Clinical and Haematologic Features of 300 Patients Affected by Hereditary Spherocytosis as a Function of the Type of the Membrane Protein Defect.

Abstract Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.

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Erythrocyte cellular and membrane deformability in hereditary spherocytosis

Blood ◽

10.1182/blood.v53.3.481.bloodjournal533481 ◽

1979 ◽

Vol 53 (3) ◽

pp. 481-485 ◽

Cited By ~ 1

Author(s):

K Nakashima ◽

E Beutler

Keyword(s):

Cell Membrane ◽

Hereditary Spherocytosis ◽

Volume Ratio ◽

Osmotic Fragility ◽

Red Cell ◽

Red Cell Membrane ◽

Relative Rigidity ◽

Altered Surface ◽

Red Cell Membranes ◽

Expected Increase

In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.

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Erythrocyte cellular and membrane deformability in hereditary spherocytosis

Blood ◽

10.1182/blood.v53.3.481.481 ◽

1979 ◽

Vol 53 (3) ◽

pp. 481-485 ◽

Cited By ~ 16

Author(s):

K Nakashima ◽

E Beutler

Keyword(s):

Cell Membrane ◽

Hereditary Spherocytosis ◽

Volume Ratio ◽

Osmotic Fragility ◽

Red Cell ◽

Red Cell Membrane ◽

Relative Rigidity ◽

Altered Surface ◽

Red Cell Membranes ◽

Expected Increase

Abstract In order to determine whether the relative rigidity of the hereditary spherocytosis (HS) red cell is due to membrane rididity or merely to an altered surface/volume ratio, we investigated the deformability of resealed red cell membranes from patients with HS. Whereas the osmotic fragility of intact red cells of HS patients showed the expected increase, the osmotic fragility of resealed HS membranes was normal, thus indicating that their surface/volume ratio was normal. Measurements with an ektacytometer showed that deformability of intact HS cells was markedly diminished, whereas deformability of resealed HS membranes was normal. These findings indicate that the HS red cell membrane is not intrinsically abnormally rigid, as has been suggested, but that the lack of deformability of the erythrocyte is primarily a function of the altered surface/volume ratio.

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Diminished osmotic fragility of human erythrocytes following the membrane insertion of oxygenated sterol compounds

Blood ◽

10.1182/blood.v58.2.317.bloodjournal582317 ◽

1981 ◽

Vol 58 (2) ◽

pp. 317-325

Author(s):

RA Streuli ◽

JR Kanofsky ◽

RB Gunn ◽

S Yachnin

Keyword(s):

Cell Membrane ◽

Hereditary Spherocytosis ◽

Osmotic Fragility ◽

Human Erythrocytes ◽

Fragility Curve ◽

Membrane Insertion ◽

Red Cell ◽

Red Cell Membrane ◽

Mean Cell Volume ◽

Osmotic Lysis

Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625- 5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.

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Diminished osmotic fragility of human erythrocytes following the membrane insertion of oxygenated sterol compounds

Blood ◽

10.1182/blood.v58.2.317.317 ◽

1981 ◽

Vol 58 (2) ◽

pp. 317-325 ◽

Cited By ~ 30

Author(s):

RA Streuli ◽

JR Kanofsky ◽

RB Gunn ◽

S Yachnin

Keyword(s):

Cell Membrane ◽

Hereditary Spherocytosis ◽

Osmotic Fragility ◽

Human Erythrocytes ◽

Fragility Curve ◽

Membrane Insertion ◽

Red Cell ◽

Red Cell Membrane ◽

Mean Cell Volume ◽

Osmotic Lysis

Abstract Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625- 5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.

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Red cell membrane protein abnormalities in hereditary spherocytosis in Brazil

British Journal of Haematology ◽

10.1111/j.1365-2141.1994.tb05021.x ◽

1994 ◽

Vol 88 (2) ◽

pp. 295-299 ◽

Cited By ~ 22

Author(s):

Sara T. O. Saad ◽

Fernando F. Costa ◽

Dilmara L ◽

Vicentim Tereza ◽

S. I. Salles ◽

...

Keyword(s):

Cell Membrane ◽

Membrane Protein ◽

Hereditary Spherocytosis ◽

Red Cell ◽

Red Cell Membrane ◽

Cell Membrane Protein

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Hereditary Spherocytosis

Journal of Lumbini Medical College ◽

10.22502/jlmc.v6i1.202 ◽

2018 ◽

Vol 6 (1) ◽

pp. 41-43

Author(s):

Surendra Koju ◽

Ramesh Makaju

Keyword(s):

Abdominal Pain ◽

Cell Membrane ◽

Blood Smear ◽

Hereditary Spherocytosis ◽

Osmotic Fragility ◽

Peripheral Blood Smear ◽

Laboratory Evaluation ◽

Red Cell ◽

Red Cell Membrane ◽

Cell Disorder

Introduction: Hereditary spherocytosis is a red cell membrane disorder that causes hemolytic anemia. Due to defective cell membrane, red cells are spherical shaped and result in their early lysis. Osmotic fragility of spherocytic red cell is increased. Case report: A 22 year old female presented with chief complain of abdominal pain. Initially she was diagnosed as cholelithiasis. Under laboratory evaluation she was found to be anemic with reticulocytosis. In peripheral blood smear, spherocytes were moderately distributed. Antihuman globulin test was negative but osmotic fragility was high. Hence, she was confirmed as case of hereditary spherocytosis. Conclusion: Hereditary spherocytosis is a rare red cell disorder and its diagnosis can be made by osmotic fragility test.

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A Case of Congenital Red Cell Pyruvate Kinase Deficiency Associated with Hereditary Spherocytosis

Blood ◽

10.1182/blood.v118.21.5272.5272 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5272-5272

Author(s):

Cristina Vercellati ◽

Anna Paola Maria Luisa Marcello ◽

Elisa Fermo ◽

Paola Bianchi ◽

Carla Boschetti ◽

...

Keyword(s):

Cell Membrane ◽

Pyruvate Kinase ◽

Hemolytic Anemia ◽

Peripheral Blood ◽

Blood Smear ◽

Hereditary Spherocytosis ◽

Osmotic Fragility ◽

Peripheral Blood Smear ◽

Red Cell ◽

Red Cell Membrane

Abstract Abstract 5272 Pyruvate kinase (PK) deficiency, transmitted as an autosomal recessive trait, is the most common erythroenzymopathy of glycolytic pathway (prevalence of 1:20,000) associated with chronic non spherocytic hemolytic anemia from mild to severe. More than 180 mutations in the PK-LR gene have been so far reported, and genotype-phenotype correlation has been established for some of them. Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The main clinical features are hemolytic anemia from compensated to severe, variable jaundice, splenomegaly and cholelythiasis. The molecular defect is highly heterogeneous, caused by proteins involved in the attachment of cytoskeleton to the membrane integral domain (spectrin, ankyrin, band 3 and protein 4.2). We describe a case of PK deficiency associated with HS. The propositus was a 13 years-old Italian male with neonatal jaundice and need of blood transfusion (Hb 5.8 g/dL) during an infectious episode. At the time of the study Hb was 13.9 g/dL, MCV 81.8 fL, reticulocytes 207×109/L, unconjugated bilirubin 2.16 mg/dL, LDH 605 U/L, haptoglobin <20 mg/dL. The peripheral blood smear examination showed the presence of spherocytes (16%) and some ovalocytes (2%). The study of the most important red cell enzymes revealed reduced PK activity (59% of normal). Direct sequencing of PK-LR gene showed compound heterozygosity for the 994A mutation (Gly332Ser) and the −148T variant localized the erythroid specific promoter region. The presence of spherocytes in peripheral blood smear prompted us to investigate for the coexistence of HS. Erythrocyte osmotic fragility was decreased and SDS–PAGE analysis of red cell membrane proteins revealed a 30% spectrin reduction. Family study demonstrated a heterozygous condition for the 994A mutation in the father, who also displayed comparable enzyme deficiency, whereas promoter variant −148T was detected in the mother and in the brother. No red cell membrane abnormalities were present in the family members, although positive EMA binding test and increased osmotic fragility were found in the father and brother. The co-existence of HS and PK deficiency is very rare event, only few cases are described to date. Clinical, family and molecular studies allowed the determination of the interrelationship between the two RBC abnormalities in the patient and his relatives. The reduced PK activity in the propositus and his father is justified by heterozygous 994A mutation. The more severe clinical picture in the propositus could be caused by the coexistence of HS and by the presence of −148T mutation, that although it seems not to have effects on PK-LR mRNA expression, is often detected in PK deficient subjects with heterozygous PK mutations. Disclosures: No relevant conflicts of interest to declare.

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Cation dependence of the phosphorylation of specific residues in red cell membrane protein band 3

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(87)90232-x ◽

1987 ◽

Vol 899 (1) ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

C. Vasseur ◽

J.P. Piau ◽

E. Bursaux

Keyword(s):

Cell Membrane ◽

Membrane Protein ◽

Protein Band ◽

Band 3 ◽

Red Cell ◽

Red Cell Membrane ◽

Cell Membrane Protein

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Hereditary Spherocytosis: How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging

Blood ◽

10.1182/blood.v120.21.5163.5163 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5163-5163

Author(s):

Susanna Barella ◽

Ramon Simon-Lopez ◽

Nicola Di Gaetano ◽

Renzo Galanello

Keyword(s):

Research Funding ◽

Roc Analysis ◽

Hereditary Spherocytosis ◽

Osmotic Fragility ◽

Peripheral Blood Smear ◽

Band 3 ◽

Screening Tests ◽

New Information ◽

Related Proteins ◽

Best Parameters

Abstract Abstract 5163 Introduction: Hereditary Spherocytosis (HS) is one of the most common inherited hemolytic anemias. Many of them are autosomal dominant, being about 25% of the cases transmitted recessively. Diagnostic of HS: Classic testing for HS includes: Hematologic testing of red blood cell indices (RDW, MCHC, Reticulocyte count), peripheral blood smear (presence of spherocytes), osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms that may be used as screening tests for hereditary spherocytosis. Objective: Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV (@Mean Sphered Cell Volume), @RSF, @MAF, @ LHD%. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Hereditary Spherocytosis. There are previous studies showing the possible benefit of using MCV minus @MSCV for the detection/flagging of cases with spherocytes. Patient and Methods: We have collected 28 patients with Hereditary Spherocytosis. All of them were confirmed by red cell morphology, osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms. Results: Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from the normals were: RET% (AUC 0. 996), MCV - @MSCV (AUC 0. 996), @MSCV (AUC 0. 969), RDW(AUC 0. 892), MCHC (AUC 0. 860), HGB (AUC 0. 787). Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from other anemias (excluding normals)were: MCV - @MSCV (AUC 0. 991), MCHC (AUC 0. 987), RET% (AUC 0. 857). Disclosures: Simon-Lopez: Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Novartis: Research Funding, Speakers Bureau; Apopharma: Research Funding, Speakers Bureau; Ferrokin: Research Funding.

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