Lymphopenia acted as an adverse factor for severity in patients with COVID-19: a single-centered, retrospective study

Purpose: The outbreak of SARS-CoV-2 began in December and rapidly caused a pandemic. To investigate the significance of lymphopenia for the severity of the disease, this study was performed. Methods: 115 patients confirmed COVID-19 from a tertiary hospital in Changsha, China were enrolled. The clinical, laboratory, treatment and outcome data were collected and compared between patients with lymphopenia or not. Results: The median age was 42 years (1-75). 54 patients (47.0%) of the patients had lymphopenia on admission. In the group of lymphopenia, more patients had hypertension (30.8% vs 10.0%, P=0.006) and coronary heart disease (3.6% vs 0%, P=0.029) and more patients with leucopenia (48.1% vs 14.8%, P<0.001) and eosinophilia (92.6% vs 54.1%, P<0.001) were observed. Lymphopenia was also correlated with severity grades of pneumonia (P<0.001) and C-reactive protein (CRP) level (P=0.0014). Lymphopenia was associated with a prolonged duration of hospitalization (17.0 days vs 14.0 days, P=0.002). Moreover, the recovery of lymphocyte appeared the earliest before CRP and chest radiographs in severe cases, suggesting its predictive value for disease improvement. Conclusion: Our results showed the clinical significance of lymphopenia for predicting the severity of COVID-19 and the recovery of the disease, emphasizing the need to monitor the lymphocyte count dynamically. P values comparing patients with lymphopenia or not are from χ² test, Fisher’s exact test, or Mann-Whitney U test. COPD=chronic obstructive pulmonary disease; WBC=white blood cell; ALT=alanine aminotransferase; LDH=lactate dehydrogenase; ESR=erythrocyte sedimentation rate. 115 patients with COVID–19, our results showed that lymphopenia was common and correlated with the severity of COVID–19. To the best of our knowledge, this is the first retrospective study revealing the significance of the normalizing of lymphocyte count for predicting disease improvement, putting emphasis on the need to monitor the


Background
Coronaviruses (CoV) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) [1] and Severe Acute Respiratory Syndrome (SARS-CoV). [2] Recently, a novel coronavirus (SARS-CoV-2) has been first detected in Wuhan, China and caused pandemic spread of Coronavirus disease  all over the globe. The control of spreading, early diagnosis of COVID-19 and effective treatment still remains a great challenge.
SARS-CoV-2 is a highly infectious zoonotic coronavirus, which has been reported to be 96% identical at the whole-genome level to a bat coronavirus. [3] Common signs of infection include respiratory symptoms, fever, dry cough, shortness of breath and breathing difficulties. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, septic shock, multiorgan failure and even death. [4,5] Although COVID-19 shares much in common with SARS and MERS, its epidemiologic and clinical features has not been fully illustrated.
Among the laboratory abnormalities reported in patients with SARS, lymphopenia is prominent, which is more frequent in those patients with severe disease compared to those with non-severe disease. [6] Similarly, Lymphopenia is also observed in about 60% of patients with SARS-CoV-2 infection at the initial presentation. [4] However, the clinical significance and underlying mechanism are still not clear.
Here, we have retrospectively analyzed the frequency and association of lymphocytopenia and the severity of COVID-19.

Material And Methods Study design and participants
For this single-centered, retrospective study, a total of 115 patients diagnosed of COVID-19 from The First Hospital of Changsha were included. All patients were admitted from January 17, 2020 to February 14, 2020. Written consent was obtained from all patients and the study was approved by the Ethical Committee of The First Hospital of Changsha. The diagnosis was based on clinical criteria and laboratory features according to the WHO interim guidance. [7] The final date of follow-up was March 13, 2020.

Data Collection
Epidemiological, clinical data were collected from the patients with COVID-19 upon hospitalization.
The illness severity was defined according to the Chinese management guideline for COVID-19 (version 7.0). [8] Mild grade was defined as patients who had mild clinical manifestations and the imaging tests identified no sign of pneumonia. Severe grade was defined as cases who met any of the following: a. respiratory rate exceeded 30 times per minute; b. blood oxygen saturation less than 93%; c. oxygenation index (PaO 2 /FiO 2 [pressure of oxygen in arterial blood/fraction of inspire oxygen]) less than 300. Any patients who needed mechanic ventilation because of respiratory failure, who presented as shock or who were monitored in intensive care unit (ICU) because of multiple organ failure were critical cases. The other cases were classified of general grade. Blood counts, blood biochemistry, chest radiographs and computed tomographic (CT) scans were performed on initial days after admission. Therapeutic measures and outcomes data were collected from the electronic medical network of The First Hospital of Changsha. All of the information was reviewed and checked by two independent doctors to ensure the accuracy.

Laboratory procedures
As previously reported, [4] throat-swap specimens were taken from upper respiratory tract and the confirmation experiments for SARS-CoV2 were performed by real-time RT-PCR following the recommendation of China National Center for Disease Control. A cycle threshold value (Ct value) less than 37 was defined as a positive test, and a Ct value over 40 was defined as a negative record.

Statistical analyses
Continous variables were presented as mean (SD) if they are normally distributed or median (IQR) if they are not. Categorical variables were described as count (%). Statistical analyses were performed using the Pearson χ2 test, Fischer's exact test, and Mann-Whitney U-test. Kaplan-Meier methods and log-rank tests were used to compare the time to recover (TTR) in patients with lymphocytopenia or not. TTR was defined as the length of time from admission to the date when lung image showed signs of improvement. Analyses were conducted using the SPSS statistical package (SPSS, Chicago, IL, USA) and GraphPad Prism (GraphPad Software, San Diego, CA, USA). P-values of <0.05 were considered significant.

Clinical characteristics of patients
The clinical characteristics were summarized in Table 1  years (IQR 8-67), which was significantly younger than other categories (P<0.001). In patients with lymphopenia, 22 patients (40.8%) were in severe or critical category, which was significantly more than patients with no lymphopenia (9.8%, P<0.001). Meanwhile, the more severe the illness was, the higher the incidence of lymphopenia was. As shown in Figure 1A, the median lymphocyte count of We further compared the association of eosinophil count and the severity. As shown in Figure Figure 4D). However, eosinophilia, elevated CRP and comorbidity had no impact on the duration of hospitalization ( Figure   S1).
We further compared the recovery time of lymphocyte, eosinophil, CRP level and chest radiograph before clearance of SARS-CoV2 RNA. Since many patients still presented abnormal lung image when they were discharged, the recovery time of chest radiograph was defined as the length of time from

Treatment and outcome
Of the 115 patients, 95 patients' treatment records were available, which were summarized in Table   1. As for respiratory support, more patients in the group of lymphopenia acquired higher levels of To investigate whether patients would benefit from the treatment of IVIG or corticosteroid, Kaplan-Meier curves were conducted according to the therapeutic choice and lymphocyte count ( Figure 6). In the group of patients with lymphopenia, the median TTR of patients treated with IVIG was 11.0 days (95% CI 7.801-14.199), which was even significantly longer than patients without IVIG treatment (median 7.0 days [95% CI 5.533-8.467], P = 0.001, Figure 6A). So we compared the patients composition and found out that there were more severe or critical cases in IVIG group than that in  Figure 6C). Likewise, no significant benefit from the treatment of corticosteroid was observed, no matter in the group of lymphopenia ( Figure 6D) or in patients without lymphopenia ( Figure 6E) Figure 6F).

Discussion
This study retrospectively analyzed the characteristics of patients infected with SARS-CoV-2 and identified that lymphopenia acted as a good predictor for severity of COVID-19. In particular, more patients had hypertension and coronary heart disease in the group of lymphpenia. Lymphopenia was associated with inflammatory markers, grades of pneumonia severity and prolonged hospitalization.
Additionally, normalization of lymphocyte count indicated the recovery of COVID-19. Especially in severe and critical cases, lymphocyte count normalizing was the first appeared predictor for disease improvement. In mild and general cases, the normalizing of CRP was the most sensitive indicator for disease improvement.
Unlike the previous report that SARS-CoV-2 may infect more men than women, [5] the sex ratio was balanced in the present study. The patients with chronic underlying disease were about one third and the severe or critical cases accounted for about one fourth, which were less than other cohorts reported in Wuhan. [5,4,9] The differences could be attributed to the early insufficiency of medical resources in Wuhan, which gave priority of admission to those patients who were weak and had comorbidities. Interestingly, in patients with lymphopenia, there were more patients having hypertension and coronary heart disease ( Table 1). On the contrary, more cases with hepatitis B were observed in the group of no lymphopenia.
Angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV [10] and SARS-CoV2, [11] is a surface molecule localized on arterial and venous endothelial cells, arterial smooth muscle cells and respiratory tract, [12] which is a homologue of ACE. ACE and ACE2 play different roles in the renninangiotensin system (RAS). ACE generates angiotensin II, the increase of which was reported to be correlated to pathogenesis of heart failure and hypertension, [13] whereas ACE2 negatively regulates the level of angiotensin II. [14] ACE inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) decrease the level of ACE and increase the level of ACE2, [15] which could increase the risk of SARS-CoV-2 infection. We infer that the imbalance of ACE/ACE2 axis contributed to the severity of disease, which could be the reason why more patients had hypertension and coronary heart disease were in the group of lymphopenia, since ACEIs and ARBs are commonly used drugs under these circumstances.
The characteristics and pathogenesis of COVID-19 are both similar and different to the prior severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The major clinical symptoms resulting from these coronavirus-infected diseases include fever, cough, fatigue, myalgia and gastrointestinal symptoms. [4,16,2] However, the clinical manifestation of COVID-19 could be more asymptomatic. Of this cohort of patients, about 25% had no fever on admission (Table 1), which implies that it is more challenging to identify COVID-19 patients and control the pandemic over the globe.
Risk factors for adverse outcomes in patients with SARS and MERS have been investigated in previous studies. [17,1,16,18,19] Specifically, older age, comorbidity, low serum albumin, and concomitant infectious were identified as factors associated with poor outcomes. Lymphopenia was common in both SARS and MERS patients and has also been reported as an important predictor for severe disease in SARS [20] and MERS [21]. In the current study, about half of the patients presented with lymphopenia on admission, which was comparable with the frequency in SARS as reported, [20] but it was lower than the frequency of lymphopenia reported in COVID-19 patients in Hubei Province. [22] Our results revealed that lymphopenia was associated with disease severity, which is consistent with the study by Liu Y, et al [23] that lymphopenia was positively correlated with the severity of acute lung injury in patients with COVID-19. As shown in Figure 5, the recovery of lymphocyte count was the first sign to show up in severe and critical cases before they improved and discharged, suggesting that normalizing of lymphocyte was a more sensitive indicator than CRP or CT scan for prediction of disease recovery in patients of severe and critical grades.
Lymphopenia was reported in varies types of virus-infected diseases, such as SARS [20,6,24], MERS [18,21] and respiratory syncytial virus [25]. As for the underlying mechanism of lymphopenia, previous study reported that lymphopenia in SARS may be caused by enhanced vascular sequestration associated with increased soluble vascular cell adhesion molecule-1 levels, [26] but it remains unclear in patients with COVID-19. It has been well known that the treatment of glucocorticoid results in lymphopenia by causing the migration of lymphocytes from the peripheral blood. [27] Meanwhile, viral infections would inevitably lead to the activation of hypothalamicpituitary-adrenal axis under stress, resulting the up-regulation of endogenous corticosteroids [28], which might involve in the immunopathogenesis of lymphopenia of COVID-19.
Eosinophils only count for 0.4%-8% of leucocytes in the peripheral blood, but act as rather important inflammatory mediators and involve in innate immunity, allergies, parasitic infection and virus infection. [29] In the current study, we found that the frequency of eosinophilia (72.2%) was higher than lymphopenia. Eosinophilia was associated with lymphopenia and its recovery also acted as an indicator for the improvement of the disease. The median recovery time of eosinophils was shorter than CRP and CT scan in severe and critical cases. Thrombocytopenia was rarely observed in our study and had no significant impact on the outcome.
Currently, no specific antiviral treatment is available for SARS, MERS and COVID-19. A range of treatments including lopinavir/ritonavir, interferon-β2b and recombinant human cytokine derived protein was used in this cohort of patients, but no improvement for outcome by any of them was observed. As a previous therapeutic measure for SARS, IVIG has not been confirmed effective for outcome improvement. [30] In our study, no benefit from the approach of IVIG was observed, no matter patients were with lymphopenia or not. Given the high cost of IVIG and economic burden for public health systems, more investigations of this approach are needed to provide evidence for a large-scale use.
Our study has some limitations. For example, some of the laboratory examination records and treatment records were not available since this is a retrospective study. In condition, an examination of lymphocyte subsets is unavailable in most of the patients. So there was still no evidence for which subset contributed the profound lymphopenia in COVID-19 patients. On this basis, a more comprehensive and thorough investigation is necessary in the future. Lymphocyte count and eosinophil count in different groups of illness severity of COVID-19.
(A) lymphocyte count in severe and critical cases were significantly lower than that in mild cases. (B) eosinophil count in severe and critical cases were significantly lower than that in mild cases. ***, P<0.001; **, P<0.01; *, P<0.05.

Figure 2
The correlation of lymphocyte count and laboratory test. Lymphopenia was highly correlated with CRP level (A), serum albumin level (B) and neutrophil count (D). No significant correlation was found between lymphocyte count and Ct value (C). Spearman rank correlation analysis (r) and P values are provided in each graph.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download. Figure S1.tif