key: cord-0973200-hhsfq8bz authors: Tan, Li; Wang, Qi; Zhang, Duanyang; Ding, Jinya; Huang, Qianchuan; Tang, Yi-Quan; Wang, Qiongshu; Miao, Hongming title: Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study date: 2020-03-03 journal: nan DOI: 10.1101/2020.03.01.20029074 sha: f19e2df932f90f387fe71533f3802875d9042160 doc_id: 973200 cord_uid: hhsfq8bz Background: Coronavirus disease-2019 (COVID-19) is a rapidly escalating epidemic caused by SARS-CoV-2. Identification of a simple and effective indicator to assess disease severity and outcome is urgently needed. Methods: This study retrospectively analyzed dynamic changes of lymphocyte percentage (LYM%) in 15 death cases, 15 severe cases as well as 40 moderate cases of COVID-19 patients. Next, prognostic role of lymphopenia in COVID-19 were verified in 92 hospitalized cases. Results: Our results from death and severe cases showed that LYM% in blood test were inversely associated with the progression and severity of COVID-19. LYM% in patients with moderate COVID-19 remained higher than 20% 10-12 days after symptom onset. In contrast, LYM% was lower than 20% in severe cases. However, LYM% in severe cases was higher than 5% 17-19 days after the onset of the disease, while it fell below 5% in death cases. Therefore, we established a reliable Time from symptom onset-LYM% model (TLM), which could be used to evaluate disease severity and predict the outcomes of hospitalized patients with COVID-19. Conclusion: Lymphopenia can be used to indicate clinical course, treatment effect and outcomes of COVID-19 patients. Coronaviruses are a large family of viruses that cause both common cold and serious respiratory illnesses, such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) [1] [2] [3] . An outbreak of an unknown infectious pneumonia has recently occurred in Wuhan, China 4 . The pathogen of the disease was quickly identified as a novel coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2), and the disease was named coronavirus infection disease-19 (COVID-19) 5 . the virus has so far caused 78959 confirmed cases and 2791 deaths in China according to WHO. COVID-19 has been spreading in many countries such as Japan, Korea, Singapore, Iran and Italia The clinical manifestation of COVID-19 include fever, cough, fatigue, muscle pain, diarrhea, and pneumonia, which can developed to acute respiratory distress syndrome, metabolic acidosis, septic shock, coagulation dysfunction, and organ failure such as liver, kidney and heart failure 4, 6, 7 . Unfortunately, There is no effective medication other than comprehensive support. However, the mild type of COVID-19 patients can recover shortly after appropriate clinical intervention. The moderate type patients, especially the elderly or the ones with comorbidity, can worsen and became severe, indicating high mortality 6,7 . However, efficient indicators for the disease severity, therapeutic response and disease outcome are still incompletely investigated. Once such indicators are present, reasonable medication and care can be inclined, which is believed to significantly reduce the mortality of severe patients. Routine examinations include complete blood count, coagulation profile, and serum biochemical test (including renal and liver function, creatine kinase, lactate dehydrogenase, and electrolytes). Complete blood count is the most available, efficient and economic examination. This study aimed to retrospect and analyze the time-courses of complete blood count of cured and dead patients, in order to obtain key indicators of disease progression and outcome and to provide guidance for subsequent clinical practice. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The mild clinical symptom were mild with no pulmonary inflammation on imaging. The moderate is the overwhelming majority, showing symptoms of respiratory infections such as fever, cough, and sputum, and pulmonary inflammation on imaging; when symptoms of dyspnea appear, including any of the following: shortness of breath, RR ≥ 30bpm, blood oxygen saturation ≤ 93% (at rest), PaO 2 / FiO 2 ≤ 300 mmHg, or pulmonary inflammation that progresses significantly within 24 to 48 hours> 50%, it was classified as severe; respiratory failure, shock, and organ failures that require intensive care were critically ill. Among them, mild patients were not admitted in this designated hospital. In this study, the basic information, complete blood count, coagulation profile, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint and serum biochemical test (including renal and liver function, creatine kinase, lactate dehydrogenase, and electrolytes) and disease outcome of all included patients were collected. In this study, GraphPad 6.0 software was used for data statistics and mapping. The presentation of dynamic changes of blood markers is descriptive. The consistence between Guideline and TLM-based disease classification was tested using kappa statistic. Kappa≧0.75 indicates a high consistence and 0.75﹥Kappa≧0.4 indicates a general consistence. In order to explore the relationship between tested blood markers and disease conditions in COVID-19 patients, we first randomly selected 5 death cases and monitored dynamic changes in blood tests for each patient from Among all factors, LYM% showed the most significant and consistent trend ( Fig.1F ), indicating that this indicator might reflect the disease progression. In addition, we also investigated the indicators reflecting liver, kidney and myocardial functions. Significant changes in these indicators were usually accompanied by organ failure and cytokine storm syndromes (data not shown), which occurred at the end of disease course and thus could not reflect disease progression. Therefore, we only focused on lymphopenia in COVID-19 in the present study. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint 6 To further confirm the relationship between blood LYM% and patient's condition, we increased our sample size to 12 death cases (mean age: 76 years; average therapeutic time: 20 days) (Supplementary Table 1 ). Most cases showed that LYM% was reduced to lower than 5% within 2 weeks after disease onset ( Fig. 2A) . We also randomly selected 7 cases (mean age: 35 years, average therapeutic time: 35 days) with severe symptoms and cured outcome (Supplementary Table 2 ) and 11 cases (mean age: 49; average therapeutic time: 26 days) with moderate symptoms and cured outcomes (Supplementary Table 3 ). LYM% of severe patients fell down initially and then rose to higher than 10% until discharged (Fig. 2B ). In contrast, LYM% of moderate patients fluctuated very little after disease onset and was higher than 20% when discharged (Fig. 2C) . These results suggest that lymphopenia is a predictor of prognosis in COVID-19 patients. By summarizing all the death cases, severe-cured cases and moderate-cured cases in our hospital to depict the time-LYM% curve ( We further collected information from all COVID-19 patients who were admitted to hospital, and validated the consistency between TLM and the existing guideline. As shown in Figure 4 , 92 COVID-19 patients were currently . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint hospitalized in light of the classification criteria of the Chinese New Coronavirus Pneumonia Diagnosis Program (5th edition): 55 moderate patients, 24 severe patients and 13 critically ill patients. According to the TLM we proposed, LYM% in 24 out of 55 moderate cases was lower than 20% at TLM-1; LYM% of all these patients was above 5%, indicating that these patients would recover soon. Regarding other 24 patients with severe symptoms, LYM% at the TLM-1 was lower than 20% in 20 out of 24 cases. LYM% at TLM-2 in 6 cases was less than 5%, indicating a poor prognosis. LYM% in 12 out of 13 critically ill patients at the TLM-1 was lower than 20%. LYM% of these patients at TLM-2 in 6 cases was lower than 5%, suggesting a poor prognosis (Fig. 4A ). Furthermore, with kappa statistic test, we further verified the consistence between TLM and the existing guideline in disease typing (Fig. 4B ). By retrospectively tracking the dynamic changes of LYM% in death cases and cured cases, this study suggests that lymphocyte count is an effective and reliable indicator for disease classification and prognosis in COVID-19 patients. We also established TLM innovatively and verified its reliability in a considerable number of hospitalized COVID-19 patients. A high correlation of blood lymphocytes with the disease progression suggested that lymphocyte deficiency or incapacity is the key cellular pathology of COVID-19. The protection, maintenance or promotion of lymphocyte levels might have a good effect on the prevention and treatment of COVID-19. The classification of disease severity in COVID-19 is very important for the grading treatment of patients. In particular, when the outbreak of an epidemic occurs and medical resources are relatively scarce, it is necessary to conduct grading severity and treatment, thus optimizing the allocation of rescue resources, and prevent the occurrence of overtreatment or undertreatment. According to the latest 5 th edition of the national treatment guideline, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This study suggested that LYM% can be used as a reliable indicator to classify the moderate, severe and critical ill types independent of any other auxiliary indicators. During the development of mild disease into severe condition, the proportion of lymphocytes in the blood gradually decreased and maintained at a low level. By the time the disease began to improve, LYM% in the blood gradually rose to normal or nearly normal levels. Patients with persistently low levels of blood lymphocytes, especially less than 5%, often had a poor prognosis. Therefore, we suggested that LYM% should be used as an indicator for evaluating the effectiveness of clinical drugs or therapies. We suggest more medical staff apply and improve our proposed TLM to give patients more timely and appropriate treatments. Lymphocytes play a decisive role in maintaining immune homeostasis and inflammatory response throughout the body. Understanding the mechanism of reduced blood lymphocyte levels is expected to provide an effective strategy for the treatment of COVID-19. We speculated four potential mechanisms leading to lymphocyte deficiency. (1) The virus might directly infect lymphocytes, resulting in lymphocyte death. Lymphocytes express the coronavirus receptor ACE2 and may be a direct target of viruses. 9 (2) The virus might directly destroy lymphatic organs. Acute lymphocyte decline might be related to lymphocytic dysfunction, and the direct damage of novel coronavirus virus to organs such as thymus and spleen cannot be ruled out. This hypothesis needs to be confirmed by pathological dissection in the future. (3) Inflammatory cytokines continued to be disordered, perhaps leading to . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint lymphocyte apoptosis. Basic researches confirmed that TNFα, IL-6 and other pro-inflammatory cytokines could induce lymphocyte deficiency 10 . (4) Inhibition of lymphocytes by metabolic molecules produced by metabolic disorders, such as hyperlactic acidemia. The severe type of COVID-19 patients had elevated blood lactic acid levels, which might suppress the proliferation of lymphocytes 11 . Multiple mechanisms mentioned above or beyond might work together to cause lymphopenia, and further research is needed. The clinical data in the present study came from a single center and the sample size was limited. The TLM we established was based on the specific treatment condition in the hospital and might not be fully applicable to some patients who were treated in light of a different guideline. Lymphopenia is an effective and reliable indicator of severity and hospitalization in COVID-19 patients. We suggest that the TLM should be included in the diagnosis and therapeutic guidelines of COVID-19. The authors declare no competing interests. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . https://doi.org/10.1101/2020.03.01.20029074 doi: medRxiv preprint 14 at TLM-2, those pre-severe patients are reclassified as moderate. If 5%