key: cord-0857524-vknxzzgm authors: Chen, Wanxin; Li, Ziping; Yang, Bohan; Wang, Ping; Zhou, Qiong; Zhang, Zhiguo; Zhu, Jianhua; Chen, Xuexing; Yang, Peng; Zhou, Hao title: Delayed‐Phase Thrombocytopenia in Patients of Coronavirus Disease 2019 (COVID‐19) date: 2020-05-26 journal: Br J Haematol DOI: 10.1111/bjh.16885 sha: 7ff1d5c7e68d082ee1f2e6fdfbb6ae1ff931e0db doc_id: 857524 cord_uid: vknxzzgm COVID‐19 can affect the hematopoietic system. Thrombocytopenia at admission was prevalent, while late‐phase or delayed‐phase thrombocytopenia (occurred 14 days after symptom onset) is obscure. This retrospective single‐center study screened 450 COVID‐19 patients and enrolled 271 patients at the Union Hospital, Wuhan, China, from January 25th to March 9th, 2020. COVID‐19 associated delayed‐phase thrombocytopenia occurred in 11.8% percent of enrolling patients. The delayed‐phase thrombocytopenia in COVID‐19 is prone to develop in elderly patients or patients with low lymphocyte count on admission. The delayed‐phase thrombocytopenia is significantly associated with increased length of hospital stay and higher mortality rate. Delayed‐phase nadir platelet counts demonstrated a significantly negative correlation with B cell percentages. We also presented bone marrow aspiration pathology of three patients with delayed‐phase thrombocytopenia, showing impaired maturation of megakaryocytes. We speculated that immune mediated platelet destruction might account for the delayed‐phase thrombocytopenia in a group of patients. Besides, clinicians need to pay attention to the delayed‐phase thrombocytopenia especially at 3‐4 weeks after symptom onset. In December 2019, the outbreak of coronavirus disease 2019 (COVID-19) was initially reported in Wuhan, China [1] . Common symptoms of COVID-19 include fever, cough, and shortness of breath, since the lung is the major target of SARS-CoV-2. Cardiac, digestive and neurologic complications were also found in COVID-19. These extra-pulmonary manifestations imply diverse target organs in addition to the lung. Hematopoietic system can also be affected by COVID-19. A multicenter study by our hospital and others demonstrated that on admission, lymphocytopenia was present in 83.2% of the patients and thrombocytopenia in 36.2% [2] . With increased hospitalization capacity and prolonged isolation period, we are now able to study the disease longitudinally apart from cross-sectionally on admission. In particular, our initial observations showed there were sudden dramatic decline in platelet count in several COVID-19 patients without evidence of other coagulation abnormalities, which happened three weeks or more after symptoms onset. COVID-19 related early-phase thrombocytopenia was prevalent [3] , while late-phase or delayed-phase thrombocytopenia is obscure. In the current study, we reported the incidence, characteristics, and outcomes of patients with delayed-phase thrombocytopenia. We also presented bone marrow aspiration pathology of three patients with delayed-phase thrombocytopenia. This was a retrospective, descriptive, longitudinal study, conducted from January 25 th to March complete panel of routine laboratory tests. The exclusion criteria were: (1) monitoring period less than 21 days from symptom onset; (2) autoimmune or hematological disease history; (3) previous HIV, hepatitis B or hepatitis C infection; (4) dialysis patients. The demographics data, clinical characteristics, laboratory data and treatment were obtained from patients' medical records. The clinical outcomes (ie, discharges, mortality, and hospital stay time) were monitored up to April 15 th , 2020, the final date of follow-up. Any uncertain information was clarified through direct communication with their families. We collected the continuous monitored laboratory data including blood routine, blood coagulation function, inflammatory cytokines, and lymphocytes subset analysis. For some patients, the SARS-CoV-2 IgG results were recorded when available. The date of disease onset was defined as the day when the symptom was noticed. The severity of COVID-19 was defined according to the diagnostic criteria of "COVID-19 diagnosis and treatment Plan (trial seventh edition)" [5] . Throat swab samples were collected for viral detection. SARS-CoV-2 was confirmed with nucleic acid detection kit (Shanghai bio-germ Medical Technology, Shanghai, China). The IgG antibody was detected with SARS-CoV-2 IgG antibody detection kit (YHLO biotechnology, Shenzhen, China). Descriptive data were presented as means (± standard deviation [SD]) for normally distributed continuous variables and as medians with interquartile range (IQR) for non-normally distributed data. Categorical variables were presented as percentages. Proportions for categorical variables were compared using the χ2 test, and the Fisher exact test was used when data were limited. The quantized variables of parameters are tested by t-test. Nonparametric variables are tested by Mann Whitney U test. The Spearman rank correlation coefficient were used for correlation analysis. All statistical analysis was performed using SPSS v.19.0 (IBM Corp., Armonk, NY, USA). A two-tailed P value <0.05 was considered statistically significant. Based on the dynamic of antibody response [6], duration of the viremia phase after SARS-CoV-2 infection [7] and our preliminary clinical observations, we propose to set 14 days as the rough while reasonable and feasible cutoff value. Therefore, delayed-phase thrombocytopenia is defined as thrombocytopenia beginning after 14 days post symptoms appearance. We found 32 patients (11.8 %) developed delayed-phase thrombocytopenia (Table1). The mean time for delayed-phase thrombocytopenia nadir appeared at 28.3 days, from illness onset. The mean duration time for delayed-phase thrombocytopenia was 4.32 (SD = 2.15). We also found that the mean platelet count at nadir to be 86.0 × 10 9 /L (SD = 37.48). Delayed-phase thrombocytopenia is more prevalent in elderly persons (71.9% in over 60-years-old patients). The clinical outcomes for these 32 patients with delayed-phase thrombocytopenia were 9 patients remained in hospital, 19 discharged, and 4 died. Additionally, there were 8 cases have been admitted to ICU (delayed-phase thrombocytopenia patients). Notably, mortality was markedly higher in patients with delayed-phase thrombocytopenia than in patients without delayed-phase thrombocytopenia (4 [12.5%] vs 4 [1.7%]) ( Table 1 ). The representative platelet count curves were shown in Supplemental Data. On admission, the delayed-phase thrombocytopenia cases demonstrated lower lymphocyte count (0.745 [0.62-1.03], P = 0.001), comparing with the group without delayed-phase thrombocytopenia ( Table 2 ). The level of the lymphocyte subsets (CD3 + T, CD4 + T and B cells) and inflammatory cytokines (IL-4, IL-6 and TNF-α) showed significant alterations from their counterpart. Furthermore, we conducted time-correlated data analysis of the cytokines and lymphocyte subset results, at around the time delayed-phase thrombocytopenia occurred. Delayed-phase nadir platelet counts demonstrated a significantly negative correlation with B cell percentages (r s = 0.509, P < 0.001) and serum IL-6 levels (r s = 0.443, P < 0.001). We specifically studied three patients underwent bone marrow aspiration in the Union Hospital. All these three patients developed rapid and dramatic decline in platelet count at delayed phase, without evidence of other coagulation abnormalities. The pathology results from each of the three patients shared common features: (1) bone marrow showed no obvious abnormities in the myeloid or the erythroid cells; (2) number of atypical or reactive lymphocytes increased; (3) maturation of megakaryocyte was impaired, the mature platelet-producing megakaryocytes were rare, and most megakaryocytes were immature granular megakaryocytes. Two patients' bone marrow smear images were presented in Supplemental Data. The present study was conducted by reviewing the medical records of patients with COVID-19 from January 25th to March 9th, 2020, in a heavily affected hospital during the initial outbreak in China. We found that COVID-19 associated delayed-phase thrombocytopenia occurred in 11.8% percent of enrolling patients. The delayed-phase thrombocytopenia in COVID-19 is prone to develop in elderly patients or patients with low lymphocyte count on admission. The delayed-phase thrombocytopenia is significantly associated with increased length of hospital stay Accepted Article and higher mortality rate. Previous cross-sectional studies have shown that, at admission time, thrombocytopenia was prevalent in acute COVID-19 infected patients [3, 8, 9] . Besides, patients infected by other coronaviruses, severe acute respiratory syndrome (SARS) or Middle East respiratory syndrome (MERS), also frequently suffered thrombocytopenia at admission [10, 11] . However, the acute viral infection associated with delayed-phase thrombocytopenia has merely been reported as rare case reports without enough patients for statistical analysis [12, 13]. Our retrospective study showed that thrombocytopenia might occur at delayed phase in COVID-19 patients and at a significant percentage in the enrolled patients. To clarify the pathogenesis of the delayed-phase thrombocytopenia, we longitudinally reviewed the results of cytokines and lymphocyte subsets. Our results suggest that IL-6 might be an active player in the delayed-phase platelet decline. Since antibody production by B cells is crucial in virus protection, our results imply, but not confirm, that antibodies might play an important role in the delayed-phase platelet decrease. We also found most of the delayed-phase thrombocytopenia lasted less than 7 days, implying the delayed-phase thrombocytopenia is transient. Furthermore, we sought to obtain evidence from bone marrow. All bone marrow aspiration pathology showed common features as impeded megakaryocyte maturation, and mature platelet-producing megakaryocytes were rare (less than 5 in the bone marrow smear). These bone marrow features were as similar as that in immune thrombocytopenia. Based on the antibody curve after SARS-CoV-2 infection [6,14], we speculated that the delayed-phase platelet decrease in these three patients might be immune mediated. This study has several limitations. First, our retrospective study was based on a relatively small sample. Second, our hospital-based study no doubt missed patients who were mild cases. Third, regression and survival analysis are recommended. Forth, the relationship between prognosis and thrombocytopenia remains to be investigated since a few patients were still hospitalized. This article is protected by copyright. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved A Novel Coronavirus from Patients with Pneumonia in China Clinical Characteristics of Coronavirus Disease 2019 in China Clinical Characteristics of 138 Hospitalized Patients With Alanine aminotransferase We acknowledge all health care workers involved in the diagnosis and treatment of patients at Union Hospital. Wanxin Chen and Ziping Li, drafting or revision of the submitted article.Qiong Zhou and Bohan Yang, health care providers of the patient.Ping Wang and Zhiguo Zhang, constructive suggestions and data analysis. Jianhua Zhu and Xuexing Chen, data collection and analysis.Hao Zhou and Peng Yang, design of the study and revision of the submitted article.Written informed consent was obtained from the patients in our study. This work was supported by the Clinical Innovation Funds of Union Hospital (No. 2019-125). All authors declare that they have no conflicts of interest. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved