Combining microfluidics with machine learning algorithms for RBC classification in rare hereditary hemolytic anemia.

Combining microfluidics technology with machine learning represents an innovative approach to conduct massive quantitative cell behavior study and implement smart decision-making systems in support of clinical diagnostics. The spleen plays a key-role in rare hereditary hemolytic anemia (RHHA), being the organ responsible for the premature removal of defective red blood cells (RBCs). The goal is to adapt the physiological spleen filtering strategy for in vitro study and monitoring of blood diseases through RBCs shape analysis. Then, a microfluidic device mimicking the slits of the spleen red pulp area and video data analysis are combined for the characterization of RBCs in RHHA. This microfluidic unit is designed to evaluate RBC deformability by maintaining them fixed in planar orientation, allowing the visual inspection of RBC's capacity to restore their original shape after crossing microconstrictions. Then, two cooperative learning approaches are used for the analysis: the majority voting scheme, in which the most voted label for all the cell images is the class assigned to the entire video; and the maximum sum of scores to decide the maximally scored class to assign. The proposed platform shows the capability to discriminate healthy controls and patients with an average efficiency of 91%, but also to distinguish between RHHA subtypes, with an efficiency of 82%.

Differential diagnosis of hereditary hemolytic anemias in a single multiscreening test by TGA/chemometrics.

A multi-screening test based on the coupling of thermogravimetry and chemometrics was optimized for the differential diagnosis of hereditary hemolytic anemias. The novel test is able to simultaneously perform a simple and fast diagnosis of sickle cell anemia, thalassemia, hereditary spherocytosis and hereditary elliptocytosis in a single analysis of a few microliters of non-pretreated whole blood. The thermogravimetric profile of blood from patients affected by such disorders was found to be characteristic of a specific anemic status or a disorder due to membrane defects. In addition, chemometric tools were used to validate a model of prediction to process the thermogravimetric curves and to obtain in 1 hour an accurate diagnosis. The effectiveness of the novel test was evaluated by comparing results with the confirmatory analyses specific for each disorder. The TGA/chemometric test made it possible to perform a first level test of congenital erythrocyte defects, including the hemoglobinopathies and disorders due to membrane defects with the same accuracy of confirmatory analyses obtained by molecular investigation. In addition, the novel test was used for the diagnosis of a number of Italian difficult cases, including neonatal patients for which the conventional screening tests did not manage to obtain a diagnosis confirming the high prediction ability of the single multiscreening test.

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1.

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

Hereditary stomatocytosis and cation-leaky red cells--recent developments.

The hereditary stomatocytoses (HSt) are a diverse group of conditions. Common features include hemolytic anemia, a red cell cation leak and morphological changes, but the severity of the condition can vary enormously. We have previously shown that one form of HSt (cryohydrocytosis), where the monovalent cation leak is increased at low temperature, results from amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1). These substitutions appear to convert band 3 from an anion exchanger into a cation channel. More recently we found that over-hydrated hereditary stomatocytosis (OHSt) results from amino acid substitutions in Rh-associated glycoprotein (RhAG), a putative gas channel protein. Both band 3 and RhAG associate in the red cell membrane to form a macrocomplex that is thought to be involved in red cell gas exchange. In this paper I will review the data that has been published so far on the molecular basis of HSt. I will mention other similar conditions that cause either a cation leak or stomatocytosis or both, and consider the mechanisms of red cell shape change and permeability.

Heterogeneity of the epsilon gamma delta beta-thalassaemias: characterization of three novel English deletions.

We have characterized three novel epsilon gamma delta beta-thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire beta-globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98-kb deletion extended 90-kb upstream of the epsilon gene to 8 kb upstream of the G gamma-gene, leaving the gamma,delta and beta-genes intact. The 439 kb deletion is the largest deletion reported so far to cause epsilon gamma delta beta-thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.

MRI evaluation of cranial bone marrow signal intensity and thickness in chronic anemia.

BACKGROUND AND PURPOSE: The aim is to assess the magnetic resonance imaging (MRI) findings for cranial bone marrow (CBM) signal intensity and thickness in patients with chronic anemia and compared these with findings in healthy subjects. We also investigated the relationships between CBM changes and age, type of anemia (hemolytic versus non-hemolytic), and severity of anemia. METHODS: We quantitatively evaluated CBM signal intensity and thickness on images from 40 patients with chronic anemia (20 with congenital hemolytic anemia (HA) and 20 with acquired anemia) and compared these to findings in 28 healthy subjects. The intensity of CBM relative to scalp, white matter (WM), gray matter (GM), and muscle intensity was also investigated in patients and subjects in the control group. The sensitivity and specificity of CBM hypointense to GM and CBM hypointense to WM as markers of anemia were evaluated. Relationships between age and CBM thickness/intensity, and between anemia severity (hemoglobin (Hb) level) and CBM thickness/intensity were evaluated. RESULTS: Cranial bone marrow signal intensity was lower in the chronic anemia patients than in the controls (P<0.001). In the control group, CBM intensity was higher than GM intensity, whereas the opposite was true in the patient group. The finding of CBM hypointense to GM was 85% sensitive and 67% specific as a marker of anemia. The corresponding statistics for CBM hypointense to WM were 90 and 46%. The patients had thicker CBM than the controls (temporal, P<0.05; parietal, P<0.005). The subgroup with hemolytic anemia had thicker parietal CBM than the subgroup with non-hemolytic anemia (NHA) (P<0.05) and exhibited thicker temporal and parietal CBM than the controls (temporal, P<0.05; parietal, P<0.001). The CBM thicknesses in the non-hemolytic anemia subgroup were similar to control values (P>0.05 for both). There were no correlations between age and CBM intensity or thickness, or between anemia severity and CBM intensity or thickness. CONCLUSION: Patients with chronic anemia exhibit lower CBM signal intensity on MRI than healthy subjects. Patients with hemolytic anemia have thicker CBM than patients with non-hemolytic anemia or healthy individuals. Decreased CBM intensity may indicate that the patient has anemia, and increased CBM thickness may specifically point to hemolytic anemia. These MRI findings may signal the need for further evaluation for the clinician.

The hereditary stomatocytoses and allied disorders: congenital disorders of erythrocyte membrane permeability to Na and K.

The hereditary stomatocytoses and allied disorders are a set of dominantly inherited haemolytic anaemias in which the plasma membrane of the red cell 'leaks' sodium and potassium. There are about 10 different forms of these conditions, ranging from a moderately severe haemolytic anaemia to minor conditions in which the haematology is essentially normal, but where the patients present with pseudohyperkalaemia, due to leakage of K from the red cells on cooling to room temperature. Frequently misdiagnosed as atypical hereditary spherocytosis, these conditions can show marked thrombotic complications after splenectomy, which should be avoided. Laboratory studies of these conditions have drawn attention to a 32 kDa membrane protein, stomatin, which seems to act as a regulator of Na and K transport in human and animal tissues generally, but mutations in this gene do not cause these diseases. Genetic mapping in some kindreds, but not all, points to a mutation locus on chromosome 16.

Novel mutations and structural implications in R-type pyruvate kinase-deficient patients from Southern Italy.

Deficiency of the R-type pyruvate kinase (R-PK) causes an autosomal recessive, hereditary, nonspherocytic hemolytic anemia (HNSHA). We screened seven unrelated patients from the south of Italy for the known mutations and found one patient homozygous for the 1529A (R510Q) mutation, two others bearing the 1456T (R486W) mutation, one homozygous and another heterozygous, and two heterozygotes for the 994A mutation (G332S). We also found three novel mutations at the heterozygote status: a G to C transversion in position 1010 (1010C; R337P) and a C to T transition in position 1492 (1492T; R498C), which are missense, and a T to G transversion in position 1523 (1523G; L508Z), which produces a stop codon with a subsequent loss of the C-terminal protein domain. The structural features of R-PK in the mutation-bearing regions were examined. In all cases the mutations altered the local conformation of the enzyme. Both G332S and R337P are in highly conserved sequence regions. In particular, the R337P mutation significantly affects the intersubunit interactions, because it is located in a region subjected to a large conformational change that occurs during the R-->T allosteric transition, which is essential for the enzyme activity. The R486W mutation affects an external pocketlike region, producing only a local conformational change; the R498C mutation changes the interactions among neighbouring residues; the R510Q mutation involves the loss of interdomain interactions that may reduce enzyme stability and activity. Our data also indicate that in patients from Southern Italy, pyruvate kinase deficiency is heterogeneous, the 1529A mutation, which is the most frequent mutation in the U.S. Caucasian population, having a lower frequency.

[Atypical congenital dyserythropoietic anemia]. / Atypische kongenitale dyserythropoietische Anämie.

The case is presented of a 26-year-old male patient suffering from chronic anemia since infancy. At age 12 (1972), congenital dyserythropoietic anemia (CDA) was diagnosed which could not be grouped with one of the three known variants of CDA. From onset the disease was complicated by thrombocytopenia with severe hemorrhagic diathesis. After splenectomy (1972) an extremely enlarged accessory spleen was removed in 1985 disclosing, histologically, excessively developed extramedullary hematopoiesis. Since bone marrow biopsy showed massive hyperplasia, the cellular phase of a myeloproliferative syndrome could not be excluded by histology alone.--Only a few cases of CDA with involvement of granulocytes and megakaryocytes have been reported so far. It is not clear whether or not they should be classed with CDA. In any event, involvement of granulopoiesis and/or thrombopoiesis indicates a genetic lesion of very early precursors, perhaps stem cells. It is proposed that such variants would be more correctly designated "congenital dyshematopoietic syndromes".

Variants of congenital dyserythropoietic anaemia: an update.

During the past decade a large number of variant congenital dyserythropoietic anaemias (CDA) have been described. The morphological, genetical, clinical and serological features of the classical types (I, II and III) are not well delineated, so, there are no exact borders between classical and variant forms. In the present review attention will be given to variant serological features, variant globin chain synthesis, variant genetical and morphological features and variant clinical presentations. Finally, it is emphasized that the existence of transitional forms between dysplastic and hypoplastic affections of bone marrow function points to the stem cell origin of these disorders.

Characteristics of peripheral blood monocytes in hereditary xerocytosis and spherocytosis.

Peripheral blood monocytes isolated from patients with congenital hemolytic anemia, hereditary xerocytosis and spherocytosis, demonstrated in vivo engulfment of red cell and platelet fragments. In addition, morphometric studies performed on these monocytes showed an increase in cytoplasmic/nuclear ratio as well as lysosome and phagosome volumes. The production of carbon dioxide from glucose-1-14C in abnormal monocytes was increased (15-80%) but the intracellular values of beta-glucuronidase and esterase activity were similar to control monocytes. Monocyte locomotion assessed in the presence of chemotactic stimuli was found significantly increased (73 +/- 12 monocytes/oil immersion fields vs. 46 +/- 5 for control monocytes). We concluded that the monocytes in hemolytic anemias associated with increased in vitro red cell fragmentation have some features resembling the 'stimulated' monocytes and that this alteration may be due to red blood cell fragment ingestion.