key: cord-0997822-ght7b9v9
authors: Azhdari Tehrani, Hamed; Darnahal, Maryam; Vaezi, Mohammad; Haghighi, Shirin
title: COVID-19 associated thrombotic thrombocytopenic purpura (TTP) ; A case series and mini-review
date: 2021-01-22
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2021.107397
sha: 24e0034c962eb1652db1201a4265635258695597
doc_id: 997822
cord_uid: ght7b9v9

Introduction : Thrombotic microangiopathies are a group of disorders that are mainly related to endothelial dysfunction. This category of endothelial dysfunction results of several imbalances between platelets, endothelium and immune system, also cytokine production. Aim of this study : To report cases with thrombotic thrombocytopenic purpura (TTP) and COVID-19 and review COVID-19 endothelial dysfunction literature. Methods : Primary laboratory data, peripheral blood smear, ADAMTS13 antigen activity level, and antibody ordered for each of these four patients. Treatments for COVID-19 administered for all patients. Traditional treatments for TTP also were administered. Results : There were numerous schistocytes (more than 5%) in peripheral blood smears for each patient. ADAMTS13 antigen activity level was below 10%, and ADAMTS13 antibody was elevated for each patient. COVID-19 PCR was positive for all patients, and CT-Scans were indicative of the involvement of COVID-19. Conclusion : In this case series, we reported four COVID-19 patients who presented with signs and symptoms of anemia and thrombocytopenia, resulting in thrombotic thrombocytopenic purpura.

Thrombotic microangiopathies (TMA) are coagulopathies, that presented with two significant manifestations of hemolytic anemia, thrombocytopenia [1] . The most proven pathophysiology of this entity is endothelial dysfunction. TMAs are dividing further into primary and secondary [2] . Primary TMAs result from the genetically intrinsic deficiency of one or several factors such as complement regulating proteins deficiency in hemolytic uremic syndrome (HUS) or primary ADAMTS13 deficiency in primary thrombotic thrombocytopenic purpura (TTP) [3] . Secondary forms result from extrinsic triggers such as autoimmune disorders, drugs, bone marrow transplantation, pregnancy, viruses that result in acquired deficiency, and starting the TMA process such as acquired TTP, HELLP syndrome, or catastrophic anti-phospholipid syndrome [4] . However, the secondary forms do not have merely acquirable. These conditions are characterized by extremely high lethality and mortality rate due to thrombosis and organ dysfunction.

Soon after the detecting of the first case of COVID-19 in Wuhan, this virus spread throughout the world. Up to now, many facts and pieces of evidence released about the pathogenicity of this virus [5, 6] . There are several manifestations of COVID-19 coagulopathy, such as deep venous thrombosis, pulmonary thromboembolism, and thrombotic microangiopathies. This paper reports four cases of COVID-19 associated thrombotic thrombocytopenic purpura (TTP), and then we discuss the probable pathogenicities of this phenomenon. Thrombotic thrombocytopenic purpura (TTP) is a rare blood disorder characterized by clotting in small blood vessels (thromboses), resulting in a low platelet count. The main manifestations of TTP are hemolytic anemia, thrombocytopenia, neurologic abnormalities. In the full-blown form, fever and renal dysfunction are also manifested. Most sporadic TTP cases appear to be associated with severe deficiency of ADAMTS13 activity due to auto-antibodies against this protease. The table below, summarizes the difference between thrombotic thrombocytopenic purpura (TTP), and Disseminated intravascular coagulation (DIC). In this study, we reported four cases of TTP associated with COVID-19 disease.

This article reviews the pathophysiology, clinical presentation, diagnosis, and evaluation of thrombotic microangiopathies, especially thrombotic thrombocytopenic purpura (TTP) in patients with COVID-19 infection. A search of MEDLINE , Google-Scholar, from January 2020 up to December 2020 was done. Terms searched included combinations of COVID-19, microangiopathies, Thrombotic thrombocytopenic purpura, pathogenesis, clinical manifestations. Randomized clinical trials, observational studies, meta-analyses, and review articles were all included in the search. All reports were in the English language.

This study aims to describe patients with anemia and thrombocytopenia in association with TTP. Anemia and thrombocytopenia are common findings among patients with COVID-19 disease, especially in severe ones. However, in these cases, we describe below, ADAMTS13 antigen activity level and antibody were correlated with TTP. These laboratory data distinguish these patients from typical findings of anemia or thrombocytopenia in COVID-19 patients.

A 25-year-old pregnant woman in her third trimester of pregnancy, with severe respiratory symptoms, was referred to our emergency department. Vital signs in the admission were : Temperature: 38 However, the gynecologist decided to terminate the pregnancy. We also checked the ADAMTS13 antigen activity level and antibody. Results were shown in table 2. So we conclude that the TTP would be the primary cause of her Bicytopenia. After the cesarean section, plasma exchange started, and gradually patient's hemoglobin level, platelet counts began to increase [7] .

A 56-year-old woman with a previous history of locally advanced breast cancer now in remission, a triple negative breast cancer treated by surgical resection and adjuvant chemotherapy twenty years ago, was admitted to our hospital for severe respiratory symptoms and also Bicytopenia and leukocytosis. In peripheral blood smear, several schistocytes were seen. With a normal coagulation test, we ordered ADAMTS 13 antigen activity level and antibody. Results were shown in table 2. With regard to these findings, plasma exchange started daily; however, hemoglobin and platelet counts did not change significantly after intensifying plasma exchange. We started Rituximab 375mg/m2 beside plasma exchange. After two weeks, hemoglobin and platelet count became normal as shown in table 2. With regard to deterioration of the consciousness level, a brain CT-scan showed a hemorrhagic stroke in her left frontal lobe, which led to death [8] .

A 57-year-old woman with severe respiratory symptoms and Bicytopenia with normal White blood cells count was referred to the emergency room. She had no past medical history and medication use. Lung CT-Scan has shown patchy bilateral infiltration suggestive of COVID-19 infection. In peripheral blood smear, schistocytes about 5% were seen. We planned to start the plasma exchange regarding ADAMTS 13 antigen activity and antibody shown in table 2. However, only we infused fresh frozen plasma divided twice daily regarding the shortage of plasmapheresis setting. Also, Intravenous immunoglobulin (IVIG) for five days was administered. After one week of administration, the Hemoglobin level and platelet count began to rise and became normal after fourteen days (shown in table 2).

A 38-year-old man who refereed to emergency department by emergency service with complaining of rectorrhagia. Since a week ago, he had a COVID-19 infection with patchy infiltration in the right upper lobe in his lung CT-Scan. He had not any complain of respiratory symptoms. Oxygen saturation was normal. In laboratory data, there was Bicytopenia with a leukocytosis. In peripheral blood smear, schistocytes were seen in about 2% of each high-power field. However, with this schistocytes count less than 5%, the definitive diagnosis of TTP is not conclusive, and other diagnoses such as idiopathic thrombocytopenic purpura would be considered. Coagulation studies were normal. Lactate Dehydrogenase (LDH) was mildly elevated. Due to poor general condition, we started the plasma exchange daily. Intravenous Dexamethasone started 8 mg twice daily. Because of suspicion of idiopathic thrombocytopenic purpura (ITP) we administered IVIG daily for five consecutive days. There was no change in platelet and hemoglobin status following a plasma exchange, and hemoglobin dropped to three in the first week of admission. ADAMTS13 antigen activity level and antibody were sent after three days of plasma exchange. The results were shown in table 2. We decided to start Rituximab 375mg/m 2 weekly for four consecutive weeks. By continuing plasma exchange therapy and administering Rituximab platelet count and hemoglobin level, it began to rise after two weeks of admission and reached 9 g/dl for hemoglobin and 88000/µl for platelets count. After three weeks of admission, Hb and platelets reached near-normal levels (shown in table 2). 

There are several manifestations of COVID-19 coagulopathy, such as deep venous thrombosis, pulmonary thromboembolism, and thrombotic microangiopathies. Out of 1008 patients with COVID-19, who underwent pulmonary CT-Angiography, Up to 40% of patients had small branches pulmonary thromboembolism [9] .

There is a link between inflammation and organ damage by cytokines such as interleukin 6, interleukin 17A, tumor necrosis factor α, which are released during the cytokine storm phase of infection [10, 11] . Hypercoagulability is an essential hallmark of inflammation. Pro-inflammatory cytokines lead to platelet activation and also coagulation cascade in which thrombosis occur [12] . In patients especially with moderate to severe disease especially in patients admitted to ICU, there may be several abnormalities in lab data such as thrombocytopenia, elevated prothrombin time (PT) and partial thromboplastin time (PTT), elevated D-Dimer, LDH, Fibrin degradation product (FDP), Fibrinogen and also several pro-inflammatory cytokines such as interleukin 6 [13, 14] . It is also assumed that patients with more lung involvement have higher pro-inflammatory markers and, therefore, more abnormalities in platelet count and coagulation pathways [15] . Pro-inflammatory cytokines activate tissue macrophages, which express tissue factor (TF) and then platelets and factor VIII bind to TF, So the thrombin formation occurs. Also, anti-coagulation proteins such as tissue factor pathway inhibitor (TFPI) and protein C,S are functionally impaired by these cytokines [16] . The recruited platelets release further vascular endothelial growth factor (VEGF), So TF would be further expressed, and coagulation cascade amplified [17] .

On the other arm, it is proven that the COVID-19 virus binds to angiotensin-converting enzyme 2 (ACE2) and then down-regulates this enzyme. So Angiotensin II increased. This increment results in vasoconstriction and also the expression of tissue factors, which leads to thrombosis [18, 19] .

The initiating point of all of the thrombotic microangiopathies is endothelial damage that is caused by the virus. It has been proposed that by binding SARS-COV-2 to the ACE-2 receptor, which is expressed in airway epithelial cells, vascular endothelial cells are damaged. Proinflammatory chemo-attractants, including C3a and C5a, are released, leading to recruiting more leukocytes [20, 21] . Leukocytes being activated and release more cytokines (IL-6, TNF, IL-1). This cytokine release leads to more endothelial damage and more platelet aggregation. This is a vicious cycle. Garlinski et al. introduced SARS-COV to mice, which causes lung damage and deposition of complement proteins C3b, C4d, and C5b-9 in damaged tissues. However, when the virus was introduced to mice deficient in C3, lung damage was reduced [22] .

ADAMTS13 is a protease that cleaves von Willebrand factor (VWF) multimers into small ones. This protease's inherited or acquired deficiency leads to VWF giant multimers that bind to platelets, and coagulation factors promote the coagulation cascade [23, 24] . This endothelial dysfunction may lead to one of the worst outcomes, disseminated intravascular coagulation (DIC). Other COVID-19 associated thrombotic microangiopathies such as TTP or HUS may also occur, usually with the thrombotic presentation.

There are several strategies for the standard of care in patients with TMA, especially in TMAs other than DIC. All of these strategies work to modulate the immune response and balance the coagulation and anti-coagulation axis. These are high dose corticosteroids, plasma exchange, IVIG therapy, Rituximab, complement inhibitors [25] [26] [27] . Early reports in China indicated that treatment with methylprednisolone decreased the death risk in patients with COVID-19 [28] . In the RECOVERY trial, the use of Dexamethasone for patients with severe COVID-19 infection, resulted in a significantly lower 28-day mortality rate. Also, there was a trend, showing the greatest absolute and proportional benefit of Dexamethasone in patients who received mechanical ventilation [29] . In a systemic meta-analysis of seven trials, using different doses of Dexamethasone, Hydrocortisone, or Methylprednisolone, the Odds Ratio (OR) for 28 days of allcause mortality decreased [30] .

Plasma exchange is a safe procedure that removes inflammatory cytokines and thrombogenic molecules, also replenishes any deficiency in natural anti-coagulant proteins. Sometimes recovery occurs with IVIG after plasma exchange [31] [32] [33] . The use of IVIG for treatment for COVID-19 had been reported, which resulted in rapid recovery. An early study suggested that anti-coagulation with unfractionated heparin or low molecular weight heparin may have antiinflammatory properties leading to the reduction of the 28-day mortality rate [30, 34] . Glas et al. proposed the administration of anti-coagulants and protein C to reverse pulmonary microthrombi [35] . Heparin exhibits anti-inflammatory effects by neutralizing damage associated cellular proteins, thereby stabilizing endothelial cell membrane, decreasing vascular leakage, and thrombus formation [36, 37] . A report from China, administration of low molecular weight heparin reduced the IL-6 levels and improved anti-coagulation indices [38] .

In this case series, we reported four cases in our hematology center with anemia and thrombocytopenia. In evaluating these cases, peripheral blood smears had shown numerous schistocytes, and they had normal coagulation tests. We measured ADAMTS13 antigen activity and antibody in doubt for thrombotic thrombocytopenic purpura. All of them were positive for antibody and low level or lower limit of normal in antigen activity. Indeed, we found associated thrombotic microangiopathy except for DIC. All of the patients responded to traditional treatment of TTP, such as plasma exchange with FFP, FFP infusion, and also Rituximab therapy. So, we conclude that the main triggering factor in TTP development in these patients might be COVID-19, since this novel virus is far from completely understood. COVID-19 disease was confirmed by lung CT imaging and PCR test. It is proposed that Rituximab administration in patients, impair B lymphocytes activation, function, and antibody production [39] . Hindilerden et al. reported a 74-year-old woman with a diagnosis of COVID-19 and associated TTP.

ADAMTS13 antigen activity level and antibody confirmed the diagnosis of TTP. She received daily plasma exchange and methylprednisolone [40] . Capecchi et al. reported a 55-year-old woman with a previous TTP history associated with bacterial pneumonia, thirty years ago. In a recent infection with COVID-19, she developed anemia and thrombocytopenia and schistocytes' appearance in the peripheral blood smear. ADAMTS13 antigen activity level was low and ADAMTS13 antibody was high. They started methylprednisolone and plasma exchange and also Caplacizumab [41] . Albiol et al. reported a 57-year-old woman with a previous history of breast cancer in complete remission, presented with COVID-19, anemia, and thrombocytopenia. In laboratory analysis, ADAMTS13 antigen activity level was decreased, and ADAMTS13 antibody was detected. She received plasma exchange therapy [42] .

Antibodies play a role in neutralizing viral antigens. However, it may have an inverse role in the phenomenon of antibody-dependent enhancement, which leads to the production of nonneutralizing IgGs, and therefore induced more cytokine releases by macrophages [43] . [44] . After Rituximab therapy, case number 2 had no exacerbating COVID-19 signs or symptoms, however in case number 4 and case with classic TTP with COVID-19 infection after four weeks, they had an exacerbation in signs and symptoms of COVID-19. So, it is impossible to clearly define that Rituximab therapy may delay cytokine release or promote infection by inducing immunosuppression [45] . It needs further studies to clearly define the potential role of Rituximab therapy in COVID-19 pathogenesis, especially in patients who had disorders that fundamentally need targeting immunotherapy.

In conclusion, we reported four cases of COVID-19 who developed Bicytopenia, which was associated with thrombotic thrombocytopenic purpura. They received anti-COVID therapies besides traditional therapies for thrombotic thrombocytopenic purpura. In COVID-19 associated microangiopathies, autoimmunity besides endothelial damage, may have a crucial role in these manifestations. Plasma exchange may further improves this condition.

The most important limitation of this study was that we had a low population of patients with COVID-19 and associated thrombotic thrombocytopenic purpura. Therefore, no conclusions can be drawn from this treatment process.

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: All procedures performed in this study were in accordance with the ethical standards of the Helsinki declaration.

Informed Consent: Informed consent was obtained from all individuals.

Funding : The authors received no financial support for the research, author-ship, and/or publication of this article. 

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Hamed Azhdari Tehrani : Acquisition of research data

Acquisition of clinical data Mohammad Vaezi : Revising the article Shirin Haghighi : Writing original draft