Introduction: Severely ill COVID-19 patients receiving prophylactic-dose anticoagulation exhibit high rates of thrombosis and mortality. The escalation of anticoagulation also does not reduce mortality and has an uncertain impact on thrombosis rates. The reasons why escalated doses fail to outperform prophylactic doses in reducing risks of thrombosis and death in severely ill COVID-19 patients remain unclear. We hypothesized that escalated anticoagulation would not effectively prevent hypercoagulability and, consequently, would not reduce the risk of thrombosis and death in some severely ill patients. Methods: We conducted a prospective multicenter study that enrolled 3860 COVID-19 patients, including 1654 severely ill. They received different doses of low-molecular-weight or unfractionated heparin, and their blood coagulation was monitored with activated partial thromboplastin time, D-dimer, and Thrombodynamics. A primary outcome was hypercoagulability detected by Thrombodynamics. Blood samples were collected at the trough level of anticoagulation. Results: We found that escalated anticoagulation did not prevent hypercoagulability in 28.3% of severely ill patients at the trough level of the pharmacological activity. Severely ill patients with such hypercoagulability had higher levels of inflammation markers and better creatinine clearance compared to severely ill patients without it. Hypercoagulability detected by Thrombodynamics was associated with a 1.68-fold higher hazard rate for death and a 3.19-fold higher hazard rate for thrombosis. Elevated D-dimer levels were also associated with higher hazard rates for thrombosis and death, while shortened APTTs were not. The simultaneous use of Thrombodynamics and D-dimer data enhanced the accuracy for predicting thrombotic events and fatal outcomes in severely ill patients. Conclusions: Thrombodynamics reliably detects hypercoagulability in COVID-19 patients and can be used in conjunction with D-dimer to assess the risk of thrombosis and death in severely ill patients. The pharmacological effect of LMWH at the trough level might be too low to prevent thrombosis in some severely ill patients with severe inflammation and better creatinine clearance, even if escalated doses are used.

Diagnosis of pyruvate kinase deficiency (PKD) remains challenging in clinical practice. The pyruvate kinase (PK) to hexokinase (HK) activity ratio (PK/HK) was proposed to reduce the confounding effect of reticulocytosis on PK activity measurement. However, decreased PK activity and PK/HK ratios have also been observed in other anemias, raising doubts about their diagnostic value. We assessed the diagnostic accuracy of PK/HK ratio versus PK activity in differentiating PKD from other hereditary anemias. This study included 41 patients with molecularly confirmed PKD and 62 patients with other anemias. We also evaluated the influence of reticulocytosis and transfusions on erythrocyte PK activity. The PK/HK ratio showed 73% specificity, while PK activity alone achieved 95%. In PKD patients, reticulocytosis did not affect PK activity because reticulocyte PK activity was already markedly reduced (23-fold) compared with controls. In other anemias, decreases in PK activity were present in both reticulocytes and erythrocytes, but to a lesser extent. Transfusions contribute more to the false-normal result of PK activity than reticulocytosis. Measuring reticulocyte-specific PK activity during regular transfusions provided reliable results, as only patient-derived reticulocytes are present in the blood. PK activity demonstrates higher specificity than PK/HK ratio in diagnosing PKD. Reticulocytosis is not a confounder, while transfusions remain the main limitation. Reticulocyte-specific PK activity measurement may improve diagnostic accuracy in transfused patients.

Diagnosis of pyruvate kinase (PK) deficiency remains challenging in clinical practice. To avoid the influence of reticulocytosis when measuring PK activity, was proposed to measure PK:hexokinase (HK) enzyme activity ratio. However, decreased PK activity and PK:HK ratio were observed in patients with other anemias, so use of this ratio in differential diagnostics remains questionable. Here, we assessed the specificity and sensitivity of this ratio in the differential diagnosis of PK deficiency (PKD) and other anemias. Under these conditions, the specificity of the ratio was 73% while the specificity of PK activity was 95%. In a cohort of PKD (n=41) and other anemias (n=62) patients with molecularly confirmed anemias, we demonstrated the absence of PK activity dependence on reticulocytosis in PKD patients and showed the reason for this absence: PK activity is already significantly (23-fold) reduced in reticulocytes in these patients. A decrease in PK activity in both reticulocytes and erythrocytes was also found in patients with other anemias, but to a much lesser extent. Transfusions contribute more to the false-normal result of PK activity than reticulocytosis and we suggested measuring specific PK activity in reticulocytes during regular transfusions, since after the transfusion the patient's blood contains only his own reticulocytes.

Hereditary hemolytic anemias (HHAs) have quite similar clinical manifestations, complicating differential diagnosis and determining treatment, such as splenectomy. For the differential diagnosis of hereditary spherocytosis (HS), pyruvate kinase deficiency (PKD), and hereditary stomatocytosis (HSt), we proposed to measure erythrocyte filterability through membrane filters with a pore diameter of 3 or 3.5 μm. Our modified method provides improved reproducibility reducing the variation in results caused by differences in the filters used. The filterability of erythrocytes significantly differs in patients with HS, HSt and PKD (medians and 1.5 IQR were: 0.045 [0; 0.51] (n=88); 0.685 [0.46; 0.79] (n=12) and 0.75 [0.55; 0.86] (n=28) rel. un., respectively). The specificity of the filterability test for HS diagnosis was 100%, comparable to the best available tests, while its sensitivity (85.2%) was somewhat lower than that of some alternative methods. Only the filterability test could identify a second subgroup in HS patients (14.8%) with higher filterability (close to normal), but their HS diagnosis was confirmed by the decreased filterability by 3-µm pore filter. This subgroup requires further detailed study. The proposed filterability measurement method is promising for diagnosing HS. It has high sensitivity and specificity and can be performed quickly without expensive equipment.

Hereditary hemolytic anemias (HHAs) have very similar clinical manifestations, complicating differential diagnosis and determining treatment strategies, such as splenectomy. To differentiate between hereditary spherocytosis (HS), pyruvate kinase deficiency (PKD), and hereditary stomatocytosis (HSt), we proposed measuring erythrocyte filterability using membrane filters with a pore diameter of 3 or 3.5 μm. Our modified method provides improved reproducibility, reducing the scattering in results caused by differences in the filters used. Erythrocytes’ filterability differs significantly between patients with HS, HSt, and PKD (medians and 95% confidence intervals were 0.025 [0.004; 0.100] (n = 86); 0.645 [0.520; 0.730] (n = 10), and 0.760 [0.700; 0.782] (n = 26), respectively). The specificity of the filterability test for the HS diagnosis was 99.1%, comparable with the best available tests, while its sensitivity (85.4%) was slightly lower than that of some alternative methods. Only the filterability test was able to identify a second subgroup of HS patients (14.0%) with higher filterability (close to normal), but the diagnosis of HS in these patients could be confirmed by reduced filterability on a 3-μm filter. This subgroup requires further detailed study. The proposed method for measuring filterability is promising for diagnosing HS. It has high sensitivity and specificity and can be performed quickly without expensive equipment.