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Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37+/-3.2 vs. 27+/-1.4, mean+/-SD; p0.001), in association with moderate thrombocytopenia (505+/-49 x 10(3)/microl vs. 1151+/-162 x 10(3)/microl; p0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell-Jolly bodies and "pocked" erythrocytes (p0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5+/-1% vs. 1+/-1%; p0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.

A congenital dyserythropoietic anaemia (CDA) was recognised in a French Caucasian male patient. Blood smears showed a pronounced aniso-poikilocytosis. Bone marrow light microscopy showed signs of dyserythropoesis, but no internuclear chromatin bridges. Electron microscopy disclosed erythroblast nuclei with the Swiss cheese aspect and the presence of cytoplasmic organelles, assessing the diagnosis of CDA I. The presence of internuclear chromatin bridges may thus be missing in CDA I. The patient proved to be homozygous for the Arg1042Trp mutation in codanin-1 (the 'Bedouin mutation'). By the age of 25, the patient's vision started to deteriorate as a result of retinal angioid streaks and macular abnormalities. Evolution was controlled and the patient, being nearly 50 yr old now, still has a partial use of his eyes. This second case of retinal angioid streaks reported in CDA I adds to the non-haematological features likely to be associated with this condition.

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.

Key words Name of the disease and synonyms Definition/diagnostic criteria Etiology Biological methods of diagnosis Incidence Management and treatments Unresolved questions and comments References Abstract Congenital dyserythropoietic anemias (CDA) result from diverse erythropoietic disorders; they lead to the defective production of red blood cells (RBC) and often mild hemolysis that attests to a qualitative defect of these RBC released into the circulation. Three forms of CDA have been characterized: types I, II and III. The shared symptoms include anemia of variable severity, intermittent jaundice, splenomegaly and hepatomegaly. Iron overload is progressive in types I and II. CDA I is often associated with dysmorphisms, particularly affecting the digits. Diagnosis of a CDA relies on light and electron microscopy examinations of the erythroblasts in a bone-marrow biopsy, which will demonstrate the presence of chromatin bridges between erythroblast nuclei and a spongy 'Swiss cheese' appearance of the condensed chromatin in CDA I; binucleated cells and endoplasmic reticulum remnants are seen in CDA II. Electrophoresis of erythrocyte membrane proteins also provides a sure diagnosis. The protein 4.1 level is low in CDA I, further associated with an unusual appearance of band 3 and the presence of reticular endoplasmic proteins - calreticulin, glucose regulatory protein 78 and isomerase disulfide - in CDA II. According to available estimations, CDA I and II incidence do not exceed 1/100 000 births per year. These forms are transmitted by recessive inheritance. CDA III results from dominant inheritance and is extremely rare. The genes responsible for CDA I, II and III have been localized to chromosomes 15q15.1-15.3, 20q11.2 and 15q21-q25, respectively. The gene implicated in CDA I was recently identified but the type and function of its product, codanine-1, remains to be established. The treatment of these diseases is essentially symptomatic. However, interferon-alpha attenuates the anemia of CDA I. The proteins causing these diseases and the underlying molecular mechanisms are still unknown.

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.

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.

Constitutional deficit in the erythroid protein 4.1 (4.1R), a structural component of the erythrocyte membrane, is implicated in hereditary elliptocytosis. Acquired deficit in protein 4.1R have been rarely described in myelodysplastic syndromes. Here, we report a series of six patients presenting a myelodysplastic or a myeloproliferative disease in association with an elliptocytosis curve on osmotic gradient ektacytometry and a significant decrease in protein 4.1R level. We confirm that deficit in protein 4.1R is recurrent in myeloid malignancies and should be particularly investigated when deletion del (20q) is present, since we found this chromosomal abnormalityin four out of six patients. Am. J. Hematol., 2008. © 2007 Wiley-Liss, Inc.

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 are seen in the clinical manifestations of hereditary elliptocytosis (HE). To define the cellular alteration(s) that best reflect the variable expression of the disease, we evaluated the pathobiologic features of red blood cells in a series of 18 patients with HE, 15 persons from six families with HE as a result of defects in spectrin, and 3 persons from one family with HE caused by partial or total deficiency of protein 4.1. We found that decreased cellular deformability is a distinguishing feature of red blood cells in all patients studied. Comparison of volume and hemoglobin content histograms of red blood cells and reticulocytes revealed that cell fragmentation is a feature of mature red blood cells. The extent of red blood cell fragmentation as reflected by increased percentage of microcytic red blood cells was the best indicator of the severity of hemolytic anemia. Furthermore, we found that the observed variations in cellular properties of HE red blood cells in different persons is the consequence of varying amounts of mutant protein assembled into the membrane. These findings enabled us to define the mechanistic basis for cellular changes in this red blood cell membrane disorder better and also to obtain insight into the cellular basis for variable clinical expression.

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.

We describe the second case of a homozygous mutation in band 3 (anion exchanger 1 (AE1), SLC4A1) causing both hereditary spherocytosis (HS) and distal renal tubular acidosis (dRTA). This new variant differed from the previous homozygous variant (Band 3 Coimbra, Ribeiro et al, Blood2000: 96, 1602) in that a significant amount of band 3 was present in the red cell membrane and the dRTA was incomplete. In the proband, an Algerian male baby, a severe hemolytic anemia rapidly developed following birth. Low hemoglobin (3.5 g Hb/dL) at D12 demanded a first transfusion. Hepato-splenomegaly, marked palor and jaundice were noted. Eight transfusions were administered in the following months. Subtotal splenectomy, performed at the age of 9 months, cancelled the transfusional needs. Veinous blood examined after splenectomy showed band 3 to be reduced to ~35% of normal, as shown by immunoblotting. The other known proteins of the band 3/Rh macrocomplex were also found to be reduced. The parents were first cousins. Both showed mild spherocytosis associated with a mild band 3 deficiency. DNA sequence analysis revealed a novel homozygous mutation: TCC to TTC at codon 667 in exon 16, leading to an amino acid substitution: Ser667Phe, located in proposed transmembrane helix 8. Both parents were heterozygous for the same mutation. Anion transport (sulphate uptake) in the patient’s red cells was ~40% normal, showing that transport specific activity of the mutant band 3 was not affected. The mutant red cell band 3 and kidney band 3 were expressed in Xenopus oocytes, with and without co-expression of glycophorin A (GPA). There was very little chloride transport detected in oocytes expressing either mutant red cell or kidney protein alone, but transport was partially rescued by co-expression of GPA. After birth the child showed a temporary acidosis which spontaneously receded. No nephrocalcinosis has been noted to date. At 2 years of age, an ammonium chloride challenge suggested that the child has incomplete dRTA; over the seven hours of the test the blood bicarbonates decreased down to 15.6 mmoles/L, but urinary pH remained above 5.90. Stable expression of mutant kidney band 3 in non-polarised Madin-Darby canine kidney (MDCK) cells showed that the mutant protein was retained in the endoplasmic reticulum. We are currently investigating the effects of this mutant in polarized MDCK cells. Overall our results suggest that the Ser667Phe does not affect the anion transport function of band 3 but causes a trafficking defect in both red blood cells and kidney cells. The trafficking defect may be less severe in red blood cells where it is probably attenuated by the chaperone-like effect of GPA, which is not expressed in kidney cells. The fact that the hematological manifestations are far more conspicuous than their nephrologic counterpart will be discussed.

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.