Lymphopenia in hospitalized patients and its relationship with severity of illness and mortality.

BACKGROUND: Lymphopenia is associated with various pathologies such as sepsis, burns, trauma, general anesthesia and major surgeries. All these pathologies are clinically expressed by the so-called Systemic Inflammatory Response Syndrome which does not include lymphopenia into defining criteria. The main objective of this work was to analyze the diagnosis of patients admitted to a hospital related to lymphopenia during hospital stay. In addition, we investigated the relationship of lymphopenia with the four levels of the Severity of Illness (SOI) and the Risk of Mortality (ROM). METHOD AND
FINDINGS: Lymphopenia was defined as Absolute Lymphocyte Count (ALC) <1.0 x109/L. ALC were analyzed every day since admission. The four levels (minor, moderate, major and extreme risk) of both SOI and ROM were assessed. A total of 58,260 hospital admissions were analyzed. More than 41% of the patients had lymphopenia during hospital stay. The mean time to death was shorter among patients with lymphopenia on admission 65.6 days (CI95%, 57.3-73.8) vs 89.9 (CI95%, 82.4-97.4), P<0.001. Also, patients with lymphopenia during hospital stay had a shorter time to the mortality, 67.5 (CI95%, 61.1-73.9) vs 96.9 (CI95%, 92.6-101.2), P<0.001.
CONCLUSIONS: Lymphopenia had a high prevalence in hospitalized patients with greater relevance in infectious pathologies. Lymphopenia was related and clearly predicts SOI and ROM at the time of admission, and should be considered as clinical diagnostic criteria to define SIRS.

Introduction

Lymphopenia is associated with various pathologies such as sepsis, burns, trauma, general anesthesia and mayor surgeries. This leads to a life-threatening period of immunosuppression [1-5]. This situation represents a serious public health problem, since both incidence and mortality from these processes are increasing [6-8]. All these pathologies have in common the clinical expression of the so-called Systemic Inflammatory Response Syndrome (SIRS), which includes a number of clinical and laboratory criteria such as leukocytosis and leukopenia, but does not include lymphopenia [9].

There is a need for a consensual definition of SIRS and sepsis, since there is sometimes difficulty, with the current criteria, to define pathological cases with SIRS caused by infection, since it is not easy to identify the infectious focus in some situations. Therefore, those criteria should be better adjusted to avoid erroneous diagnoses. Persistent lymphopenia is an independent predictor of increased mortality in critically ill emergency general surgical patients [10], septic patients [11], and outpatients [12, 13]. However, to our knowledge, lymphopenia has not been studied in all the pathologies that make up the repertoire of diagnoses of patients admitted to hospital.

The All Patients Refined Diagnosis Related Groups (APR-DRGs) are a patient classification scheme by means of several attributes which include severity of illness, risk of dying, prognosis, treatment difficulty, need for intervention, and resource intensity. Severity of Illness (SOI) refers to the extent of physiologic decompensation or organ system and Risk of Mortality (ROM) refers to the likelihood of dying. There are four SOI and ROM subclasses which are numbered sequentially from 1 to 4 indicating respectively, minor (1), moderate (2), major (3), or extreme (4) severity of illness or risk of mortality [14-16].

The main objective of this work was to analyze the diagnosis of patients admitted to hospital related to lymphopenia on admission and during hospital stay. In addition, we investigated the relationship of lymphopenia with the four levels of SOI and ROM according to DRGs.

Methods

Study design and obtaining information

This retrospective cohort study was conducted at the Arnau de Vilanova–Llíria Health Department in Valencia, Spain, including two hospitals: Arnau de Vilanova’s Hospital and Llíria’s Hospital, with a total of 450 beds, which cover an area of 300,000 inhabitants. Patients older than 14 years were admitted from January 1, 2016, to December 31, 2019, from Emergency Department (80%) and Scheduled Admission (20%). A file was generated with all analytical parameters of patients seen in the emergency room and hospitalization in the four years of the study. A cross was made with hospital episodes and emergency data. The resulting file was crossed, in turn, with the Database of DRGs from those same years. We obtained a Database of 72,984 records that combines data related to the hospitalization episode (date of admission, date of discharge, reason for admission, diagnoses, procedures); data related to the patient (age, sex); data related to DRGs (DRG, CMD, Severity, Risk of Mortality). Finally, patients who had undergone in-hospital laboratory tests were selected, ordered by evolution of the laboratory results in the admission episode, obtaining a sample of 58,860 patients. Clinical diagnoses were coded using International Classification of Diseases (ICD-10, 2016 version). The study protocol was approved by the Ethics and Investigation Committee of the Arnau de Vilanova-Llíria Hospital (approval number 2019–12), Valencia City, Spain.

Study variables

Demographic

Age and gender. Hospital stay, up to 15 diagnoses associated with each patient and at each admission, all blood counts performed in each episode, and in-hospital mortality.

Lymphopenia was defined as Absolute Lymphocyte Count (ALC) <1.0 x109/L. ALC were analyzed every day from admission. The mean of the analyses performed each day of hospitalization was calculated. We assessed the Severity of Illness (SOI) and the Risk of Mortality (ROM), with its four levels, minor (1), moderate (2), major (3), and extreme risk (4).

Statistical analysis

When normality was assumed (Kolmogorov-Smirnov test), Student t test was used to compare the means between two different groups of patients. When the hypothesis of normality was not accepted, the Mann-Whitney U test was used. Two-tailed Fischer’s exact test was used to compare lymphopenia with diagnosis (Odds Ratio). Logistic regression (Exp B) and Cox regression (Hazard Ratios) were used to compare the two groups (with lymphopenia and without lymphopenia) taken into account analytical variables. Repeated Measures ANOVA were used to assess the differences of lymphocytes frequency between dead and alive patients over time. The Kaplan-Meier survival method was used to estimate the cumulative probability of mortality according to lymphopenia on admission and lymphopenia during hospital stay. Differences between curves were tested using the Log-Rank test (Statistical program R, version 3.3). A Cox proportional hazards regression model was used to assess whether lymphopenia variables were independently associated with the risk of mortality. P value <0.05 was considered statistically significant.

Results

Characteristics of admitted patients and diagnosis

A total of 58,260 hospital admissions were analyzed. The mean age was 67.8 ± 18.4 years (66.4 ± 17.7 in males and 69.5 ± 19.0 in females, P<0.001). The mean age of patients with lymphopenia was 73.5 ± 15.6 years vs 63.9 ± 19.2 in patients without lymphopenia, p<0.001.

The mean of hospital stay was 6.5 ± 6.6 days (6.8 ± 6.9 in males and 6.1 ± 6.1 in females, p <0.001). The mean of hospital stay in lymphopenic patients was 7.7 ± 7.6 days vs 5.6 ± 5.7 days in no lymphopenic patients (p<0.001). A total of 23,892 (41.0%) patients presented lymphopenia during hospital stay. Eighteen thousand three patients (30.9%) presented lymphopenia on admission. The characteristics of the admitted patients and the relationship of the variables studied according to lymphopenia are shown in Table 1.

Table 1

Characteristics of admitted patients and their relationship with lymphopenia (univariant analysis).

	Cases	Lymphopenia	OR (CI 95%)	Sig. p
	n = 58,260	n = 23,892 (41.0%)
	n (%)	n (%)
Gender
Male	30,597 (52.5)	12,987 (42.4)	1.13 (1.09–1.17)	< 0.001
Female	27,663 (47.5)	10,905 (39.4)	0.88 (0.85–0.91)	< 0.001
Origin of Hospital Admission
Emergency Admission	46,598 (80.0)	19,891 (42.7)	1.43 (1.37–1.49)	< 0.001
Scheduled Admission	11,662 (20.0)	4,001 (34.3)	0.70 (0.67–0.73)	< 0.001
Diagnosis Related Groups
Surgical APR-DRG	11,820 (20.3)	5,094 (43.1)	1.11 (1.07–1.16)	< 0.001
Medical APR-DRG	46,440 (79.7)	18,798 (40.5)	0.90 (0.86–0.94)	< 0.001
Severity of Illness
SOI-1	22,329 (38.3)	6,613 (29.6)	0.45 (0.44–0.47)	< 0.001
SOI-2	26,583 (45.6)	11,489 (43.2)	1.18 (1.14–1.22)	< 0.001
SOI-3	8,788 (15.1)	5,348 (60.9)	2.59 (2.48–2.72)	< 0.001
SOI-4	560 (1.0)	442 (78.9)	5.47 (4.46–6.71)	< 0.001
Risk of Mortality
ROM-1	30,052 (51.6)	8,553 (28.5)	0.33 (0.32–0.35)	< 0.001
ROM-2	19,449 (33.4)	9,821 (50.5)	1.79 (1.73–1.86)	< 0.001
ROM-3	7,674 (13.2)	4,705 (61.3)	2.59 (2.47–2.72)	< 0.001
ROM-4	1,085 (1.9)	813 (74.9)	4.42 (3.85–5.07)	< 0.001
Mortality	3,213 (5.5)	2,345 (73.0)	4.20 (3.88–4.55)	< 0.001

APR-DRG: Refined Diagnosis Related Group. SOI: Severity of Illness. ROM: Risk of Mortality. Significance: Two-tailed Fischer’s exact test was used.

Relationship of lymphopenia with diagnosis

Table 2 shows the relationship of lymphopenia according to Major Diagnostic Categories (MDC).

Table 2

Relationship of lymphopenia according to Major Diagnostic Categories (MDC) (n = 58,260).

	Cases	Lymphopenia	OR (CI 95%)	Sig. p
		n = 23,892 (41.0%)
	n	n (%)
01 - Nervous System	3,823	954 (25.0)	0.46 (0.42–0.49)	< 0.001
02 - Eye	216	30 (13.9)	0.23 (0.16–0.34)	< 0.001
03 - Ear, Nose, Mouth and Throat	965	411 (42.6)	1.07 (0.94–1.22)	NS
04 - Respiratory System	10,530	5,658 (53.7)	1.88 (1.80–1.96)	< 0.001
05 - Circulatory System	8,868	3,003 (33.9)	0.70 (0.67–0.73)	< 0.001
06 - Digestive System	7,461	2,952 (39.6)	0.93 (0.89–0.98)	0.007
07 - Hepatobiliary System and Pancreas	3,311	1,685 (50.9)	1.53 (1.42–1.64)	< 0.001
08 - Musculoskeletal System and Connective Tissue	5,950	2,725 (45.8)	1.24 (1.18–1.31)	< 0.001
09 - Skin, Subcutaneous Tissue and Breast	1,006	320 (31.8)	0.67 (0.58–0.76)	< 0.001
10 - Endocrine, Nutritional and Metabolic System	1,312	463 (35.3)	0.78 (0.70–0.86)	< 0.001
11 - Kidney and Urinary Tract	5,293	2,285 (43.2)	1.10 (1.04–1.17)	0.001
12 - Male Reproductive System	749	262 (35.0)	0.77 (0.66–0.90)	0.001
13 - Female Reproductive System	1,341	246 (18.3)	0.32 (0.28–0.36)	< 0.001
14 - Pregnancy, Childbirth and Puerperium	9	2 (22.2)	0.41 (0.85–1.98)	NS
16 - Blood and Blood Forming Organs and Immunological Disorders	946	529 (55.9)	1.84 (1.62–2.10)	< 0.001
17 - Myeloproliferative Diseases (Poorly Differentiated Neoplasms)	863	540 (62.6)	2.44 (2.10–2.80)	< 0.001
18 - Infectious and Parasitic Diseases	1,575	1,031 (65.5)	2.80 (2.52–3.12)	< 0.001
19 - Mental	1,652	162 (9.8)	0.15 (0.13–0.18)	< 0.001
20 - Alcohol/Drug Use or Induced Mental Disorders	927	75 (8.1)	0.12 (0.10–0.16)	< 0.001
21 - Injuries, Poison and Toxic Effect of Drugs	439	151 (34.4)	0.75 (0.62–0.98)	0.005
23 - Factors Influencing Health Status and Other Contacts Health Services	803	282 (35.1)	0.78 (0.67–0.90)	0.004
24 –Human Immunodeficiency Virus Infection	197	112 (56.9)	1.90 (1.43–2.52)	< 0.001
25 - Multiple Significant Trauma	22	14 (63.6)	2.52 (1.06–6.00)	0.048

The diagnoses in each MDC correspond to a single organ system or cause, fundamentally based on the main diagnosis on admission. Therefore, there is only one MDC for each hospitalization episode.

The relationships between lymphopenia and diagnostic categories were analyzed using univariate analysis. International Statistical Classification of Diseases and Related Health Problems (ICD-10) was used. The results are shown in S1 Table.

Infectious processes were the most frequently coded (21,093, 36.2%). “In patients without infectious pathology, the incidence of lymphopenia was 34.6% (n = 12,876/37,167) vs 52.2% in patients with infectious pathology (n = 11,016/21,093); ORlymphopenia without infectious pathology = 0.66 (0.63–0.65), p<0.001 " ORlymphopenia with infectious pathology = 2.06 (1.99–2.14), p<0.001".

There were 6,208/58,260 (10.7%) patients with sepsis, 3,528 (56.8%) of which presented lymphopenia vs 2,680 (43.2%) who had lymphopenia in the group without sepsis, OR = 2.1 (1.9–2.2), p<0.001.

The patients with Septic Shock were 515/58.260 (0.9%), 446 (86.6%) of which had lymphopenia. Only 69 (13.4%) patients with septic shock did not have lymphopenia, OR = 9.5 (7.3–12.2), p<0.001.

S1 Appendix shows the number of diagnoses in each patient according to disease groups.

Each patient can have multiple diagnoses, so there are more diagnoses than patients. S1 Appendix shows the number of patients who have one or more diagnoses at the same time according to disease groups. Finally, it can be seen that the sum of the totals is equal to the sum of the diagnoses.

Mortality and lymphopenia

The number of deaths was 3,213 (5.5%). Of these patients, 2,345 (73.0%) had lymphopenia during hospital stay and 1,743 (54.2%) had lymphopenia on admission, OR = 4.2 (CI95%, 3.9–4.6) and OR = 2.8 (CI95%, 2.5–3.1) respectively, P<0.001. Fig 1 shows the evolution of absolute lymphocytes counts (mean) according to alive and dead patients in the consecutive days analyzes performed (1 to 20 days) during the hospital stay, in four situations: all cases, infectious pathology, sepsis and septic shock. OA: On Admission. Lymphopenia—<1.0 x109/L. Repeated Measures ANOVA (p<0.05). T-bars show typical error. Significative differences (Mann-Whitney U test) between alive and dead: Total patients; Infectious Diseases and Sepsis (p<0.001); Septic Shock: days 1 to 10 (p<0.001), days 11 to 15 (p<0.05); days 16 to 20 no significant.

Fig 1

Evolution of absolute lymphocytes counts (mean) according to alive and dead patients in the consecutive days analyzes performed (1 to 20 days) during the hospital stay, in four situations: All cases, infectious pathology, sepsis and septic shock.

OA: On Admission. Lymphopenia—<1.0 x109/L. Repeated Measures ANOVA (p<0.05). T-bars show typical error. Significative differences (Mann-Whitney U test) between alive and dead: Total patients; Infectious Diseases and Sepsis (p<0.001); Septic Shock: days 1 to 10 (p<0.001), days 11 to 15 (p<0.05); days 16 to 20 no significant.

The Kaplan-Meier curve for the risk of mortality is shown in Fig 2. Kaplan-Meier plots illustrating Survival probability following lymphopenia on admission (A) and lymphopenia during hospital stay (B) in peripheral blood of admitted patients. Time is expressed in days (mean). Log-Rank test (p-value). Percentage of dead patients according to the number of analyzes performed with lymphopenia on admission or during admission (C). Percentage of patients with lympophenia on admission and during hospital stay according to four subclases of Severity of illness (SOI) (D) and Risk of Mortality (ROM) (E). Chi square Pearson test, p<0.001. Pearson’s R: Interval-by-interval, p<0.001. Spearman correlation: Ordinal-by-ordinal, p<0.001 (D and E panels). The mean time to death was shorter among lymphopenia on admission (A) 65.6 days (CI95%, 57.3–73.8) vs 89.9 (CI95%, 82.4–97.4), P< 0.001. In addition, lymphopenia during hospital stay in hospital admission patients (B) had a shorter time to the mortality, 67.5 (CI95%, 61.1–73.9) vs 96.9 (CI95%, 92.6–101.2), p< 0.001.

Fig 2

Kaplan-Meier plots illustrating Survival probability following lymphopenia on admission (A) and lymphopenia during hospital stay (B) in peripheral blood of admitted patients. Time is expressed in days (mean). Log-Rank test (p-value). Percentage of dead patients according to the number of analyzes performed with lymphopenia on admission or during admission (C). Percentage of patients with lymphopenia on admission and during hospital stay according to four subclases of Severity of illness (SOI) (D) and Risk of Mortality (ROM) (E). Chi square Pearson test, p<0.001. Pearson’s R: Interval-by-interval, p<0.001. Spearman correlation: Ordinal-by-ordinal, p<0.001 (D and E panels).

Furthermore, patients with lymphopenia on admission had a higher risk of mortality on a multivariate analysis, Hazard Ratio (HR) adjusted: 2.4 (CI95%, 2.2–2.5), P<0.001 and lymphopenia during hospital stay, HR adjusted: 2.8 (CI95%, 2.6–3.0) P<0.001. The percentage of patients who die increases progressively according to the number of analyzes performed with lymphopenia during their hospital admission (Fig 2C). Relationship of lymphopenia with severity and mortality risk has been described in Fig 2, panel D and E, respectively.

Table 3 shows relationship between leukocyte and platelet counts with mortality rates.

Table 3

Relationship between leukocyte and platelet counts with mortality rates.

	Cases	Deaths
	n (%)	n	Fatality rate	% in Total Deaths	Two-tailed Fischer’s exact test	Sig.	Logistic Regression	Sig.	Cox Regression	Sig. p
	58,260	3,213	(%)	100			Exp (B) (CI95%)		HR Adjusted
	(100.0)		(5.5%)
Lymphopenia	23,892 (41.0)	2,345	(9.8)	(73.0)	4.2 (3.9–4.5)	<0.001	2.84 (2.63–3.07)	<0.001	2.30 (2.12–2.50)	<0.001
(< 1.0 x 109/L)
Neutrophylia	33,570 (57.6)	2,631	(7.8)	(81.9)	3.5 (3.2–3.9)	<0.001	2.74 (2.50–3.00)	<0.001	1.75 (1.56–1.96)	<0.001
(> 7.0 x 109/L)
Leukocitosis	21,433 (36.8)	1,973	(9.2)	(61.4)	2.9 (2.7–3.1)	<0.001	2.19 (2.03–2.35)	<0.001	1.49 (1.33–1.58)	<0.001
(>12.1 x 109/L)
Thrombocytopenia	7,470 (12.8)	811	(10.9)	(25.2)	2.5 (2.2–2.7)	<0.001	1.66 (1.53–1.58)	<0.001	1.49 (1.37–1.63)	<0.001
(< 125 x 109/L)
Leukopenia	3,818 (6.6)	292	(7.6)	(9.1)	1.5 (1.3–1.7)	<0.001	0.98 (0.86–1.10)	0.977	0.79 (0.67–0.94)	0.006
(< 4.0 x 109/L)
Neutropenia	1,590 (2.7)	143	(9.0)	(4.5)	1.7 (1.5–2.1)	<0.001	1.05 (0.88–1.24)	0.152	1.07 (0.86–1.34)	0.536
(<1.4 x 109/L)

All analytical variables were included in the analysis using logistic regression and Cox regression.

Discussion

This is the first study to evaluate the association of lymphopenia with all hospital diagnoses in such a large number of patients. Highlights the high prevalence of lymphopenia in our study, since more than 30% of the patients admitted to the hospital had lymphopenia on admission. In addition, the incidence increased to 41% when including lymphopenia during hospital stay.

Lymphopenia was more common in men than in women. On the other hand, lymphopenia was higher among patients admitted for surgical pathologies vs medical processes and it was also more frequent in Emergency compared to Scheduled Admission.

The greatest decrease in lymphocytes was observed in pathological processes associated with infection. The lowest values were detected in sepsis and septic shock [17, 18]. The most probable cause of this transient phenomenon of immunoparalysis with decreased of lymphocytes has been attributed to increased apoptosis of this cells [19, 20]. Considering the importance of lymphocytes in the host’s immune defense, it is not surprising the relationship of lymphopenia with severity and mortality. This fact was previously observed in other studies, although in a smaller number of patients. A prospective Danish population-based study, in subjects without acute pathology and underwent voluntary analysis, demonstrated an association between lymphopenia and increased risk of hospitalization and infection-related death [13]. We previously observed that lymphopenia was present in 75% of patients admitted to the hospital with sepsis. An inverse relationship of γδ T cells with disease severity and mortality was observed in septic patients [21].

In this work, a significant direct relationship between SOI and ROM with lymphopenia has been demonstrated. SOI and ROM variables are not available for the clinician at the time of admission. However, lymphocyte count and other objective parameters are available at the time of admission. The definition of SIRS is based on clinical and analytical criteria easily obtained and quickly accessible to clinicians. Leukocytes and especially total neutrophils and “band” neutrophils are routinely performed in the emergency laboratory. The interpretation of leukocytosis and leukopenia is used as criteria for SIRS and infection. In this work we showed that lymphopenia has a higher predictive value for mortality than the measurement of leukocytes and total neutrophils.

The inclusion criteria for SIRS did not allow distinguishing uninfected patients from septic patients. However, as we demonstrated in our study, lymphopenia is clearly related to infectious pathology, being much more marked in sepsis and in septic shock [9].

A greater association was observed between mortality and lymphopenia during hospitalization than with lymphopenia on admission. This finding could be due to the fact that the percentage of patients who die increased progressively according to the number of analyzes performed during their hospitalization.

In summary, the decrease in lymphocytes should be valued by physicians as a factor to consider in the prognosis of their patients. In conclusion, lymphopenia should be included among the criteria that evaluate the SOI and ROM at the time of admission. Furthermore, lymphopenia should be a criterion for evaluating SIRS patients with infectious and non-infectious causes at the time of admission.

Limitations

Our study has the limitations of retrospective studies. In addition, it lacks pediatric patients and patients with large burns since these pathologies are centralized in another hospital center whose database we have not been able to access. In any case, we think that the number of patients evaluated in the present study is large enough to be able to draw solid conclusions.

Conclusion

Lymphopenia has a high prevalence in patients admitted to a general hospital, with greater relevance in subjects with infectious pathology. Furthermore, lymphopenia is related and clearly predicts the Severity of Illness and the Risk of Mortality at the time of admission, and should be considered as a clinical diagnostic criteria to define SIRS patients with infectious and non-infectious causes at the time of admission.

Significant association lymphopenia according to diagnostic (CIE-10) (n = 58,260).

(DOCX)

Click here for additional data file.

Significant association lymphopenia according to infectious diseases (CIE-10).

(DOCX)

Click here for additional data file.

Number of diagnoses in each patient according to disease groups.

Each patient can have multiple diagnoses, so there are more diagnoses than patients. S1 Appendix shows the number of patients who have one or more diagnoses at the same time according to disease groups. Finally, it can see that the sum of the totals is equal to the sum of the diagnoses.

(DOCX)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

22 Jul 2021

PONE-D-21-19890

Lymphopenia in hospitalized patients and its relationship with severity of illness and mortality.

PLOS ONE

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Reviewers' comments:

Reviewer #1: This is an interesting manuscript that explores an under-appreciated aspect of routine laboratory studies in patients. It is a welcome addition to the literature.

General comments: The manuscript at times does not quite make sense in English. I struggled to understand some parts of the paper. There are also several grammatical and spelling errors (e.g. in the abstract the first line says "mayor" when I believe the intent is "major").

Abstract: Rewrite the second sentence in the background. The current wording is confusing. There are also other spelling/grammatical errors (e.g. "mean time to dead" should probably be "mean time to death")

Introduction:

-In the first paragraph it mentions SIRS and states that includes criteria such as thrombocytopenia and neutrophilia. These are manifestations of sepsis but are not included in the SIRS definition. I would also include other criteria for sepsis (qSOFA, SOFA) in the introduction as well.

-The second paragraph needs rephrasing as well. SIRS in it's current definition is well defined but the accuracy and clinical utility are called into question. I believe the intent the authors are trying to convey with this sentence is that sepsis itself is difficult to diagnose.

-I recommend moving the definitions for SOI and ROM to the introduction from discussion.

Methods:

-Were these all patients or only adult patients?

Results:

-Again would revise some of the language to make the text more clear

-Table 2 is a bit confusing. From my understanding each admission gets one diagnosis as the number of cases equals the number of admissions captured. It would be helpful to explain how they were coded as such given that many patients should have multiple reasons for hospitalization (i.e. having both a respiratory and circulatory system issue).

-If feasible it would be interesting to compare lymphopenia in infectious diagnosis vs non-infectious diagnoses

-For Table 2 what does the acronym "DD" stand for?

-On page 8 why wwas 20 chosen as the days of lymphopenia?

-On page 8 I am unsure what the first sentence of the third paragraph is trying to convey. I believe more days of lymphopenia are linked with worse prognosis but I am unsure as the figure is unlabeled.

-In table 3 I think it would be beneficial if the significance was reported in leukopenia and neutropenia in the "bivariate analysis" given their inclusion in the multivariable analysis

Discussion:

-The first paragraph of page 10 needs to be revised heavily. I would also consider a citation of why lymphocytes are suppressed in sepsis

-The definitions of APR-DRG, SOI, and DRG should be moved to the introduction

-Why is lymphopenia during hospitalization more associated with mortality than lymphopenia on admission as show on page 8 and figure 2?

-I am not sure that the phrase "very significant direct relationship" can be used in this context on page 10. I would discuss this with a statistician to determine the appropriate language.

-On page 11 I do not think you can make the claim that "lymphopenia is clearly related to infectious pathology". Unless I missed it, the results do not specifically compare infectious vs non-infectious diagnoses so the authors do not know lymphopenia is more associated with sepsis.

Figures:

-Figure 1 needs the axes labeled on both x and y axes. It also needs a short explanation of what each table is describing

-Figure 2c needs to have the axes labeled and also an explanation of what it is conveying

-Figures 2D and 2E need to have the axes labeled and an explanation of what it is trying to convey

Overall the data presented is interesting. My main issues with the manuscript is the clarity and language, all I believe can be addressed with further revision.

Reviewer #2: Dear authors, this is a very interesting manuscript. I have some suggestions before publication:

1) In all tables you only provide information of the overall population and of the group of interest . However, to better understand the results it is important to provide the comparison group ( non limphopenia, patients who survived etc...) I presume that the p-value in these tables refer to the univariate logistic regressions. I believe that it would be more statistically sound to provide the univariate comparisons as descriptive statistics also for categorical variables (noun and %) using fisher test or qui- square test. Please add these comparisons to the statistical plan in the methods section and to the tables in the results section.

2)Please explain in the cox model which variable were included and provide (as supplement) the HR and CI for each adjusted variable.

3) Please explain further appendix S1. It is not clear to me what it represents for example 1 means that the patient only had one diagnosis within the disease subgroup?

4) The figures are very illustrative but please provide the individual p values of each comparison in figure 1. In figure 2 D and E I suggest that you provide the non limphopenia group as well and to compare both groups (limpohpenia in admission vs non limphopenia in admission and limphopenia during hospital stay and non limphopenia during hospital stay) providing a p value.

5) the manuscript needs English editing . for example in the abstract and in the introduction it is written mayor instead of major surgery . Also some sentences appear incomplete.

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Reviewer #1: No

Reviewer #2: Yes: Elisa Gouvêa Bogossian

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1 Aug 2021

Response to Reviewers

Reviewers' comments:

Reviewer #1: This is an interesting manuscript that explores an under-appreciated aspect of routine laboratory studies in patients. It is a welcome addition to the literature.

General comments: The manuscript at times does not quite make sense in English. I struggled to understand some parts of the paper. There are also several grammatical and spelling errors (e.g. in the abstract the first line says "mayor" when I believe the intent is "major")

Reply: The manuscript has been thoroughly revised. We have tried to correct spelling and grammatical errors.

Abstract: Rewrite the second sentence in the background. The current wording is confusing. There are also other spelling/grammatical errors (e.g. "mean time to dead" should probably be "mean time to death")

Reply:

The second sentence in the background has been corrected: “All these pathologies are clinically expressed by the so-called Systemic Inflammatory Response Syndrome which does not include lymphopenia as a defining criteria”.

“Dead” has been replaced by “death”

Introduction:

-In the first paragraph it mentions SIRS and states that includes criteria such as thrombocytopenia and neutrophilia. These are manifestations of sepsis but are not included in the SIRS definition. I would also include other criteria for sepsis (qSOFA, SOFA) in the introduction as well.

Reply: The sentence has been replaced by:

“which includes a number of clinical and laboratory criteria such as leukocytosis and leukopenia, but does not include lymphopenia”

-The second paragraph needs rephrasing as well. SIRS in it's current definition is well defined but the accuracy and clinical utility are called into question. I believe the intent the authors are trying to convey with this sentence is that sepsis itself is difficult to diagnose.

Reply: The sentence has been replaced by:

“There is a need for a consensual definition of SIRS and sepsis, since there is sometimes difficulty, with the current criteria, to define pathological cases with SIRS caused by infection, since it is not easy to identify the infectious focus in some situations”.

-I recommend moving the definitions for SOI and ROM to the introduction from discussion.

Reply: Definitions of SOI and ROM have been described in the introduction.

Methods:

-Were these all patients or only adult patients?

Reply: Pediatricians treat patients up to 14 years of age. The sentence has been replaced by: “Patients older than fourteen years were admitted from January 1, 2016, to December 31, 2019,….

Results:

-Again would revise some of the language to make the text more clear

Reply: The English edition has been corrected

-Table 2 is a bit confusing. From my understanding each admission gets one diagnosis as the number of cases equals the number of admissions captured. It would be helpful to explain how they were coded as such given that many patients should have multiple reasons for hospitalization (i.e. having both a respiratory and circulatory system issue).

Reply: The following text has been explained in Table 2:

“The diagnoses in each MDC correspond to a single organ system or cause, based on the main diagnosis on admission, therefore there is only one MDC per hospitalization episode”

-If feasible it would be interesting to compare lymphopenia in infectious diagnosis vs non-infectious diagnoses

Reply: We have added in results the relationship of lymphopenia with non-infectious pathology, as well as with sepsis and septic shock.

“In patients without infectious pathology, the incidence of lymphopenia is 34.6% (n = 12,876 / 37,167) vs 52.2% of lymphopenia in patients with infectious pathology (n = 11,016 / 21,093); ORlymphopenia without infectious pathology = 0.66 (0.63 - 0.65), p <0.001 " ORlymphopenia with infectious pathology = 2.06 (1.99 – 2.14), p <0.001.

There were a total of 6,208/58,260 (10.7%) patients with sepsis, 3,528 (56.8%) of which presented lymphopenia vs 2,680 (43.2%) who had lymphopenia in the group without sepsis, OR=2.1 (1.9 – 2.2), p<0.001.

The patients with Septic Shock were 515/58.260 (0.9%), of which 446 (86.6%) had lymphopenia vs 69 (13.4%) of the patients with Septic Shock who did not have lymphopenia, OR=9.5 (7.3 – 12.2), p<0.001.”

-For Table 2 what does the acronym "DD" stand for?

Reply: DD stands for Diseases and Disorders. In table 2 the acronym “DD” has been changed to “Diseases and Disorders”.

-On page 8 why was 20 chosen as the days of lymphopenia?

Reply: The first 20 days have been chosen because the number of patients with more than 20 days of admission is very low and therefore the results would not reach statistical significance.

-On page 8 I am unsure what the first sentence of the third paragraph is trying to convey. I believe more days of lymphopenia are linked with worse prognosis but I am unsure as the figure is unlabeled.

Reply: Figure 2C shows how the percentage of deaths progressively increases according to the number of analyzes with lymphopenia that the patient has during their admission. Thus, as can be seen in Fig. 2C, 8.1% of patients who have had only one analysis with lymphopenia on admission or during admission die vs 30.3% of patients who have ten analyzes with lymphopenia during their admission. It has been explained in Figure 2C.

-In table 3 I think it would be beneficial if the significance was reported in leukopenia and neutropenia in the "bivariate analysis" given their inclusion in the multivariable analysis

Reply: Table 3 has been modified to analyze the variables by Two-tailed Fischer's exact test, logistic regression and Cox regression. The method has been added in Statistical analysis.

Discussion:

-The first paragraph of page 10 needs to be revised heavily. I would also consider a citation of why lymphocytes are suppressed in sepsis

Reply: We have justified the possible causes of immunoparalysis associated with sepsis. Three bibliographic citations have been added

“The most probable cause of this transient phenomenon of immunoparalysis with decreased of lymphocytes has been attributed to increased apoptosis of this cells [19, 20]”

-The definitions of APR-DRG, SOI, and DRG should be moved to the introduction

Reply: Definitions of SOI and ROM have been described in the introduction

-Why is lymphopenia during hospitalization more associated with mortality than lymphopenia on admission as show on page 8 and figure 2?

Reply: We believe that lymphopenia during hospitalization is more associated with mortality than lymphopenia on admission because the percentage of patients who die increases progressively according to the number of analyzes performed with lymphopenia during their hospital admission, as shown in Figure 2C. These results have been commented in the discussion section.

-I am not sure that the phrase "very significant direct relationship" can be used in this context on page 10. I would discuss this with a statistician to determine the appropriate language.

Reply: After consulting a statistician as suggested by the reviewer we have removed the word “very”. The correct expression is “significant direct relationship”.

-On page 11 I do not think you can make the claim that "lymphopenia is clearly related to infectious pathology". Unless I missed it, the results do not specifically compare infectious vs non-infectious diagnoses so the authors do not know lymphopenia is more associated with sepsis.

Reply: As already indicated above, we have added in results the relationship of lymphopenia with non-infectious pathology, as well as with sepsis and septic shock.

“In patients without infectious pathology, the incidence of lymphopenia is 34.6% (n = 12,876 / 37,167) vs 52.2% of lymphopenia in patients with infectious pathology (n = 11,016 / 21,093); ORlymphopenia without infectious pathology = 0.66 (0.63 - 0.65), p <0.001 " ORlymphopenia with infectious pathology = 2.06 (1.99 – 2.14), p <0.001.

There were a total of 6,208/58,260 (10.7%) patients with sepsis, 3,528 (56.8%) of which presented lymphopenia vs 2,680 (43.2%) who had lymphopenia in the group without sepsis, OR=2.1 (1.9 – 2.2), p<0.001.

The patients with Septic Shock were 515/58.260 (0.9%), of which 446 (86.6%) had lymphopenia vs 69 (13.4%) of the patients with Septic Shock who did not have lymphopenia, OR=9.5 (7.3 – 12.2), p<0.001.”

Figures:

-Figure 1 needs the axes labeled on both x and y axes. It also needs a short explanation of what each table is describing

Reply: Figure 1 has been better explained.

-Figure 2c needs to have the axes labeled and also an explanation of what it is conveying

Reply: The axes in figure 2C have been labeled and conveniently explained.

-Figures 2D and 2E need to have the axes labeled and an explanation of what it is trying to convey

Reply: Figures 2D and 2E have been labeled and better explained.

Overall the data presented is interesting. My main issues with the manuscript is the clarity and language, all I believe can be addressed with further revision.

Reviewer #2: Dear authors, this is a very interesting manuscript. I have some suggestions before publication:

1) In all tables, you only provide information of the overall population and of the group of interest. However, to better understand the results it is important to provide the comparison group (no lymphopenia, patients who survived etc...) I presume that the p-value in these tables refer to the univariate logistic regressions. I believe that it would be more statistically sound to provide the univariate comparisons as descriptive statistics also for categorical variables (noun and %) using fisher test or qui- square test. Please add these comparisons to the statistical plan in the methods section and to the tables in the results section.

Reply: Table 3 has been modified to analyze the variables by Two-tailed Fischer's exact test, logistic regression and Cox regression. The method has been added in Statistical analysis.

2) Please explain in the cox model which variable were included and provide (as supplement) the HR and CI for each adjusted variable.

Reply: In the logistic regression analysis and COX regression, all the analytical variables have been introduced into the model (Table 3), and it´s explained at the bottom of the table.

3) Please explain further appendix S1. It is not clear to me what it represents for example 1 means that the patient only had one diagnosis within the disease subgroup?

Reply: Each patient can have several diagnoses so there are more diagnoses than patients. The appendix S1 shows the number of patients who have 1 or more diagnoses at the same time according to groups of diseases. Finally, it can be seen that the sum of the totals coincide with the diagnoses.

4) The figures are very illustrative but please provide the individual p values of each comparison in figure 1. In figure 2 D and E I suggest that you provide the non limphopenia group as well and to compare both groups (limphopenia in admission vs non limphopenia in admission and limphopenia during hospital stay and non limphopenia during hospital stay) providing a p value.

Reply: Figures 1 and 2 show the percentage of patients with lymphopenia in each subgroup (minor, moderate, major, and extreme). If we have not misunderstood, the percentage of patients without lymphopenia will be the rest of the percentage until reaching 100%, so we believe that it would be a figure with too many lines that would be confusing. Statistical analysis has been explained.

“Figure 2. Kaplan-Meier plots illustrating Survival probability following lymphopenia on admission (A) and lymphopenia during hospital stay (B) in peripheral blood of admitted patients. Time is expressed in days (mean). Log-Rank test (p-value). Percentage of dead patients according to the number of analyzes performed with lymphopenia on admission or during admission (C). Percentage of patients with lympophenia on admission and during hospital stay according to four subclases of Severity of illness (SOI) (D) and Risk of Mortality (ROM) (E). Chi square Pearson test, p<0.001. Pearson's R: Interval-by-interval, p<0.001. Spearman correlation: ordinal by ordinal, p<0.001 (D and E panels)”

5) The manuscript needs English editing, for example in the abstract and in the introduction, it is written mayor instead of major surgery. Also some sentences appear incomplete.

Reply: We have made a new edition in English.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Aug 2021

Lymphopenia in hospitalized patients and its relationship with severity of illness and mortality.

PONE-D-21-19890R1

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5 Aug 2021

PONE-D-21-19890R1

Lymphopenia in hospitalized patients and its relationship with severity of illness and mortality.

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