Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

Patients with pyruvate kinase (PK) deficiency have spiculated red cells termed echinocytes, but little is known about the basis for this characteristic morphologic abnormality. PK deficient RBC were incubated at 0°C for 15' with the membrane active agent chlorpromazine (CPZ) which reverses echinocytosis and lysolecithin which causes echinocytosis. Samples were fixed in 1% glutaraldehyde (pH 7.4) and using phase contrast microscopy the proportions of echinocytes quantitated using criteria of Bessis. In PK deficient RBC echinocytosis was exacerbated by lysolecithin and completely reversed by CPZ. Since recent experiments have suggested that echinocytic shape reversal depends on the hexose monophosphate shunt (HMPS), we incubated PK RBC with hydrogen peroxide and 1 mM methylene blue (MB) at both 20°C and 0°C. Peroxide was minimally effective at either temperature. MB, a tertiary amine similar in structure to CPZ, was highly effective instantaneously reversing echinocytosis at both 20°C and 0°C. These results indicate that echinocytic shape change in PK deficiency can be completely reversed by membrane active agents, a process which is independent of HMPS activity and can in fact occur independent of cell metabolism. These results also imply that the metabolic abnormality of PK deficient RBCs which leads to shape change is mediated by an effect on the relative surface areas of the bilayer leaflets.

Congenital spherocytic anemia is a relatively common disorder but the genetic defect has not yet been definitively mapped to a chromosome. We studied a family in which 2 of 3 children were dysmorphic, affected by severe spherocytosis requiring intermittant transfusions, and showed deletion of a portion of the short arm of chromosome 8 (8p-). Both parents and the sibling were normal and showed no evidence of hemolysis or the deletion. Assays of carbonic anhydrase II, factor VII, thryroglobulin, and glutathione reductase, genes previously assigned to 8p were unremarkable. In order to elucidate the membrane defect, red cell membranes were electrophoresed using 1-D Fairbanks SDS polyacrylamide gels and a modified O'Farrell 2-D technique. Lipids were examined using thin-layer chromatography. Coomasie blue stained 1-D and silver stained 2-D electrophoretograms were normal; in particular members of the ternary binding complex, spectrin (α and β), band 4.1, and actin were normal as were membrane cholesterol and phospholipids. The association between the gene deletion and spherocytosis in the affected family members suggests that a gene for this defect lies on 8p, but does not affect the quantities of membrane proteins conventionally associated with rec cell membrane stability.

Despite intensive investigation, no prognostic factor has yet been identified for predicting the frequency of painful crises in HbSS. Direct microscopic observation of a transfused animal model suggested that reversibly sickling cells (RSC), rahter than ISC, were more likely to obstruct vessels (Lacelle, Blood Cells, 1977). We examined the significance of this observation by following 65 children with HbSS for the frequency of painful crises. Without knowledge of the clinical course, hematologic parameters and proportion of ISC (morphologic criteria) were used to estimate the absolute number of cells capable of being RSC (100% -%ISC -%F) × RBC. The patients were followed for a mean of 27 months (144 prospective patient-years). A median of I event occurred per 12 months (range 0-10). The frequency of painful events was correlated with RSC (r=0.32, p <0.01), those patients with the highest red cell levels and fewest ISC having higher RSC levels and more frequent crises. RSC was for any given patient stable over time and inversely correlated to MCH (r= -0.64, p <0.01). These results indicate that the proportion of RSC is a prognostic factor and imply that strategies which decrease the number of ISC or increase the hemoglobin level without decreasing the number of cells capable of reversible sickling may increase the frequency of painful crises.

NHL's of children are a heterogeneous group of malignancies which when disseminated respond poorly to therapy. In CCSG 551, a stratified prospective trial for children with NHL, we randomized 314 patients to receive 4 drugs (COMP) or 10 drugs (modified LSA2L2). All patients received radiation to bulk disease and prophylactic IT methotrexate. Patients were grouped by extent of disease (regional vs. disseminated), and histologic type (lymphoblastic vs. non-lymphoblastic). Of the 60 patients with regional disease, 85% remained free of disease at 24 mos, regardless of histology or treatment arm. Of the 254 patients with disseminated disease, 76% of those with lymphoblastic histology remained disease free at 18 months using 10 drugs while only 36% were still in 1st remission on the 4 drug protocol (p < 0.05). In contrast, 61% of those patients with non-lymphoblastic morphology remained in 1st remission at 18 mos on 4 drugs, but only 29% were still in 1st remission using 10 drugs (p 0.05). As the overall toxicity of the 10 drug protocol exceeded that of the 4 drug protocol, we now treat all patients with regional disease with local irradiation and the 4 drug protocol. For patients with disseminated disease the 10 drug protocol is superior for lymphoblastic disease while the 4 drug is advantageous for other histologies.

Niemann-Pick disease is characterized by hepatosplenomegaly and progressive neurologic deterioration in the first year of life. Histiocytes of the reticuloendothelial system accumulate cytoplasmic inclusions, which results in characteristic cells. 1 In the examination of a patient with hepatosplenomegaly, discovery of these cells in bone marrow aspirates provides a valuable diagnostic clue. These cells, however, are few in number and may be difficult to recognize in standard preparations. We describe a case of Niemann-Pick disease in which foam cells were present in the spinal fluid and were easily demonstrated by cytocentrifugation. Evaluation of CSF cells by cytocentrifugation may provide a useful diagnostic tool in suspected cases of lipid storage disease. Report of a Case .—The patient was noted to have massive hepatosplenomegaly at 7 months of age. The boy's parents were first cousins. Physical examination showed a small, microcephalic infant. His height (25 cm), weight (6,130 g), and head circumference