Early postnatal diagnosis of hereditary spherocytosis by combining light microscopy, acidified glycerol lysis test and eosin-5'-maleimide binding assay.

Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

[A case of hereditary over-hydrated stomatocytosis with stomatospherocytes and spherocytes in the blood]. / Przypadek diedzicznej uwodnionej stomatocytozy z obecnoscia stomatosferocytów i sferocytów we krwi.

UNLABELLED: A patient of 31 years of age with an atypical overhydrated hereditary stomatocytosis is described. The diagnosis was established on the basis of a markedly increased red cell volume with low MCHC, high osmotic fragility of red cells, but increased binding of eosin-5-maleimide (EMA) to red cells, presence of stomatospherocytes and large spherocytes in blood and a high sodium and low potassium concentration in erythrocytes. A double band 7 was found by SDS-PAGE of the erythrocyte membrane, but even when only one them was taken into account, the level of stomatin was normal. Expression of stomatospherocytes in patient's blood was erratic: in blood films prepared in 2005, both stomatospherocytes and large spherocytes were present but in those from 2008 large erythrocytes of spherocyte morphology predominated. Clinically, the disease symptoms were typical for haemolytic anemia. When heparinized blood of the patient was kept at 0 degrees Celsius for 24 h, the haemolysis of red cells amounted only to 2%. The patient's son, 5 years old, suffers from the same disease. CONCLUSION: In spite of its rarity, hereditary stomatocytosis and allied disorders should be taken into consideration in differential diagnosis of haemolytic anemia including newborns. The diagnosis is supported by finding increased binding of eosin-5-maleimide (EMA) dye to patients' erythrocytes associated with their elevated osmotic fragility. Absence of a significant count of stomatocytes in the blood does not exclude the diagnosis of overyhydrated hereditary stomatocytosis.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

Apparently normal ankyrin content in unsplenectomized hereditary spherocytosis patients with the inactivation of one ankyrin (ANK1) allele.

The study of erythrocyte membrane protein concentration by polyacrylamide gel electrophoresis (PAGE) is the first step in approaching the primary molecular defect in hereditary spherocytosis (HS). Normal or greater than normal protein 2.1 levels were found in ten unrelated HS patients showing the inactivation of one ankyrin allele. Erythrocyte membranes from the same patients, once splenectomized, showed a homogeneous degree of protein 2.1 reduction. Thus protein 2.1 levels could misleadingly appear normal due to the high number of circulating reticulocytes. To calculate the true ankyrin level using PAGE and consequently to avoid mistakes in studying a mutated gene, a simple equation, based on the number of reticulocytes, was developed.