Positive Acidified Serum Test Research Articles

Progress in the congenital dyserythropoietic anemias: Juicy but high-hanging fruits?

Progress in the congenital dyserythropoietic anemias: Juicy but high-hanging fruits?

Congenital dyserythropoietic anemia type II - a case report of two siblings in a family.

Two children out of three siblings of a family presented with tiredness, fatigue, and breathlessness for more than 6 months. Examination of peripheral blood smear, bone marrow aspirate, and a positive acidified serum test (HEMPAS) revealed these children to be a case of congenital dyserythropoietic anemia type II. This case is reported because of its rarity.

Congenital dyserythropoietic anemia type II: exclusion of seven candidate genes

Congenital dyserythropoietic anemias (CDA) are genetic disorders characterized by anemia and ineffective erythropoiesis. Three main types of CDA have been distinguished: CDA I, CDAII and CDA III, whose loci have been already mapped. After the identification of the locus for CDA II, also known as HEMPAS (hereditary erythroblast multinuclearity with positive acidified serum test), on the long arm of chromosome 20 (20q11.2) we have analyzed by a mutational search seven candidate genes in a large series of CDA II patients. In particular, the following genes have been investigated: integrin beta 4 binding protein, ribophorin II, ubiquitin protein ligase ITCH, mannosil-oligosaccharide alpha-1,2-mannosidase like protein, erythrocyte protein band 4.1 like protein, zinc finger protein PLAGL2, and finally novel zinc finger protein. None of them resulted as the causative gene but several protein variants and DNA polymorphisms have been identified. These data exclude the role of the above mentioned genes in causing CDA II and add further information in the process of cloning the CDA II gene.

Structure of a neutral glycosphingolipid recognized by human antibodies in polyagglutinable erythrocytes from the rare NOR phenotype.

NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.

Diagnostik und Genetik der kongenitalen dyserythropoetischen Anämien (CDA)

The congenital dyserythropoietic anemias (CDA) are hereditary diseases characterized by a lifelong, mostly moderate anemia. CDA can be diagnosed already in early childhood. However, diagnosis is complicated due to poor knowledge of morphological criteria and the large number of differential diagnoses that have to be excluded. CDA type I is characterized by macrocytic anemia with megaloblastic changes in erythropoiesis and chromatin bridges between isolated erythroblasts. Type II shows a normocytic anemia with a positive acidified serum test and increased agglutination with anti-i. Erythroblasts can present with 2 or more nuclei. CDA type III presents with a macrocytic anemia and erythroblasts with up to 12 nuclei, the so called gigantoblasts. Some patients lack the typical morphological abnormalities of type I-III (variants or type IV). Besides light microscopic abnormalities, CDA type-specific changes in electron microscopy are described. The clinical picture of the patients vary between the different forms: signs of hemolysis and ineffective erythropoiesis such as icterus, splenomegaly and gall stones can be present. Most important is the tendency of a part of patients to have an increased iron absorption and iron storage. Patients with and without transfusion dependency are described. Supportive care such as iron chelation can be necessary in some patients. The CDA are inherited in an autosomal recessive manner; in type III an additional autosomal dominant variant exists. Recently, the determination of gene loci for type I, II and III was enabled by linkage analysis on different regions of chromosome 15 and 22. It is considered that CDA I and II are genetically heterogenic. Until now no gene has been identified in either type of CDA. In CDA type II, a glycosylation defect of erythrocyte membrane proteins is present. An international group plans to do further research. Therefore, identification and registration of patients in a registry is necessary. Patients' data and material would enable gene characterization. The results would allow an extended classification according to genotype and prediction of the course of the disease. Additionally, information on the regulation and control of normal and abnormal erythropoiesis could be obtained.

Protein Glycosylation and Diseases: Blood and Urinary Oligosaccharides as Markers for Diagnosis and Therapeutic Monitoring

N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease. This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration. The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer. Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.

Polylactosamines are not obligate receptors for invasion of Plasmodium falciparum malaria as shown in HEMPAS variant II-gal- erythrocytes.

A HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum test) erythrocyte, atypical Variant II (referred to herein as Variant II-gal-), lacking long-chain polylactosamine on both glycoproteins (Band 3 and 4.5) and glycosphingolipids, was characterized by the carbohydrate profile of the erythrocyte membrane according to Fukuda et al. (Blood, 73, 1331-1339, 1989). Two laboratories previously reported that polylactosamine isolated from the erythrocyte protein Band 3 inhibited invasion of red blood cells by Plasmodium falciparum in malarial culture, suggesting a role for this carbohydrate in adhesion of the parasite. Therefore, HEMPAS erythrocyte Variant II-gal- presented a unique opportunity to further examine this premise. Freshly drawn blood samples (normal and HEMPAS Variant II-gal-) were separately incubated with P. falciparum from mannitol-synchronized cultures. The parasite was found to invade HEMPAS Variant II-gal- erythrocytes at a 30% lower rate through two life cycles, as shown by microscopic evaluation of invasion and by [3H]hypoxanthine incorporation into parasite. This observation, along with the published fact that glycophorin-deficient MkMk cells are also infectable, but at a lower rate, indicates that neither sialoglycoproteins nor polylactosamines are an obligate adhesive ligand for P. falciparum, although the possibility remains that either may still contribute to adhesive events during infection.

Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II.

Congenital dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) is a genetic disease caused by membrane abnormality. Previously we have found that Band 3 and Band 4.5 are not glycosylated by lactosaminoglycans in HEMPAS erythrocytes, whereas normally these proteins have lactosaminoglycans (Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Br. J. Haematol. 56, 55-68). In order to find out where glycosylation of lactosaminoglycans stops, we have analyzed the carbohydrate structures of HEMPAS Band 3. By fast atom bombardment-mass spectrometry, methylation analysis, and hydrazinolysis followed by exoglycosidase treatments, the following structure was elucidated: (formula; see text) N-Linked glycopeptides synthesized in vitro by reticulocyte microsomes from HEMPAS were shown to be predominantly the above short oligosaccharide, whereas those from normal reticulocytes contain large molecular weight carbohydrates. The N-acetylglucosaminyltransferase II, which transfers N-acetylglucosamine to the C-2 position of the Man alpha 1----6Man beta 1----arm of the biantennary core structure, was therefore examined by using Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcol as an acceptor. N-Acetylglucosaminyltransferase II activity was demonstrated in the lymphocyte microsome fraction from normal individuals. However, this enzyme activity was found to be decreased in those from HEMPAS patients. These results suggest that the primary defect of HEMPAS lies in the lowered activity of N-acetylglucosaminyltransferase II.

Isolation and characterization of poly- N-acetyllactosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients

Congenital dyserythropoietic anemia type II or hereditary erythroblastic polynuclearity with positive acidified serum test (HEMPAS) is a rare genetic disease inherited by a recessive mode. Previous studies on HEMPAS erythrocytes have shown that Band 3 and Band 4.5 glycoproteins were not glycosylated by lactosaminoglycans, while polylactosaminyl carbohydrates are accumulated as glycolipids (P. Scartezzini et al., Br J. Haematol., 51 (1982) 569; M.N. Fukuda et al., Br. J. Haematol., 56 (1984)55). Presently, we have isolated polylactosaminyl lipids from HEMPAS blood cells and analyzed their structures by fast atom bombardment-mass spectrometry (FAB-MS), methylation analysis, endo-β-galactosidase digestion. The results indicate that polylactosaminyl lipids accumulated in HEMPAS erythrocytes are a species of poly- N-acetyllactosaminylceramides which are also present in normal erythrocytes, but at 7 ∼ 9 times lower level. Isolated polylactosaminylceramides exhibit I-, i-, H- and Le x antigenic activities which suggests that the polylactosaminylceramides are derived from both erythrocytes and granulocytes.

Mechanism of enhanced complement-dependent cytotoxicity of papain-treated lymphocytes: evidence for increased stability of classical pathway C3 convertase.

The mechanisms of enhanced antibody-mediated, complement-dependent cytotoxicity caused by proteolytic treatment of cells was studied in a model system based on HLA microlymphocytotoxicity methods. In this model, papain treatment of lymphocytes resulted in a) no change in antibody binding, b) a slight decrease in initial binding of C4, c) a marked increase in stability of cell-bound C4b, resulting in d) an increase of cell-bound C3, and e) no increase in lytic efficiency of the C5b-9 membrane attack complex. We conclude that the most important step in papain enhancement of lysis of lymphocytes is an increased stability of cell-bound C4b, possibly through decreased surface binding of C4-bp. This mechanism may be relevant to the pathologically increased lysis of cells occurring in patients with hereditary erythroblastic multinuclearity with a positive acidified-serum test (HEM-PAS).

Polyagglutinabilité due à l'antigène Hempas

A new antigen has been recently discoverd in patients with congenital dyserythropoietic anemia type II. The acronyme Hempas was proposed for this disease as a remind of the main morphological feature of erythroblasts (hereditary erythroblastic multinuclearity) and the characteristic serological findings (positive acidified serum test). The patients red cells are agglutinated and lysed by an IgM cold reacting antibody present in the serum of most normal subjects and not previously recognized. This behaviour is thus reminding of cells carrying antigens such as T, Tn, Cad or acquired B. As for T and Tn cells, sialic acid and electrophoretic mobility are reduced, but in contrast, agglutinability of Hempas cells is enhanced by enzyme treatment. Agglutination by anti H and anti Pr specific reagents is reduced. I and mainly i activity are strongly increased. The relationship between the membrane abnormalities of Hempas red cells and the failure of normoblasts to divide their cytoplasm i still largely unknown.

The action of seed and other reagents on HEMPAS erythrocytes.

Erythrocytes of a patient with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS) were tested with many seed extracts and various other reagents. Serological evidence of membrane abnormality was confirmed. Various anti-H reagents reacted relatively poorly with HEMPAS cells. HEMPAS cells have both enhanced i and depressed H antigens. A brief note on a 'new' anti-H lectin (Cytisus glabrescens) is provided.

A New Abnormal Hemoglobin O Padova, α 30 (B11) Glu → Lys, and a Dyserythropoietic Anemia With Erythroblastic Multinuclearity Coexisting in the Same Patient

Abstract A patient with a not previously described abnormal hemoglobin (α30Glu → Lys) and dyserythropoietic anemia with erythroblastic multinuclearity is reported. The heat stability and the functional properties of the new abnormal hemoglobin, named hemoglobin O Padova, are normal, although the replacement lies in the α1β1 interchain contact. The hemolytic condition, which was much improved by splenectomy, therefore appears to be linked to the hereditary erythroblastic multinuclearity similar to hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS). In addition to the leading features observed in published cases of this entity, our case exhibited some immunologic peculiarities.

Congenital dyserythropoietic anemia, type II (Hempas) first five reported cases in Italy

Five siblings from an italian family with congenital dyserythropoietic anaemia, type II, are reported. Diagnosis was established by demonstration of erythroid hyperplasia and erythroblastic multinuclearity in the bone marrow, and a positive acidified serum test.

Association of type II congenital dyserythropoietic anaemia and von Willebrand's disease.

Summary. In a patient with a history of anaemia, jaundice and splenomegaly, the bone marrow showed erythropoietic hyperplasia with 20% of multinucleated erythroblasts. His red cells gave high agglutination scores with anti‐i, and haemolysis tests with acidified iso‐group sera were positive.A type II congenital dyserythropoietic anaemia was diagnosed in this patient and this was shown to be associated with von Willebrand's disease. A brother of the propositus also had a positive acidified serum test and showed agglutination by anti‐i, but polynucleated erythroblasts were not found.

Congenital Dyserythropoietic Anemia In Jews From Morocco

To the Editor.— During the last three months, seven cases of congenital dyserythropoietic anemia (CDA) type 2 (HEMPAS) have been diagnosed in our hospital. Of these, three cases were diagnosed in retrospect in patients who had been splenectomized two, four, and six years ago for chronic hemolytic anemia with splenomegaly. In all cases, the diagnosis was confirmed by a positive acidifiedserum test and an agglutination of the erythrocytes with an anti-i-serum. Bone marrow examination was performed in five cases, and the typical picture of multinucleated normoblasts and Gaucher-like cells was found. This examination has not yet been performed in two cases. Three pairs of siblings are affected. Two or more siblings affected in the same family is not rare, and has been reported by other authors. 1,2 Our most important observation is that all the patients' country of origin is Morocco. In addition to our observation, we obtained the information

Electronmicroscopic and biochemical observations on erythroid cells in congenital dyserythropoietic anemia type II.

Erythroid cells in a patient with dyserythropoietic anemia (CDA) were examined for biochemical, serologic and ultrastructural alterations. The activity of hexokinase and 6-phosphogluconate dehydrogenase was increased and the acidified serum test was positive. These observations are consistent with CDA type II (CDA II), a diagnosis that has been said to depend on a positive acidified serum test. Electronmicroscopic analysis of the erythroid cells of the present patient indicated multinuclearity, karyorrhexis, continuity between cisternae of endoplasmic reticulum and the nuclear envelope at sites where the latter would show a complete break, extrusion of intracellular material and organelles, and excessive smooth endoplasmic reticulum. These observations may be considered the ultrastructural hallmark of CDA II. Most of them have also been made in four of five examined patients with positive acidified serum test whereas the fifth patient was reported to have markedly dissimilar erythroid ultrastructure. It appears, therefore, that diagnostic classification of CDA by the acidified serum test may not always coincide with that achieved by electronmicroscopy.

Mechanisms of Immune Lysis of the Red Cells in Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum Test and Paroxysmal Nocturnal Hemoglobinuria

The red cells of patients with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS), a form of congenital dyserythropoietic anemia, and the cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are lysed more readily than normal cells by certain antibodies, notably cold agglutinins (anti-I) and complement. With some but not other examples of anti-I, HEMPAS and PNH cells adsorbed more antibody than normal cells. Equal quantities of adsorbed antibody bound equal quantities of the first component of complement (C1) to normal, PNH, and HEMPAS cells. However, for a given quantity of bound antibody and C1, much more of the fourth component of complement (C4) was bound to HEMPAS cells than to normal cells. This resulted in the binding of proportionately larger quantities of the third component of complement (C3) to these cells. The same amount of bound C3 was found on the membranes of normal and HEMPAS cells for a given degree of lysis. Hence, the marked increase in lysis of HEMPAS cells is due to the increased adsorption of antibody and/or increased binding of C4.PNH cells bound the same amount of C4 per bound C1 as normal cells but bound more C3 than normal cells. However, the mean concentration of C3 on the membrane of PNH cells was one-third to one-fifth that on normal cells for a given degree of lysis. Hence, the increased lysis of PNH cells is due to the increased binding of C3 and increased hemolytic effectiveness of the bound C3.

Mechanisms of Increased Susceptibility to C Lysis of Abnormal Human Red Blood Cells

Abstract The cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) are abnormally susceptible to lysis by C and cold-reactive antibodies, but for different reasons. For PNH cells, the fixation of antibody, C1 and C4 is the same as for normal cells. However, the amount of C3 fixed/C4 is increased 3- to 7-fold. The amount of C3 fixed for a given degree of lysis is about one-third that of normal cells. In a less sensitive variant of PNH cells, the amount of C3 fixed/C4 was about the same as for the markedly abnormal cells, but the amount of C3 fixed for a given amount of lysis was the same as for normal cells. Normal cells treated with sulfhydryl-reactive agents resembled PNH cells in these reactions. HEMPAS cells fix 2 to 3 times as much of some, but not other, examples of anti-I. The amount of C1 fixed/Ab is normal.

CONGENITAL DYSERYTHROPOIETIC ANEMIA, TYPE II: REPORT OF TWO CASES AND A REVIEW OF THE LITERATURE

Two children with congenital dyserythropoietic anemia type II, are described and contrasted. This recently recognized disorder of unknown pathogenesis is characterized by variable degrees of anemia, splenomegaly, and jaundice. Diagnosis was established by the demonstration of erythroid hyperplasia and erythroblastic multinuclearity in the bone marrow, and a positive acidified serum test. Erythrokinetic studies revealed a significant degree of ineffective erythropoiesis. One of the patients demonstrated Gaucher cells in the marrow as a presumed consequence of the increased rate of red blood cell destruction. Means of distinguishing this autosomal recessive disorder from other refractory anemias is discussed.