Summary Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real‐time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin β gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt‐4G>A, 1502insA and IVS20nt‐2A>G.

Background: Osmotic fragility test (OFT) is widely considered as a sensitive indicator of red blood cells' sensitivity to the hypotonic solution. It is often used as a screening test for the diagnosis of hereditary spherocytosis (HS). Nowadays, the osmotic fragility test based on flow cytometric analysis (FCM OF) is widely used in laboratory practice. The purpose of this study was to optimize the assay sensitivity and to validate its clinical application in the diagnostic screening of childhood anemias.Methods: The study was conducted on 175 children suffering from various types of anemia (including 30 children with proven hereditary spherocytosis, HS) and 16 healthy subjects. All children were aged between 3 months and 17 years, including 94 boys and 97 girls. FCM OF was performed on every subject according to two different analysis time patterns (hemolysis was analyzed for 214 or 300 s) using Cytomics FC500 flow cytometer.Results: Significant higher sensitivity was demonstrated by the tests carried out according to the longer analysis time pattern (90.0 vs. 83.33%). The level of specificity of both the analysis patterns was similar. When an extended analysis time was used, the percentage of red cell survival levels in HS patients were significantly lowered compared to the same cases analyzed with shorter incubation times and all other non-HS anemic cases (9.31 6 4.69 vs. 35.59 6 15.30%, P < 0.05). During the shorter analysis time, the values obtained were 13.76 6 7.92% for HS and 48.18 6 19.04% for non-HS, P < 0.0001. The 300-s test is very useful in distinguishing thalassemia patients from patients with other types of anemias (94.74% sensitivity and 90.12% specificity) and provided the values of remaining red blood cells as 70.46 6 12.29% for thalassemia and 27.16 6 13.01% for nonthalassemia subjects, P < 0.0001. Conclusion: Flow cytometric osmotic fragility test with a longer (300-s) analysis time demonstrated an increased sensitivity in detecting HS in anemic children. V C 2017 International Clinical Cytometry Society

Summary. Congenital dyserythropoietic anaemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and multinuclearity of erythroblasts. Three main types of the disease have been described. Glycoconjugate abnormalities in erythrocyte membrane glycoconjugates, consisting of hypoglycosylation of band 3 and accumulation of certain glycosphingolipids including lactotriaosylceramide, neolactotriaosylceramide and polyglycosylceramides, have been described only in patients with CDA type II (CDA-II). We report on identical, although less pronounced, abnormalities in erythrocyte glycoconjugates from a patient with CDA-I. A low degree of hypoglycosylation of band 3 in our patient with CDA-I suggests that hypoglycosylation is not a cause, but, most probably, a consequence of dyserythropoiesis.

Innovations in laboratory equipment have enabled a widening of the spectrum of hematological parameters obtained from single measurements of peripheral blood samples, including reticulocyte parameters. The usefulness of reticulocytes indices to confirm the diagnosis of pediatric anemia was analyzed in this study. The study group consisted of 163 children, aged 1 month-17 years, with anemia. Complete blood count extended with an analysis of reticulocyte parameters were measured using a Beckman Coulter LH 750. The mean sphered corpuscular volume (MSCV) in the group of children with hereditary spherocytosis (HS) was 66.71 ± 8.45 fL, whereas in other anemic patients MSCV was 87.76 ± 11.22 fL, < 0.0001. In HS children the average mean corpuscular volume of red blood cells was higher than the MSCV value, while an inverse correlation was observed in the group of children with other anemias, < 0.0001. A significant difference was found between the ratio of absolute reticulocyte count and IRF fraction (Ret#/IRF)-0.6 ± 0.28 in the HS group and 0.23 ± 0.16 in the non-HS group, respectively. Our results suggest that analysis of reticulocyte parameters is useful in the diagnosis of anemia and should be included in the routine CBC analysis in anemic children.

Eosin-5′-maleimide (EMA) binding test is a flow cytometric test used to detect hereditary spherocytosis (HS). To perform the test sample from patients, 5–6 reference samples of red blood are needed. Our aim was to investigate how the mean corpuscular volume (MCV) of red blood cells influences on the value of fluorescence of bounded EMA dye and how the choice of reference samples affects the test result. EMA test was performed in peripheral blood from 404 individuals, including 31 children suffering from HS. Mean fluorescence channel of EMA-RBCs was measured with Cytomics FC500 flow cytometer. Mean corpuscular volume of RBCs was assessed with LH750 Beckman Coulter. Statistical analysis was performed using Graph Pad Prism. The correlation Spearman coefficient between mean channel of fluorescence of EMA-RBCs and MCV was r = 0.39, p < 0.0001. Interpretation of EMA test depends on MCV of the reference samples. If reference blood samples have lower MCV than the patients MCV, EMA test result might be negative. Due to different MCV values of RBCs in infancy and ca. Three months later, EMA test in neonates might be interpreted falsely negative. Samples from children younger than 3 months old had EMA test result 86.1 ± 11.7 %, whereas same samples that analyzed 4.1 ± 2.1 later had results of 75.4 ± 4.5 %, p < 0.05. Mean fluorescence of EMA-bound RBC depends on RBC’s volume. MCV of reference samples affects EMA test results; thus, we recommend selection of reference samples with MCV in range of ±2 fL compared to MCV of patient RBC’s.