Immature platelets in COVID-19

Abstract Platelets play a critical role in immune response. Coronavirus disease 2019 (COVID-19) patients with a severe course often show pathological coagulation parameters including thrombocytopenia, and at the same time the proportion of immature platelets increases. In this study, the platelet count and the immature platelet fraction (IPF) of hospitalized patients with different oxygenation requirements was investigated daily over a course of 40 days. In addition, the platelet function of COVID-19 patients was analyzed. It was found that the number of platelets in patients with the most severe course (intubation and extracorporeal membrane oxygenation (ECMO)) was significantly lower (111.5 ∙ 106 /mL) than in the other groups (mild (no intubation, no ECMO): 203.5 ∙ 106 /mL, p < .0001, moderate (intubation, no ECMO): 208.0 ∙ 106 /mL, p < .0001). IPF tended to be elevated (10.9%). Platelet function was reduced. Differentiation by outcome revealed that the deceased patients had a highly significant lower platelet count and higher IPF (97.3 ∙ 106 /mL, p < .0001, 12.2%, p = .0003). Plain Language Summary What is the context? Pathological coagulation is a feature of severe cases of COVID-19, with both bleeding complications and thrombosis. Patients with severe COVID-19 are frequently treated with extracorporeal membrane oxygenation (ECMO), which is often associated with bleeding complications. Platelets play an important role in blood clotting. The proportion of immature platelets has been characterized as hyperreactive and associated with high prothrombotic activity. In addition, they are discussed as predictors of COVID-19 disease severity. What is new? In grading the severity of disease in our patient cohort, we consider the required oxygenation measures. Thus, the focus is on severe cases requiring intubation and ECMO compared to moderate (intubation, no ECMO) and mild (no intubation, no ECMO) cases. What is the impact? This study focuses on severely ill patients who require ECMO treatment. Therefore, this study provides further evidence to use immature platelet fraction to predict the outcome of severe COVID-19 courses.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first appeared in 2019, has infected approximately 553 • 10 6 people worldwide to date, with approximately 6.3 • 10 6 infections resulting in a fatal outcome [1].Infection with SARS-CoV-2 often leads to mild cold-like symptoms in severe cases also to acute respiratory distress syndrome, failure of various organs and sepsis [2,3].In addition, severe coronavirus disease 2019 (COVID- 19) progressions also exhibit pathologic blood coagulation.Several pathological coagulation parameters have already been associated with the severity of COVID-19, for example, D-dimer levels [4].Pathologic coagulation is manifested by both the occurrence of thrombosis, e.g., venous thromboembolism, and bleeding complications, e.g., symptoms resembling disseminated intravascular coagulation (DIC).The latter is characterized by thrombocytopenia, increased D-dimer, decreased fibrinogen levels, and prolonged prothrombin time and is associated with both bleeding and thrombosis risk.Whether or not classical DIC is present in COVID-19 patients is currently controversial [5].Another factor affecting blood clotting in COVID-19 patients is that patients with severe courses are often treated with extracorporeal membrane oxygenation (ECMO).Therefore, the influences of ECMO also affect susceptibility to thrombosis or bleeding.Especially in patients requiring ECMO, bleeding complications are the most common adverse event [6].Nevertheless, activation of coagulation is observed in COVID-19 [7,8].A possible explanation for the increased activation of blood clotting could be due to immature platelets.These are newly formed by megakaryocytes and released from the bone marrow into the bloodstream.Immature platelets occur when platelets are consumed, and are a measure of increased thrombopoiesis.These immature cells are characterized by their comparatively larger size, high level of alpha and dense granules, and RNA content [9].The latter also leads to the name reticulated platelets.Compared with mature platelets, reticulated platelets are hyperreactive and possess prothrombotic activity [10].Immature platelets can be determined by flow cytometric methods.Some hematology analyzers, for example the Sysmex family, can determine immature platelet fraction (IPF), which refers to the proportion of immature platelets in the total platelet count.For this purpose, a fluorescent dye (phenoxazin) is used that can diffuse through the perforated membrane of the fixed platelets and labels the RNA.This staining is specific to platelets and does not interfere with, e.g.fragmented erythrocytes [11].Since immature platelets contain more RNA than mature platelets, the fluorescence signal is inversely proportional to the maturity of the platelets and therefore allows quantification of the immature platelet fraction [12,13].The IPF is approximately up to 6% in healthy individuals [14,15].In the context of sepsis, which includes the most severe COVID-19 cases, increased IPF has already been associated with increased mortality [16].In addition, IPF has been described as a possible predictor of bacteremia [17].Increased IPF has also been found in COVID-19 patients, which has been in part linked to disease severity [18][19][20][21].In this study, the course of IPF was determined and compared in patients with severe, moderate or mild disease.Categorization focused on the patients' oxygenation needs, particularly the necessity for ECMO.This study will help to understand IPF as a parameter in a severe COVID-19 course and provide an indication of its association with a fatal outcome.
The study was approved by the ethics committee of the Heart and Diabetes Center North Rhine-Westphalia in Bad Oeynhausen (Reg.No. 2019-556) and was performed with the informed consent of the patient or, in case of unconsciousness of the patient, of the ward physician if additional blood was needed outside of routine diagnostics.

Results
Platelet count and IPF of COVID-19 patients were determined daily.Measured values influenced by transfusion of platelet concentrates (within 3 days of transfusion) were excluded.A period of 40 days after hospitalization or intubation was considered because the density of readings was highest during this period (≥15 readings per day).From day 41, the number of measurements was below the number of measurements on day 0 (<15 readings per day).In addition, all patients with readings less than 10% (<4 readings) of the included days were excluded.The 77 remaining patients were divided into different groups according to the oxygenation measures required.Patients in the "mild" group were hospitalized but not invasively ventilated.The "moderate" group was invasively ventilated but did not require ECMO.The severe group was treated with ECMO.44 patients with a severe COVID-19 course (intubation and ECMO), 22 patients with a less severe course (intubation, no ECMO) and 15 patients with a mild COVID-19 course (no intubation, no ECMO) were finally included in this study.In addition, patients were also grouped according to mortality (Figure 1).The patients' characteristics, as well as their clinical and laboratory data are summarized in Table I.About 75% of the patients were male, with a median age of 61 years.The ratio was similar within the different groups.The mortality rate increased with the severity of the disease.In total, 28 patients died, including 52% of the patients from the severe group.This group also received the most platelet concentrates.88.3% of all patients were anticoagulated with the direct thrombin inhibitor argatroban.The remaining patients received heparin or one patient did not receive anticoagulation.Argatroban has no effect on platelet function, especially light transmission aggregometry [22].In addition, 18.2% of patients received an antiplatelet drug predominantly acetylsalicylic acid.This limits platelet function in light transmission aggregometry with the agonists arachidonic acid and ADP and to a lower extend with collagen.However, this has no effect on platelet count [23,24].The collective we used for the analysis of platelet function by light transmission aggregometry includes one patient in each of the two COVID-19 groups (+ and -ECMO) who was also treated with an antiplatelet drug.An integration of all measured values of a patient (median 16 values per patient) showed a highly significant decreased platelet count (median 111.5 • 10 6 /mL) for the group of severe cases compared with the mild (median 203.5 • 10 6 /mL, p < .0001)and moderate (median 208.0 • 10 6 /mL, p < .0001)courses.The median IPF increased with disease severity, showing a sharp tendency toward elevated IPF in the severe group (median 10.9%), whereas those with less severe disease had median normative IPF (median mild: 5.8%, moderate: 5.6%) (Figure 2A).Comparison of platelet count and IPF between deceased and surviving patients showed a highly significant lower platelet count (median 97.3 • 10 6 /mL, p < .0001)and highly significant increased IPF (median 12.2%, p = .0003)in deceased patients (Figure 2B).
In a small additional collective, the platelet function of patients with a severe course was also investigated.In addition to healthy controls, ECMO patients without COVID-19 were also studied.Light transmission aggregometry showed a decreased platelet function, which was on the level of patients with ECMO or even below.In particular, no aggregation could be detected with the inductor PAF C-16 and TRAP (Figure 3A, Table S1).The latter was also confirmed by flow cytometry analysis (Figure 3B and Figures S1 and S2).Furthermore, only slight differences in the presence of the analyzed platelet surface proteins (CD36, CD42a, CD42b, CD29, CD61, GPVI) could be detected (Figure S3).
Patients' D-dimers were evaluated over the same period as IPF.Here, as well, the median of all measured values was calculated (median 23 values per patient).The group of mildly ill patients had the lowest D-dimer levels (median 2.15 µg/mL).

Discussion
Platelets, in addition to their classic role in hemostasis, also play a crucial role in the immune response.Due to their presence in the bloodstream, various surface receptors and granular contents, platelets are able to interact with immune cells and pathogens and contribute to host defense.Especially in the context of sepsis, platelets are of essential importance [25].Platelets are also considered to have a fundamental role in COVID-19.The disease progresses in different phases.Severe cases of COVID-19 reach the prothrombotic phase, which is characterized by abnormal coagulation.One of the main features of this phase is thrombocytopenia, which is also associated with mortality [26].The platelets of these patients exhibit a hyperreactive, procoagulant phenotype, which is typical of young, immature platelets [10].Several studies show that IPF can be interpreted predictively for mortality in sepsis [16].
In our study, we determined the IPF of 77 hospitalized patients on a daily basis.These patients were grouped according to their need for oxygenation.We found that the most severely ill group (intubation and ECMO) had the highest percentage of immature platelets and suffered from thrombocytopenia.This suggests that thrombopoiesis is upregulated in severely ill patients.Furthermore, the platelet count and IPF in the deceased patients were significantly lower and higher, respectively, than in the surviving patients.Several groups have suggested IPF as a predictor of COVID-19 severity and have emphasized different Subsequently, all values between day 0 and 40 after intubation or hospital admission were considered.All patients who yielded <4 readings during this period were excluded.The 77 included patients were grouped according to their need for oxygenation, i.e. mild: neither intubation nor ECMO; moderate: intubation only, no ECMO; severe: intubation and ECMO.In addition, the 77 patients were grouped according to their outcome.N: number of measurements.ECMO: extracorporeal membrane oxygenation, IPF: immature platelet fraction, PC: platelet concentrate.Immature platelets in COVID-19 3 aspects when categorizing patients [19,21,27].Incir et al. [27] and Welder et al. [21] divided the patients studied into two groups.Incir et al. [27] subdivided based on the severity of pneumonia.Welder et al. [21] differentiated patients based on ICU admission.Cohen et al. [19] divided patients into three groups (mild, moderate, and severe disease) based on COVID-19 treatment guidelines.Our study focuses on the need for ECMO treatment and classifies patients based on oxygenation measures, as described above.This makes our study unique, but at the same time makes analysis more difficult, because platelets are affected by ECMO [28].This is also evident when looking at platelet function, which was studied in a small equivalent cohort.
D-dimers were discussed as another parameter of coagulation being a marker of disease outcome in COVID-19 [4].A significant increase in D-dimer levels was also observed in the group with the most severe disease in our study.The deceased patients exhibited significantly increased D-dimer levels as well.However, we did not find a direct correlation of D-dimer level and IPF.
In the present study, platelet function showed significant impairment.This is contrary to the results of other groups [29,30] which found that platelets of COVID-19 patients show a hyperreactive phenotype.In part, these impairments could be due to ECMO treatment [28], but also the patients of the moderate group (intubation, no ECMO) showed functional limitations, especially in light transmission aggregometry with the agonists TRAP and PAF, and accordingly also in the flow cytometric analysis of activation markers upon activation with TRAP.Further investigation is needed to determine if this is a specific functional impairment due to COVID-19.
Our study has a few limitations.First, 57% of the included patients were treated with ECMO.This treatment modality affects platelet count in terms of reduction.In a meta-analysis, Jiritano et al. [28] found that the prevalence of thrombocytopenia with venovenous-ECMO, which is typically used for COVID-19, was 25.4%, and there was a correlation between the duration of ECMO and thrombocytopenia.In addition, the platelet function of these patients is also impaired.Second, most platelet concentrates were transfused in the group with severe courses and, therefore, a high number of measurements from this group had to be excluded since these samples do not represent the patient platelets.
In conclusion, this study shows that in severely ill patients suffering from COVID-19, the platelet count is lower and IPF is higher than in less severely ill patients.In addition, platelet function is also impaired in these patients.Interestingly, the deceased patients show thrombocytopenia, moreover the IPF is significantly above normal.These values could be indicative of the outcome of severe disease.However, further studies are needed to evaluate these parameters for the quality of their predictive function.

Figure 1 .
Figure 1.Procedure for selecting and clustering patients.87 patients admitted to Heart and Diabetes Center North Rhine-Westphalia with COVID-19 between December 2020 and July 2021 were enrolled.Blood counts and IPF were determined daily.Measured values influenced by PC transfusion (within 3 days of transfusion) were excluded.Subsequently, all values between day 0 and 40 after intubation or hospital admission were considered.All patients who yielded <4 readings during this period were excluded.The 77 included patients were grouped according to their need for oxygenation, i.e. mild: neither intubation nor ECMO; moderate: intubation only, no ECMO; severe: intubation and ECMO.In addition, the 77 patients were grouped according to their outcome.N: number of measurements.ECMO: extracorporeal membrane oxygenation, IPF: immature platelet fraction, PC: platelet concentrate.

Figure 2 .
Figure 2. Platelet count and IPF were aggregated from day 0-40, and medians were compared between the different groups' (A) oxygenation requirements, (B) outcomes.A: Platelet count is significantly lower in the severe group than in the mild and moderate group.IPF is increased in severely ill patients.Deceased patients are indicated in red.One-way ANOVA, Tukey post-hoc test.B: Comparison between survivors and decedents shows a highly significant increase in IPF or decrease in platelet count.Unpaired t-test.IPF: immature platelet fraction.

Table I .
Patients' baseline characteristics.BMI: body mass index, ICU: intensive care unit.