For the first time, the influence of COVID-19 on blood microrheology was studied. For this, the method of filtering erythrocytes through filters with pores of 3.5 μm was used. Filterability was shown to significantly decrease with the increasing severity of the patient’s condition, as well as with a decrease in the ratio of hemoglobin oxygen saturation to the oxygen fraction in the inhaled air (SpO₂/FiO₂). The filterability of ≤ 0.65, or its fast decrease during treatment, were indicators of a poor prognosis. Filterability increased significantly with an increase in erythrocyte count, hematocrit and blood concentrations of hemoglobin, albumin, and total protein. The effect of these parameters on the erythrocyte filterability is directly opposite to their effect on blood macrorheology, where they all increase blood viscosity, worsening the erythrocyte deformability. The erythrocyte filterability decreased with increasing oxygen supply rate, especially in patients on mechanical ventilation, apparently not due to the oxygen supplied, but to the deterioration of the patients’ condition. Filterability significantly correlates with the C-reactive protein, which indicates that inflammation affects the blood microrheology in the capillaries. Thus, the filterability of erythrocytes is a good tool for studying the severity of the patient’s condition and his prognosis in COVID-19.

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.

The review is devoted to one of the main regulatory enzymes of glycolysis in erythrocytes – pyruvate kinase, a deficiency of which is often the cause of hereditary nonspherocytic hemolytic anemia. The article presents data on the structure and function of pyruvate kinase and the currently known mutations of coding this enzyme gene. Authors analyzed associations between various genetic types and impaired enzyme function and the severity of the hemoly sis.

Introduction. A common cause of hereditary hemolytic anemia is mutations of erythrocyte membrane proteins, which lead to a reduced ability of red blood cells (RBCs) to deform and pass through narrow capillaries and interendothelial slits of the spleen. This shortens the lifespan of erythrocytes and may result in hemolytic anemia. Therefore, the ability of RBCs to pass through narrow capillaries may serve as a good prognostic marker of a shortened RBC lifespan due to premature RBC hemolysis. The measurement of RBC filterability through artificial filters with 3–5 μm pore size (which is slightly smaller than the diameter of RBCs) might be the most suitable diagnostic method. However, the use of this method is hampered by poor reproducibility of its previously described variants. A recently proposed modified method for measuring RBC filterability using the IDA-01 device has demonstrated good sensitivity and specificity in the diagnosis of hereditary spherocytosis. However, its analytical performance (accuracy and specificity) has not yet been studied. Aim: to evaluate the analytical performance of the erythrocyte filterability measuring method using the IDA-01 filtrometer. Materials and methods. In this study, we used RBCs from healthy donors isolated from venous blood and washed using a standard procedure. The following parameters were analyzed: repeatability of the results (mean relative error and coefficient of variation) when measuring buffer flow time through the same filter (n = 10); reproducibility of the donor erythrocyte filterability measurements in two independent series of experiments conducted at different sites on different days (n = 15 and n = 31); and the reference range for normal filterability. The biological sensitivity of the method was also measured using suspensions containing varying amounts of non-filterable cells (RBCs exposed to 0.08% glutaraldehyde). All statistical calculations were performed using OriginPro 8.1 (OriginLab Corp., USA). Results and discussion. The IDA-01 device demonstrated good repeatability of measurements (relative error 2.79%, coefficient of variation 3.5%); the detection limit was 0.2 s; the reproducibility of erythrocyte filterability measurements in the two experimental series did not differ significantly (Fisher's test); the mean filterability values in these series also did not differ significantly (modified Student's t-test). The method is not biased. The reference range of filterability for donors was 0.81–0.87 (mean ± 1 standard deviation). The detection limit for pathologically altered cells was 0.003%. The accuracy of the results depends only on correct sample preparation (the degree of leukocyte removal and a constant hematocrit level in erythrocyte suspension). When all necessary conditions are met, the device specificity approaches 100%. Conclusion. The erythrocyte filterability test performed using the IDA-01 device has high sensitivity and specificity. It is very simple, does not require expensive equipment, and can be easily performed in any laboratory, making it a promising method for the diagnosis of hereditary spherocytosis.

The differential diagnosis of hereditary spherocytosis is a great challenge because of the similar clinical and laboratory signs it shares with other hereditary hemolytic anemias as well as due to the limited availability of molecular genetic testing. The development of easy-to-perform laboratory tests for the differential diagnosis of hereditary hemolytic anemias remains as relevant as ever. Here, a method of measuring red blood cell filterability for the diagnosis of hereditary spherocytosis is proposed for the first time. The aim of our study was to compare red blood cell filterability measurement with other diagnostic tests for hereditary spherocytosis as well as to assess its specificity and sensitivity. We included 30 patients (18 girls and 12 boys, with the median age of 8.6 years) with hereditary spherocytosis and 15 patients (9 girls and 6 boys, with the median age of 10 years) with other hereditary hemolytic anemias (pyruvate kinase deficiency (n = 14) and stomatocytosis (n = 1)). The diagnostic work-up for hereditary hemolytic anemia included a complete blood count test using an automated hematology analyzer, an osmotic resistance analysis before and after 24 hours of incubation at 37°С, erythrocytometry with sphericity index calculation, EMA test (eosin-5-maleimide binding assay) and red blood cell filterability measurement using artificial filters with cylindrical pores 3-5 μm in diameter. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The parents of all of the enrolled children signed a voluntary informed consent form for peripheral blood collection and diagnostic testing. In all the cases of hereditary spherocytosis diagnosed in accordance with the relevant clinical recommendations, red blood cell filterability was very low (0–0.31 units). It was higher only in 3 instances, reaching 0.47, 0.64 and 0.82 units, but in two of these cases there were no genetic data available, and the remaining patient was found to harbor the SPTA1 c.433999CT mutation which was characterized both as spherocytosis and elliptocytosis. Red blood cell filterability in the group of the patients with other hemolytic anemias equalled 0.55 to 0.86 units (with the median of 0.77 units). The sensitivity of the RBC filterability measurement method in diagnosing hereditary spherocytosis was 93% (with 100% specificity), while the EMA test had a sensitivity of 89% and specificity of 95%. Our comparative study showed that red blood cell filterability measurement and the EMA test have similar sensitivity and specificity in diagnosing hereditary spherocytosis but the former is much cheaper and easier to perform since it does not require expensive equipment and can be carried out at any laboratory.

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.