape A, Voirin N, Bohé J, Venet F, Debard AL, Thizy H, Bienvenu J, Gueyffier F, Vanhems P: Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock. Intensive Care Med 32 (8): 1175–1183, 2006.
37. Monneret G, Elmenkouri N, Bohé J, Debard AL, Gutowski MC, Bienvenu J, Lepape A: Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: application to monitoring of patients with septic shock. Clin Chem 48 (9): 1589–1592, 2002.
38. Volk HD, Reinke P, Krausch D, Zuckermann H, Asadulla K, Müller JM, Dōcke WD, Kox WJ: Monocyte deactivation—rationale for a new therapeutic strategy in sepsis. Intensive Care Med 22 (Suppl 4): S474–S481, 1996.
39. Dōcke WD, Randow F, Syrbe U, Krausch D, Asadulla K, Reinke P, Volk HD, Kox W: Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med 3 (6): 678–681, 1997.
40. Unsinger J, McGlynn M, Kasten KR, Hoekzema AS, Watanabe E, Muenzer JT, McDonough JS, Tschoep J, Ferguson TA, McDunn JE, et al.: IL-7 promotes T cell viability, trafficking, and functionality and improves survival
in sepsis. J Immunol 184 (7): 3768–3779, 2010.
41. Unsinger J, Burnham CA, McDonough J, Morre M, Prakash PS, Caldwell CC, Dunne WM Jr, Hotchkiss RS: Interleukin-7 ameliorates immune dysfunction and improves survival
in a 2 hit model of fungal sepsis. J Infect Dis 206 (4): 606–616, 2012.
42. Brahmamdam P, Inoue S, Unsinger J, Chang KC, McDunn JE, Hotchkiss RS: Delayed administration of anti–PD-1 antibody reverses immune dysfunction and improves survival
in sepsis. J Leukoc Biol 88 (2): 233–240, 2010.
43. Zhang Y, Zhou Y, Lou J, Li J, Bo L, Zhu K, Wan X, Deng X, Cai Z: PD-L1 blockade improves survival
in experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction. Crit Care 14 (6): R220, 2010.