RBC MEMBRANE DEFECTS final presentation.pptx

rbc membrane defects

1. RBC MEMBRANE DEFECTS PRESENTED BY: Dr. PUMMI KUMARI JR (ACADEMICS) MODERATED BY: Dr. JITEDRA SINGH NIGAM ASSISTANT PROFESSOR AIIMS PATNA

2. OVERVIEW RBC membrane deformability and structure. Classification of RBC membrane defects. Hereditary RBC membrane defects. - Hereditary spherocytosis -Hereditary elliptocytosis syndrome - Stomatocytosis Acquired RBC membrane defects. - Paroxysomal nocturnal hemoglobinuria . Other - Acanthocytosis - Echinocytosis -Target cell defects

3. RBC MEMBRANE DEFORMABILITY Biconcave 7 - 8   μ m in diameter. Volume range of 80 to 100 fl (90 fl). Average surface area is 140 mm 2 . This excess surface area-to-volume ratio enables RBCs to stretch undamaged up to 2.5 times their resting diameter as they pass through narrow capillaries and through splenic cords 2 μ m in diameter.

4. WHAT ENABLES RBC TO BE DEFORMABLE?? RBCs biconcave, discoid geometry : Depends on vertical and horizontal interactions between the transmembrane and cytoskeletal proteins. Elasticity of its membrane Cytoplasmic viscosity : Depends on the Concentration of hemoglobin . Maintenance of cell volume by the normal functioning of various channels and pumps on the surface of RBCs. Aquaporin 1 ATP-dependent cation pumps like Na + K + - ATPase pump and Ca 2+ - ATPase pumps.

5. RBC plasma membrane organization The RBC membrane consists of approximately 52% proteins 40% lipids. 8% carbohydrate. *Membrane protein composed of: Transmembrane (integral) Cytoskeletal (skeletal, peripheral)

6. Diagram of Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-Page) of Red Blood Cell (RBC) Membrane Proteins Stained with Coomassie Blue and Periodic AcidSchiff (PAS).

7. CYTOSKELETAL PROTEIN GENE BAND % TOTAL PROTEIN FUNCTION Alpha- spectrin SPTA1 1 16 Filamentous antiparallel heterodimer , primary cytoskeletal proteins. Beta— spectrin SPTB 2 14 Filamentous antiparallel heterodimer , primary cytoskeletal proteins. Adducin ADD1,ADD2 4.9 4 Caps actin filament, binds Ca 2+ / calmodulin Ankyrin ANK1 2.1 4.5 Anchors band 3, protein 4.2, and other proteins in the ankyrin complex to spectrin Beta- actin ACTB 5 5.5 Binds b- spectrin Protein 4.1 EPB41 4.1 5 Anchors the actin junctional complex to spectrin tetramers, RBC cytoskeleton shape Protein 4.2 (protein kinase ) EPB42 4.2 5 Part of ankyrin complex, ATP binding protein Tropomodulin TMOD1 5 Caps actin filament Tropomyosin TPM3 7 1 Stabilizes and regulates actin polymerization

8. TRANSMEMBRANE PROTEIN GENE BAND % OF TOTAL PROTEIN FUNCTION Aquaporin 1 AQP1 Water transporter, Colton antigen Band 3 (anion exchanger, AE1) SLC4A1 3 27 Anion transporter, location of ABH antigens Ca 2+ - ATPase ATP2B1 Ca 2+ transporter Glycophorin A GYPA PAS-1 85% of GP Sialic acid transporter, location of MN blood group antigens Glycophorin B GYPB PAS-4 10% of GP Sialic acid transporter, location of Ss blood group antigens Glycophorin C GYPC PAS-2 4% of GP Sialic acid transporter, location of Gerbich system antigens K + - Cl - cotransporter ATP1A2 K + Cl - transporter Na + K + - ATPase Na + , K + transporter Na + -K + -2Cl - cotransporter Na + , K + , Cl - transporter

9. VERTICAL INTERACTION HORIZONTAL INTERACTION DIAGRAMATIC PICTURE OF RBC MEMBRANE

10. Diagram representing abnormal cell associated with alteration of RBC membrane.

11. Classification of red blood cell membrane defects Hereditary RBC membrane defects Acquired RBC membrane defects Mutations that alter membrane structure: Hereditary spherocytosis Hereditary elliptocytosis syndrome. Mutations that alter membrane transport proteins: Hereditary stomatocytosis Acquired stomatocytosis Paroxysomal nocturnal hemoglobinuria Others: Acanthocytic disorders,Echinocytic disorders, Target cell disorders

12. CASE A 27-year-old woman presented with recurrent abdominal swelling, stunted growth relatively to her siblings and age groups. She has history of blood transfusion in childhood. She also has recurrent yellowish discoloration of the eyes. On examination : palpable spleen tip. Lab investigation : Hb 9g/dl WBC- 6.2x10 9 /L Platelet count - 201x10 3 / dL MCV - 75 fl (80-97) MCH- 29 pg MCHC - 37g/dl RDW- 17% Reticulocyte - 6% Increased S.LDH Increased total bilirubin . Peripheral Blood smear

13. What could be Differential diagnosis??? Hereditary spherocytosis Autoimmune hemolytic anemia Oxidant damage G6PD deficiency Unstable hemoglobin Action of bacterial toxins (Clostridium perfringes lecithinase ) Snake venom. Spherocytes in PBS Direct Coombs test Positive Negative Autoimmune hemolytic anemia Provisional diagnosis: HS Confirms HS Family history Eosin 5 – maleimide (EMA) binding test Membrane protein analysis. Osmotic fragility test Increased Normal or Decreased Rule out enzymopathies and other causes. + -

14. DIRECT COOMB’S TEST PERFORMED = NEGATIVE OSMOTIC FRAGILITY TEST

15. HEREDITARY SPHEROCYTOSIS Hemolytic disorder characterized by Anemia Intermittent jaundice Splenomegaly Responsiveness to splenectomy . Inheritance pattern -75% autosomal dominant. -25% nondominant . Extravascular , intracorpuscular hemolytic anemia . Splenomegaly is rule .

16. PATHOPHYSIOLOGY Occurs due to gene mutations in which the defective proteins disrupt the vertical linkages between the lipid bilayer and the cytoskeletal network. Gene mutations : Ankyrin (ANK1)(most common) Band 3 (SLC4A1) alpha- Spectrin (SPTA1)(most severe) beta- Spectrin (SPTB) Protein 4.2 (EPB42 ). Also excess activity of the Na + -K + ATPase resulting in reduction of intracellular cations.This results in an increase in viscosity and cellular dehydration.

17. LABORATORY FINDINGS Complete blood count results : Decreased hemoglobin Increased MCHC Increased Reticulocyte count Increased RDW Hyperchromic ( hyperdense ) RBCs Peripheral blood film findings: Spherocytes Polychromasia Mild anisopoikilocytosis . Bone marrow: mild erythroid hyperplasia Direct antiglobulin test result -Negative

18. Indicators of hemolysis Mildly decreased serum haptoglobin Increased serum lactate dehydrogenase Increased serum indirect bilirubin Osmotic fragility and incubated osmotic fragility tests Additional tests: Eosin-5’-maleimide binding test by flow cytometry Ektacytometry SDS-PAGE analysis of membrane proteins (most reliable) Molecular studies. Other tests Glycerol lysis test Autohemolysis test Cryohaemolysis test

19. ICSH guidelines for diagnosis of HS Mode of inheritance : dominant in 75% cases, and 25% recessive or nondominant HS. Presentation and observation Neonatal period/Infancy Neonatal jaundice: common in a few days of life. Risk of kernicterus for those with spherocytosis and requiring transfusion. Anemia Splenomegaly B. Childhood/Adulthood Persistent jaundice, anemia , splenomegaly , gallstones Hemolysis : can be compensated in adults Sudden onset of acute anemia : aplastic crisis (Parvovirus B19); acute increase of hemolysis due to viral  infections (e.g. CMV, Herpes 6, EBV) Pregnancy in mild HS: aggravated anemia and return to baseline after delivery  C. Elderly: diagnosed in the 7–9th decade in life, often postinfection   D. Chromosome 8p11 translocation or deletion (close to ankyrin gene): spherocytosis and dysmorphic or neurologic features. E. Fatal hydrops fetalis : rare, more likely to happen with a family history of HS 

20. Red cell indices, morphology, and other parameters Hemolysis indicators: low or no haptoglobin ; raised LDH; hyperbilirubinemia ( unconjugated ) Red cell morphology: Predominantly spherocytes . Mild HS shows occasional spherocytes or only during hemolytic crisis. Anisocytosis in patients with severe hemolytic anemia Red cell indices: Hb : variable, often lower than the normal range. May be normal Increased % hyperdense cells. MCHC in the high range or increased, reflecting RBC dehydration. Reticulocyte count: increased. Low percentage of immature reticulocyte fraction (increased RET/IRF% ratio). Exclusions: Acquired spherocytosis : autoimmune hemolytic anemia or ABO incompatibility (DAT positive) Patients with CDAIIoften presenting with a nondominant HS‐like phenotype Gilbert's syndrome: elevated hyperbilirubinemia : no hemolytic feature except if when co‐inherited with HS (increased risk of gallstones) Infantile pyknocytosis : may require top‐up transfusion in 6–9 months after birth. Resolved with no further intervention. Occasional spherocytes in conditions unrelated to membranopathy : Pyrimidine‐5′‐nucleotidase deficiency (also shows basophilic stippling), G6PD deficiency in crisis; PK deficiency or unstable hemoglobin

21. OSMOTIC FRAGILITY TEST Principle It is the measure of resistance of RBC to hemolysis when exposed to increasingly dilute saline solution. Red blood cells are suspended in a series of tubes containing hypotonic solutions of NaCl , from 0.9% to 0.0%, incubated at room temperaturefor 30 minutes, and centrifuged. The percent hemolysis in the supernatant solutions is measured using photoelectric calorimeter or spectrometer.and plotted for each NaCl concentration. Ax % is the absorbance in the tube being measured, A0.85% is the absorbance in the 0.85% NaCl tube ( 0% hemolysis ) A0.0% is the absorbance in the 0.0% NaCl tube (100% hemolysis )

22. METHOD Heparinised venous blood or defibrinated blood is used. The test should be carried out within 2 h of collection with blood stored at room temperature or within 6 h if the blood has been kept at 4 °C. 5.0ml of each of the 11 saline solutions is delivered into test tubes. Add 5.0ml of water to the 12th tube. Add to each tube 50μl of well-mixed blood . Leave the suspensions for 30min at room temperature. Remove the supernatants and estimate the amount of lysis in each using a spectrometer at a wavelength setting of 540nm or a photoelectric colorimeter. Assign a value of 100% lysis to the reading with the supernatant of tube 12 (water) and express the readings from the other tubes as a percentage of the value of tube 12. Plot the results against the NaCl concentration

23. OSMOTIC FRAGILITY CURVES

24. INCUBATED OSMOTIC FRAGILITY TEST Incubating the blood at 37° C for 24 hours before performing the test allows HS cells to become more spherical and is often needed to detect mild cases. The effect of incubation on erythrocyte osmotic fragility

25. OSMOTIC FRAGILITY IN HAEMOLYTIC ANAEMIAS Decreased OF Increased OF Thalassemia Sickle cell anemia Enzyme abnormalities Hereditary xerocytosis South asian ovalocytosis . Iron deficiency Hereditary spherocytosis (HS) Hereditary elliptocytosis (HE) Hereditary hydrocytosis Autoimmune haemolytic anaemia

26. COMPLICATION OF HS Exacerbation of anemia : occur in almost all HS patients, even in patients who have mild or clinically silent disease associated with various stresses Infection Major surgery Trauma Pregnancy Exhaustion of folate reserves by the sustained increase in net DNA synthesis. Aplastic / hypoplastic crises: from parvovirus B19 infection. Cholelithiasis Hemosiderosis and multiple endocrine disorders resulting from transfusion-induced iron overload.

27. TREATMENT Symptomatic treatment. Splenectomy Management of postsplenectomy complications. Red Blood Cell Morphology in Hereditary Spherocytosis after splenectomy .

28. CASE 12-year-old boy presented with a history of easy fatigability accompanied by breathlessness on exertion for 6 months and fever for 5 days. H/O: 2 Blood transfusion. Family history of anemia and jaundice. O/E: poorly built, Pallor ++ with mild icterus , mild hepatomegaly and moderate splenomegaly but no evidence of clubbing, edema or lymphadenopathy . CBC report: Hb %-8.2g/dl MCV-94.7 fl MCH- 28.9 pg MCHC- 30.5 g/dl TLC and Platelet count : WLN RDW: normal Corrected Reticulocyte count -7 %. LFT : increased both conjugated and unconjugated bilirubin with normal AST & ALT levels and mild hypoproteinemia . USG: moderate splenomegaly . PBS OFT was normal with fresh red cells, but when red cells incubated for 24 hours at 37 degree Celsius showed increased OFT. DIAGNOSIS????

29. HEREDITARY ELLIPTOCYTOSIS SYNDROME Group of genetically determined erythrocyte disorders characterized by elliptical red cells on the PBS.

30. PATHOPHYSIOLOGY Membrane protein defects: α - and β - spectrin Protein 4.1 Glycophorin C Defective horizontal interaction between cytoskeletal membrane protein Decreased RBC ability to withstand mechanical stress. RBCs are biconcave at first but become elliptical after repeated distortions by passage through small capillaries Membrane loss and a decrease in surface area-to-volume ratio. Damaged RBCs become trapped or acquire further damage in the spleen. Extravascular hemolysis and anemia .

31. CONDITION DEFECTIVE PROTEIN PATHOPHYSIOLOGY TYPICAL RBC MORPHOLOGY CLINICAL FINDINGS/COMMENTS HEREDITARY ELLIPTOCYTOSIS ( Autosomal dominant) a- Spectrin (SPTA1) b- Spectrin (SPTB) Protein 4.1 (EPB41) Mutation in protein that disrupts the horizontal linkages in the cytoskeleton; loss of mechanical stability of membrane 90% of cases asymptomatic; 10% of cases show moderate to severe anemia HEREDITARY PYROPOIKILOCYTOSIS (rare subtype of hereditary elliptocytosis ) (AR) a- Spectrin (SPTA1) b- Spectrin (SPTB) Homozygous or compound heterozygous Mutation in spectrin that disrupts horizontal linkages in cytoskeleton Severe anemia

32. CONDITION DEFECTIVE PROTEIN PATHOPHYSIOLOGY TYPICAL RBC MORPHOLOGY CLNICAL FINDINGS/COMMENT SOUTHEAST ASIAN OVALOCYTOSIS ( Autosomal dominant) Band 3 (SLC4A1) Mutation in band 3 that causes excessive membrane rigidity; only exists in heterozygous state Asymptomatic or mild hemolysis ; prevalent in some areas of Southeast Asia

33. LABORATORY VALUES 1.CBC Hb Reticulocyte Mcv 2.Peripheral smear- elliptocytes 3.RBC destruction (increased LDH, bilirubin ) 4.OF test 5.EMA Binding test 6.Ektacytometry

34. ICSH GUIDELINES FOR HE: Ethnicity: higher prevalence in countries in the malaria-endemic regions: probable protection against malarial infection. . Classification based on clinical severity : Asymptomatic carrier state, nonhemolytic HE: normal Hb level and normal reticulocyte count. HE with varying degrees of hemolysis : normal Hb but raised reticulocyte count in mild hemolysis ; reduced Hb and raised reticulocyte counts in severe hemolysis . HE with transient infantile poikilocytosis : transfusion dependent up to the first year, and evolving quickly into the milder phenotype of the HE parent. Hereditary pyropoikilocytosis (HPP): severe transfusion-dependent hemolytic anemia , starting in the neonatal period with Hb of 60–100 g/L and poikilocytosis . Fatal hydrops fetalis : only one case due to homozygosity in b- spectrin mutation Sor mild elliptocytosis can be exacerbated by an infection (e.g. CMV) Red cell morphology: Elliptocytes present in asymptomatic and nonhemolytic HE Hemolytic HE: increasing severity in hemolysis results in red cell fragmentation, showing poikilocytosis due to a decreased membrane mechanical stability HPP: severe microspherocytosis , micropoikilocytosis . MCV between 50 Exclusions: Iron deficiency anemia, thalassemia , and other causes of acquired elliptocytosis . Blood group Leach phenotype (Ge-2,-3,-4) in the blood group Gerbich ( Ge ) system: Individuals with Gerbich null phenotype are hematologically well and healthy. Their elliptocytic RBCs lack both GPC/D and 20% reduction of protein 4.1.

35. A: Micropoikilocytes and elliptocytes in a neonate with transient poikilocytosis and an a- spectrin gene mutation. B: Same child at 7 months of age, now exhibiting morphology of common hereditary elliptocytosis . C: Compound heterozygous hereditary elliptocytosis due to two a- spectrin self-association–site structural mutations D: Hereditary pyropoikilocytosis .

36. STOMATOCYTOSIS Stomatocytes are erythrocytes with a central slit or mouth-shaped (stoma) area of central pallor. HEREDITARY: Hereditary hydrocytosis Hereditary xerocytosis Cryohydrocytosis Rh -Null disease Familial pseudohyperkalemia Familial deficiency of HDL(Tangier disease) ACQUIRED: Acute alcoholism Hepatobiliary disease Vinca alkaloid administration Artifactual

37. CONDITION DEFECTIVE PROTEIN PATHOPHYSIOLOGY TYPICAL RBC MORPHOLOGY CLNICAL FINDINGS/COMMENT Overhydrated hereditary stomatocytosis (hereditary hydrocytosis ) (AD) Rh -associated glycoprotein (RHAG), Stomatin ( transmembrane protein) Increased membrane permeability to Na + & K + High intracellular Na causes influx of water, inc cell volume (inc. MCV), and decreased cytoplasmic viscosity (decreased MCHC) Stomatocytes (5%–50%), macrocytes Moderate to severe hemolytic Anemia,splenomegaly . Dehydrated hereditary stomatocytosis (hereditary xerocytosis ) (AD) Piezo -type mechanosensitive ion channel component 1 (PIEZO1) Increased membrane permeability to K +’ low intracellular potassium causes loss of water from cell, decrease in cell volume (decreased MCV), and increased cytoplasmic viscosity (increased MCHC) Target cells, burr cells, stomatocytes (<10%), RBCs with “ puddled ” hemoglobin at periphery, desiccated cells with spicules Mild to moderate anemia, splenomegaly ; may lead to fetal loss, hydrops fetalis ; may be accompanied by pseudohyperkalemia

38. HEREDITARY XEROCYTOSIS HEREDITARY HYDROCYTOSIS

39. CASE A 24-year-old woman presented to physician for evaluation of new tea- colored urine noticed intermittently over the past five days and abdominal pain and yellowish discolouration of eyes. Her last menstrual cycle was two weeks ago and regular. No history of renal stones, NSAID abuse, weight loss, night sweats, fever, melena or hemoptysis .   CBC -WBC count- 3600/mm 3 , Hb %- 4 g/ dL Platelet count- 60,000/ mm 3 MCV -75 fl RDW 28, Retic %- 10.9 LFT- Total S.bilirubin - 7.5 mg/ dL ( indirect -5.5) AST- 213 U/L serum LDH 1500 U/L serum haptoglobin -10 mg/ dL .  Urinalysis- hemoglobinuria . Direct Coomb test is negative. USG: no flow in hepatic veins, compressed IVC, enlarged caudate lobe, splenomegaly , and varices at the splenic hilum .

40. FLOW CYTOGRAM CD 55 PE CD 59 FITC

41. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Chronic intravascular hemolytic anemia caused by an acquired clonal hematopoietic stem cell mutation of PIGA gene(Xp22.1). Both are inhibitors of complement on the surface of RBC, WBC and platelet. This result in increased complement mediated lysis Intracorpuscular intravascular hemolysis . Codes for GPI anchored protein MIRL (CD-59) DAF (CD-55)

42. PATHOPHYSIOLOGY

43. ***Patients with PNH also display phenotypic mosaicism resulting in three RBC phenotype. Phenotypic Designation Complement Sensitivity GPI-AP Expression by Flow Cytometry Type of PIGA Mutation PNH I NORMAL NORMAL NONE PNH II Moderately sensitive (3–4 times normal) DIM POSITIVE Missense (partial PIGA inactivation) PNH III Markedly sensitive (15–20 times normal) NEGATIVE Nonsense, frameshifts , deletions, insertions (complete inactivation of PIGA)

44. CLASSIFICATION OF PNH

45. CLINICAL TRIAD OF PNH Related to Intravascular Hemolysis • Anemia • Hemoglobinuria • Chronic renal failure • Cholelithiasis Esophageal spasm Erectile dysfunction Related to Thrombosis • Venous thrombosis • Abdominal vein thrombosis: hepatic (Budd- Chiari syndrome), splenic , renal veins • Portal hypertension • Cerebral vein thrombosis • Retinal vein thrombosis and loss of vision • Deep vein thrombosis, pulmonary emboli Arterial thrombosis (less common) • Stroke • Myocardial infarction Related to Bone Marrow Failure • Pancytopenia : fatigue, infections, bleeding • Myelodysplastic syndrome ???????? Free Hb released during intravascular hemolytic episodes rapidly scavenges and removes nitric oxide (NO). The decreased NO can manifest as smooth muscle dystonia or platelet activation and thrombosis. Hemoglubinuria – Nocturnal (25% of cases) ???? At night acidic pH activates complement. Bone marrow failure ?? Due to complement mediated lysis of hematopoietic cells.

46. LAB EVALUATION AND DIAGNOSIS CBC and PBS Tests for evidences of intravascular hemolysis - S.haptoglobin , LDH,S.bilirubin . Urine analysis Iron studies Direct antiglobulin test. Bone marrow examination. Flow cytometry FLAER Others: Sugar water test (sucrose hemolysis test) Hamtest (acidified serum lysis test)

47. Flow cytogram of blood from a normal individual. Flow cytogram of blood from a patient with PNH.

48. PNH is considered under following conditions : (1) Signs and symptoms of intravascular hemolysis (abnormally high LDH) of undefined cause (Coombs-negative) with or without macroscopic hemoglobinuria often accompanied by iron deficiency. (2) Pancytopenia in association with hemolysis . (3) Venous thrombosis affecting unusual sites, especially intra-abdominal, cerebral, or dermal locations accompanied by evidence of hemolysis . (4) Unexplained recurrent bouts of abdominal pain, low backache, or headache in the presence of chronic hemolysis . (5) Budd- Chiari syndrome. Differential diagnosis: Antibody-mediated hemolytic anemias , especially paroxysmal cold hemoglobinuria . HEMPAS (Hereditary Erythroblastic Multinuclearity with a positive acidified serum lysis test, or Congenital Dyserythropoietic Anemia type II).

49. High sensitivity flow cytometry for diagnosis of PNH Disadvantages: They underestimate the percentage of type III cells because RBCs lacking CD59 undergo rapid complement lysis in the circulation. Cannot accurately determine the percentage of PNH RBCs after recent transfusion.

50. Diagnosis of PNH is made by u sing multiparameter flow cytometry with FLAER( fluorescein -labeled proaerolysin variant ) in combination with monoclonal antibodies to GPI-anchored antigens and lineage-specific antigens on WBC (two lineages (usually granulocytes and monocytes )). Four-color protocol using FLAER, CD24, CD15, and CD45 for granulocytes & FLAER, CD14, CD64, and CD45 for monocytes .

51. (A)Fluorescence intensity of RBC from a healthy control after staining with anti-CD59. (B) Fluorescence intensity of RBC from an patient with PHN after staining with anti-CD59. (C) Fluorescence intensity of granulocytes from a healthy control stained with FLAER. (D) Fluorescence intensity of granulocytes from the same patient with PHN as in B after staining with FLAER.

52. ACANTHOCYTIC DISORDER (SPUR CELLS) Acanthocytes – dense contracted RBCs, irregularly spaced thorny projections CAUSES – 1.ACQUIRED- Liver diseases Malnourished states Hypothyroidism Post- splenectomy 2.HEREDITARY – Neuroacanthosis syndromes comprising of ( i ) Abetalipoproteinemia (AR) (ii) Mcleod phenotype(X linked) (iii) Chorea acanthosis (AR)

53. ECHINOCYTIC DISORDERS (BURR CELLS/CRENATED CELLS) ECHINOCYTES – RBCs with numerous uniform spicules distributed equally through out the cell surface Preferential expansion of outer lipid bilayer relative to inner layer CAUSES – Hypophosphatemia Liver disease Pyruvate kinase deficiency Uremia (Chronic renal disease) Drugs – Calcium, Other drugs(ATP depletion) Artifactual

54. TARGET CELL DISORDER Target cells are discoid RBCs with a centralized hemoglobinized area in the clear center , resembling a bull’s eye or target. Increase in cell surface area to volume ratio CAUSES – 1.Increased surface area Obstructive jaundice 2.Decreased volume Decreased Hb synthesis – Thalassemia ,IDA Structural mutations of Hb (S, C, D, E) Primary disorders of cell hydration Decreased OF

55. ALETRATION OF ERYTHROCYTE IN LIVER DISEASE