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The ability of red blood cells (RBC) to deform under vascular conditions is essential for circulation. RBC deformability, measured at different shear rates and osmolalities, provides a useful way to evaluate RBC function and is used to diagnose several hereditary blood disorders. In clinical practice, ektacytometry has been used as a routine automated technique for measuring RBC deformability under shear stress at known osmolalities. RBC suspension is exposed to laser light and the resulting diffraction pattern is recorded. The mean deformability of the cells is characterized by the diffraction pattern. Our study is the first to compare the correspondence between two methods that measure diffraction simultaneously, on the same apparatus. Additionally, while others conducted studies under varying shear we used varying osmolalities. A laser beam splitter produced two identical diffraction patterns, evaluated by synchronous data acquisition and analysis: One pattern was acquired by a digital camera and analyzed by image processing software. The other was analyzed using photodiode measurement at four fixed points and a microcontroller interface. Data analysis resulted in two deformability vs. osmolality curves. Comparing these curves shows excellent overlap in shape with a clear difference in amplitude. Since routine Manuscript received Jan 15, 2013; revised March 1, 2013. patient curves are always compared to a normal control curve, the amplitue difference is not significant. Our results indicate that either method may be used for clinically-usable interpretation of RBC deformability, but also that additional studies are required in order to compare repeatability for both methods, and to demonstrate that the two curves overlap for a variety of pathologies.

This study defined the incidence, clinical and haematological characteristics of infantile pyknocytosis in a monocentric retrospective study of 149 blood samples referred for unexplained neonatal haemolytic anaemia. Pyknocytosis was diagnosed in 14 patients (9.4%). All patients had neonatal jaundice and severe anaemia (mean nadir haemoglobin: 6.8 g/dl) at a mean age of 21 d. The percentage of pyknocytes was 4-23%. Packed red blood cell transfusions were needed in 11 of 14 patients. Haemoglobin levels reached normal values within a mean time of 4 months. Infantile pyknocytosis is an unusual cause of neonatal haemolytic anaemia, which requires careful examination of blood smears.

As most of hereditary spherocytosis-affected individuals experience jaundice at birth, it seemed of interest to evaluate the proportion of hereditary spherocytosis in 402 severely jaundiced neonates with a bilirubinemia level prompting phototherapy. Red cell dehydration, a hallmark of spherocytosis whether constitutional or acquired, was demonstrated in 74 of them, among whom 23 disclosed a typical pattern of spherocytosis upon red cell deformability studies. Acquired spherocytosis of immune origin was diagnosed in 19/23 and hereditary spherocytosis in 4, making the proportion of hereditary spherocytosis-affected individuals among a severely jaundiced population of neonates amount to 1%, an incidence at least 30-fold that of the overall population.

Congenital dyserythropoietic anemia type I (CDA I) is a rare disorder of erythropoiesis. The objective of this study was to describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and outcome in CDA I. The 12 patients included belong to the retrospective French Multicenter Study. Clinical and biologic data were compiled. Biologic tests included light and, in some cases, electron microscopy, ektacytometry, and red cell membrane protein electrophoresis. Neonatal manifestations (anemia, early jaundice, and/or splenomegaly) and bone abnormalities were present in 11 of the 12 and 6 of the 12 patients, respectively. CDA I was initially misdiagnosed in four children. By the time of diagnosis, anemia with reticulocytosis lower than expected in a hemolytic anemia was present in all patients. Bone marrow electron microscopy examination revealed characteristic findings in all nine children. Red cell membrane protein 4.1 was reduced in all five children. At least one transfusion was required in 11 of the 12 children. Interferon alpha2 corrected anemia in the three children who received monthly transfusions. CDA I is commonly misdiagnosed in children. It should be sought in patients with unexplained chronic anemia, especially when associated with neonatal manifestations, jaundice, splenomegaly, subnormal or low reticulocytosis, and congenital bone malformations.

This report concerns congenitally Na(+)-K(+) leaky red cells of the 'hereditary stomatocytosis' class. Three new isolated cases and one new pedigree are described, and one previously reported case is expanded. In all cases, Western blotting of red cell membranes revealed a deficiency in the 32 kDa membrane protein, stomatin. All showed pronounced cation leaks at 37 degrees C with markedly abnormal intracellular Na(+) and K(+) concentrations, like all other such stomatin-deficient cases. Consistent with recent findings in two previously described British pedigrees, immunocytochemistry demonstrated that the deficiency of stomatin was not complete. On typical blood films, some red cells showed positive stomatin immunoreactivity, while most were negative, although in one case only a minority were negative. All platelets and neutrophils were stomatin positive. The cases differed markedly between themselves with regard to the temperature dependence of the passive leak to K(+). Three showed a simple monotonic temperature dependence, while two showed a minimum at around 20-25 degrees C, such that the cells were extremely leaky at 0 degrees C, giving the phenotype known as 'cryohydrocytosis'. These patients are the only two known cases of stomatin-deficient cryohydrocytosis. Both showed a congenital syndrome of mental retardation, seizures, cataracts and massive hepatosplenomegaly, probably defining a new haemato-neurological syndrome.

As part of the evaluation of the GEN.S (Coulter), we compared the Mean Corpuscular Volume (MCV) to the Mean Spherized Corpuscular Volume (MSCV) assessed during the reticulocyte count procedure under hypo-osmotic conditions. A sub-group of patients with hereditary spherocytosis (HS) was singled out: in all of them, the MSCV became smaller than the MCV. As the cell volume normally increases in red cells derived from other patients in the same conditions, we decided to further study the reason for this particular behaviour of HS red cells. Whereas normal red cells are able to undergo an osmotic expansion, the spherocytes reach a critical osmotic volume leading to cell fragmentation consistent with the decrease of MSCV. This fortuitous finding is likely to be a reliable improvement for the routine screening of HS.

Dehydrated hereditary stomatocytosis (DHS) is a rare congenital hemolytic anemia. We observed that some patients had presented with different prenatal or perinatal forms of edema in some kindreds. Within weeks or months after birth, these exhibited a spontaneous, complete and definitive resorption. We assumed that some DHS patients, who were born without edema before ultrasound was available, might nonetheless have exhibited this during the prenatal period. The present report follows up the first pregnancy in a woman with overt DHS, but not herself having a known history of perinatal effusions. Ultrasound revealed that the fetus displayed ascites that disappeared prior to birth. The neonate had DHS. Prenatal edema must therefore be more frequent in DHS than known until now. DHS is another cause of prenatal edema to be considered in the differential diagnosis.

Clinical manifestations of hereditary spherocytosis (HS) can be abrogated by splenectomy. However, concerns exist regarding exposure of patients to a lifelong risk for overwhelming infections and, to a lesser extent, to vascular complications after total splenectomy. In the search for alternative treatment modalities, we assessed, in a previous pilot study, the potential usefulness of subtotal splenectomy in a small population of patients. During a mean follow-up period of 3.5 years, subtotal splenectomy was shown to be effective in decreasing the hemolytic rate, while maintaining the phagocytic function of the spleen. In the current study, we evaluated the clinical and biologic features of 40 patients with HS who underwent subtotal splenectomy and were monitored for periods ranging from 1 to 14 years. The beneficial effect of subtotal splenectomy included a sustained decrease in hemolytic rate and a continued maintenance of phagocytic function of the splenic remnant. However, mild-to-moderate hemolysis was persistent and accounted for secondary gallstone formation and aplastic crisis in a small subset of patients. Surprisingly, regrowth of the remnant spleen did not seem to have a major impact on the beneficial outcomes of these individuals. Our results suggest that subtotal splenectomy appears to be a reasonable treatment option for management of patients with HS, especially young children.

Marked variations in the clinical manifestations of hereditary spherocytosis (HS) have long been recognized. However, neither the molecular nor the cellular basis for this variable expression has been fully delineated. To better define the cellular basis for variable expression of the disease, we evaluated the pathobiology of red cells in a large series of 55 non-splenectomized and 31 splenectomized patients with HS. Red cell membrane surface area, surface area-to-volume ratio, cell volume, and state of cell hydration were quantitated. We found that decreased membrane surface area was a distinguishing feature of red cells in all patients studied, whereas decreased surface area-to-volume ratio as reflected by increased osmotic fragility was noted in only 66% of the non-splenectomized patients. In terms of red cell indexes, the percentage of microcytes was not a good discriminator of HS phenotype but was the best indicator of the severity of the disease. In contrast, the presence of increased numbers of hyperdense cells was an effective discriminating feature of the HS phenotype but a poor indicator of disease severity. These findings have enabled us to define the dominant cellular changes that account for the variable clinical severity of this common red cell membrane disorder and have allowed development of improved approaches for its diagnosis.

Hemoglobin J (HbJ), Guantanamo, which had been described but once in the literature, was found in a family originating from Benin; this second case was found to be in association with hemoglobin C (HbC) and alpha-thalassemia. High-performance liquid chromatography (HPLC) procedures and microsequencing were used for characterization of the aminoacid substitution. The main hematological disorder, in relation with the instability of Hb J Guantanamo, seems to be a worsening of the rheological properties of the red blood cells (RBC), as demonstrated by ektacytometric studies. Oxygen-binding properties of the RBC were almost normal, but a slight decrease in cooperativity and lowered Bohr and 2,3-diphosphoglycerate (DPG) effects were observed for pure stripped Hb J Guantanamo. The expression of the electrophoretic charge difference was partly masked, as is often observed when the structural abnormality is situated in or near a contact area.