Anaemia, Pathophysiology, Types and Disorders.pptx

Hematology notes

1. Anaemia Pathophysiology of anaemia Types of Anaemia Disorder of haemoglobin

2. INTRODUCTION Anemia  is defined as a low number of red  blood cells . In a routine blood test, anemia is reported as a. low hemoglobin or hematocrit   Normal haemoglobin range differs with age and gender Physiological condition such as pregnancy have an effect on haemoglobin

3. Classification of Anaemia based on aetiology: Blood loss anaemia (acute or chronic) It takes several hours for HB to fall after excessive bleeding Hypo proliferative anaemia (low cell count) Inability to produce an adequate number of erythrocytes in response to appropriate signals; Characterized by low reticulocyte count Disorder may be due to hypo proliferation of precursor cells in the bone marrow (BM) or Due to abnormal maturation of precursors in the BM Commonly caused by iron deficiency.

4. Haemolytic anaemia::Causes vary. It is either genetical (congenital or inherited) hemoglobinopathies, enzymopathies (predominantly glucose-6-phosphate dehydrogenase [G6PD] deficiency), and membrane disorders Acquired autoimmune hemolytic anemia , microangiopathic hemolytic anemia , hemolysis related to infections, and acquired membrane disorders such as those caused by liver disease (spur cell of anemia ) and paroxysmal nocturnal hemoglobinuria ( Spur cell anemia (SCA) is an acquired form of non-autoimmune hemolytic anemia that occurs in advanced liver disease)

5. Haemolytic anaemia: Manifest with elevated reticulocyte counts. Other characteristic of haemolytic anaemia are: Elevated lactate dehydrogenase level (LDH) Increase unconjugated (indirect) bilirubin level Decreased Hb levels. In many anaemias cause by both failed bone activity and loss of RBC cause anaemia e.g chronic bleeding from the gastrointestinal tract red cells are lost from the circulation and iron deficiency prevents erythropoiesis

6. HAEMOGLOBIN Intracellular protein that give blood its characteristic red colour. In vertebrates, Hb is found in the red blood cells (erythrocytes) RBC’s major function is to encase and protect Hb . it can function as an oxygen transporter for a prolonged period. Transport CO 2 from the tissue to the lungs Comprises of two pairs of unidentical polypeptides (tetramer) with each associated with an iron group (bind O 2 ) Two alpha chains (141 amino acids) Two beta ()chains (146 amino acids)

7. Haemoglobin is encoded by two gene cluster located in chromosome 11( β –like globin ) and chromosome16 ( α -like globin ) Each gene cluster contains gene coding for different types Hb globin chain as shown below:

8. Haemoglobin :

9. Inheritance of Globin Gene Two gene of β copies are inherited, one in each chromosome 11, thus genotype of an individual presented as β/β Each chromosome 16 carries two copies of α gene, thus genotype represent as αα / αα

10. ~ Haemoglobin (HB) Expression

11. Haemoglobin (HB) Expression Different members of β –like and α -like gene clusters are produced during development. Complete Hb composition ( β –like and α -like) at during development.

12. Haemoglobin Disorders (hemoglobinopathies) Hemoglobin disorders are a group of inherited conditions that affect a person's red blood cells . Genetic mutation affect Hb synthesis in one of two ways; Quantitative: mutation reduce synthesis of Hb chain, but structure remain intact. Qualitative: Normal or near normal Hb Is produced but mutation causes a structural and functional defect , Consequence: imbalance between the production of α and β chains (thalassemia) Hb structural defects (sick cell anaemia)

13. THELASSEMIA Involves defect in globin-chain synthesis i.e. reduction gene expression. There is reduced or no production of one or more globin chains, i.e. α and β chains (most common) ε, γ, and ζ chains severe impairment is lethal for the foetus, but not yet obverse in humans

14. Different thalassemia condition have been identified They are named based on the globin chain(s) with reduced or absent synthesis Classified based on Hb chain that is adversely affected α-thalassemia β-thalassemia

15. Consequence of impaired globin chains production Reduced production of functioning Hb tetramers characterised by Hypochromia (pale RBCs) and microcytosis (small-sized RBCs) Unbalanced synthesis of the individual α- and β-subunits causes an imbalance of α/β ratio of 1:1

16. The unaffected globin chains continues to be produced and accumulate in the cell as unpaired globin chains. Unpaired chains damage RBCs or their precursors, leading to premature destruction and worsen anemia

17. β-thalassemia: molecular pathology Include all thalassemia caused by reduced expression members of the β cluster, esp. β globin Cause by mutation affecting any step of globin gene ( β chain ) expression i.e. Transcription, mRNA processing (mRNA), translation and posttranslational integrity of the β-polypeptide chain Mostly caused by point mutations (>200)in gene regulation elements e.g. promoter Absence of the synthesis of β-globin chains (β -thalassemia) a reduction in synthesis (β + -thalassemia)

18. β -thalassemia mutation denotations: β = No β chain production (β / β homozygous state) i.e No Hb A ( α 2 β 2 ) produced. β + = partial β globin chain deficiency β sillent = carrier state δ β = No δ and β chain production ( δ β / δ β : homozygous state) : )

19. β-thalassemia pathophysiology Unpaired, excess α chains precipitate in the developing RBCs, forming inclusion bodies; Oxidative stress arise and damage to cellular membranes. Apoptosis is triggered, Damaged and apoptotic RBC precursors are phagocytized and destroyed in the bone marrow by activated macrophages. This cause ineffective erythropoiesis i.e. premature death of RBC precursors in the bone marrow The bone marrow is unable sufficient viable RBCs For the few RBC released, they readily sequestered and destroyed by macrophages in the spleen

20. β-thalassemia the anaemia is multifactorial ( with different players contributing to the severity of anemia and secondary complications) and results from ineffective production and increased destruction Severe cases of β -thalassemia are asymptomatic during foetal up to 6 months after birth. Because Hb F ( α 2 γ 2 ) is the predominant circulating However, symptoms start after 6 month of age when Hb F γ is replaced by β - globin chain

21. β-thalassemia It is divided into four categories based on clinical manifestation: β- Thalassemia major (severe form) β- thalassemia intermedia: β -t halassemia minor β -t halassemia salient carrier Clinical manifestation depends on: whether one or both of the β -globin genes are affected. the extent to which the affected gene or genes are expressed i.e No production ( β ), 5 to 30% reduction ( β + ) or marginal reduction ( β salient )

22. β-thalassemia major: Reduced or No β -globin synthesis whereas α chain levels are produced. Sever anaemia requiring regular transfusion. Diagnosis between 6 months and 2 years of age when Hb A ( α 2 β 2 ) does not increase in place of HbF ( α 2 γ 2 ) Little or NO Hb A ( α 2 β 2 ) is low

23. β-thalassemia major: peripheral blood film shows marked Basophilic stippling Anisocytosis (RBCs with unequal sizes), Also observed: Reduced erythropoiesis in the Bone marrow (hyperplasia) Stippling: pptn of ribosomal RNA

24. β-thalassemia major: Treatment Mainly Blood transfusion when Hb falls <7g/dL RBCs that are less than 7 to 10 days old are used for transfusion to allow for maximum donor RBC survival in the patient Alternatively, hematopoietic stem cell transplantation (HSCT) Gene transfer (introduction of β gene using lentiviral based vector gene transfer accomplished in 2010)

25. β-thalassemia Minor ( ThalassemiaTrait ) Occurs when only one β -globin gene is affected (abolish or reduce expression) and other β -globin gene is normal (heterozygote state: β / β or β +/ β ) Is asymptomatic anemia Hb concentration is either normal or slightly reduced (1 or 2 g/dL lower than normal Hb level) RBC count is increased or normal. Red blood cells are hypochromic and microcytic.

26. Reticulocyte count is normal or slightly elevated δ- globin expression is up-regulated δ-chains will join the normally produced β -globin chains to form HbA 2 (α 2 δ 2 ) In some cases individual carry this also show slightly increased HbF levels. Severity of β-thalassemia trait increase during pregnancy Folic acid supplementation help increase Hb levels Iron supplementation may be given to avoid compounding this form of anemia

27. β-thalassemia Intermediate Has balanced globin chain synthesis decrease in α-globin chain synthesis as well as β-globin chain synthesis, or an increase in γ-globin chain synthesis Balance synthesis is attributed t the milder clinical phenotype of thalassemia intermediate Unlike thalassemia major, Individuals are to maintain a Hb level compatible with comfortable survival in the absence of regular transfusions

28. Thromboembolic (blockage of vessel by a clot released from a different clot site)events common complication: include stroke, pulmonary embolism, portal vein thrombosis, and deep vein thrombosis of the legs Hypercoagluation contribute to the pulmonary hypertension that commonly occurs in patients with thalassemia intermedia and is the primary cause of congestive heart failure Splenectomy is risk factor of Thromboembolic events (due to prolong circulation of damaged RBCs that generate increased amounts of thrombin)

29. β- thalassemia silent state includes the various heterogeneous β -globin gene mutations that produce only a small decrease in production of the b chains.) Individuals have nearly normal a-b chain ratios and no hematologic abnormalities They present with a mild b-thalassemia intermedia phenotype with an increased level of Hb F and Hb A 2

30. α - thalasemia Usually caused by large deletion of one, two, three or all four α -globin genes (involve α 1 & α 2 gene) NB: each chromosome 16 has two α -globin genes

31. Deletion of α- globin is designated as follows α + = indicate deletion of either α 1 or α 2 gene ) α = indicate deletion of both α 1 and α 2 gene (also called - - homozygous ) Although rare, α -thalassemia can also be caused by point mutations. i.e designated as α T

32. α -thalassemia There are four different kinds α- thalassemia syndromes α + - thalassemia: (one of the four α-globin genes fails to function) α - thalassemia: (two of the four α-globin genes fails to function) Hb H disease: (three of the four α-globin genes fails to function) Hb Bart: (all four four α-globin genes fails to function)

33. Clinical manifestation: silent carrier ( α + - thalassemia trait ) Only one α -globin gene is deleted leaving three functional ones. Denote as – α / αα The α / β globin chain ratio is nearly normal, and no hematologic abnormalities are present Deletion of one α -globin gene causes slight decrease in α chain synthesis No consistent hematologic manifestations. RBCs are not microcytic, and Hb A2 and Hb F are normal. common in Melanesia, as well as in Southeast Asia and in African Americans

34. Clinical manifestation: α- Thalassemia Trait Also called α-thalassemia minor. Caused by deletion of two genes It exists in two forms: homozygous either α + (_ α/_ α ) OR α ( _ _/ α α) α°-thalassemia trait characterised by Levels of Hb A 2 in the low to low normal range (1.5–2.5%) and β/α synthetic ratios averaging 1.4 : 1 It asymptomatic and characterised by mild anaemia (Hb = 12 to 13 g/dL) with microcytic, hypochromic RBCs

35. No consistent hematologic manifestations. Microcytosis is present in cord blood erythrocytes Hb H is not detected in hemolysates of peripheral RBCs, probably because of rapid proteolysis of Hb H or free β-globin chains

36. Clinical manifestation: Hemoglobin H Disease Deletion of three α-globin gene ( _ _/_ α) Associated with a moderately severe but variable anaemia It is characterized by the accumulation of excess unpaired b chains that form tetramers of Hb H in adults. clinical phenotype may be considerably milder in some patients and severe enough to cause hydrops fetalis in others. occurs predominantly in Asians and occasionally in whites (Mediterranean) but is rare in persons of African ancestry Adults develop some of the same complications as β-thalassemia including osteoporosis, cholelithiasis, and iron overload haemolysis occurs because Hb H is unstable and precipitates within the circulating RBCs

37. Concomitant iron deficiency may reduce the amount of Hb H in the patient’s RBCs It is associated with mental retardation

38. Clinical manifestation: Hydrops Fetalis With Hb Bart Caused by deletion of all α globin genes occurs almost exclusively in Asians, especially Chinese, Cambodians, Thais, and Filipinos. fetuses are usually born prematurely and are either stillborn or die shortly after birth presence of Hb Portland (ζ2γ2) prevent infant death early in pregnancy. Marked anasarca swelling of the whole body and enlargement of the liver and spleen are present Severe anemia (3–10 g/dL) RBC are microcytic and hypochromic Infants have hepatosplenomegaly (spleen and liver swelling) This is a reflection of severe congestive heart failure and hypoalbuminemia in utero caused by anemia

39. Structural haemoglobin variants There >1000 abnormal haemoglobin variants have been describe, but most are rare. Most are caused by single amino acid substitution. Sickle cell anaemia is one of the best characterised structural haemoglobin variants.

40. Sickle cell Anemia Define by a characteristic RBC shape Caused an amino acid substitution (Glutamate → Valine ) in the 6 th amine acid position of β-globin. Amino acid change cause Hb structural change since valine is hydrophobic Normal hemoglobin = the major protein which fills red blood cells Carries oxygen from the lungs to body tissues Carries carbon dioxide away from body tissues to the lungs

41. Molecular basis: Sickle cell anemia The sickle cell mutation causes aggregation/polymerization of HbS after deoxygenated state. Result: RBC with a sickle shape

42. In homozygous state (mutation in both β chains) sickling start when O 2 saturation of Hb decreases by 80% In heterozygous state (mutation in one β chains) RBC sickle when O 2 saturation of Hb decreases by 45% Meaning 80% of homozygote Hb ( HbSS ) is deoxygenated And polymerizes to form the characteristic sickled RBCs. For heterozygote only 45% HbS is available HbSS saturated by 20% of oxygen that saturate normal HbA

43. Consequence of HbS polymerization and RBC sickling: Increase blood viscosity Slow blood flow: prolong HbS carrying RBC to hypoxia. Increased blood pH: promote further RBC sickling Overall outcome Blocking of capillaries and arterioles by sickled RBCs and Infarction (blocking blood supply) of surrounding tissue

44. Two forms of Sickle cells Reversible sickle cells Circulate as normal cells when oxygenated Sickle upon deoxygenation Associated with vasoocclusive complications because they are able travel into the microvasculature. Irreversible sickle cells : do not change their shape regardless of the change in oxygen tension or degree of haemoglobin polymerization Seen in peripheral blood as elongated cells with pointed ends Removal in the spleen prevent them for entering the microcirculation.

45. Other factors RBC sickling Efflux of ions (K+, Cl, ) form HbS carring RBCs Caused by damaged RBC membrane ion channels Lead to dehydration of RBC In turn, increasing intracellular HbS concentration HbS polymerises and RBC sickle is increased redistribution of RBC membrane phospholipids (PS) HbS RBCs have increased membrane PS on surface: Promote adherence of RBC to vessel well& contribute to vasoocclusive crisis, activation of coagulation, and decreased RBC survival As well as stroke, and acute chest syndrome.

46. Sickel cell Disease (SCD) Term used describe a group of symptomatic hemoglobinopathies that have in common sickle cell formation and the associated crises. can either be: homozygous for Hb S (SS) or are compound heterozygotes expressing Hb S in combination with another hemoglobin β chain mutation like Hb C or β -thalassemia SCDs in the most common hemoglobinopathy: Most common SCD are Hb SS and Variants Hb SC and Hb S– β -thalassemia (Hb S–b- thal )

47. Sickel cell Disease (SCD) The hallmark of SCD is vasoocclusive crisis (VOC), which accounts for most hospital and emergency department visit

48. Sickle cell trait refers to the heterozygous state (Hb AS): one normal and one sickle β chain Individuals with sickle cell trait are generally asymptomatic. no significant clinical or hematologic manifestations RBC sickling and vasoocclusion occur under extreme occur under extreme hypoxia. Other cause of RBC sickling include Severe respiratory infection, unpressurized flight at high altitudes, Anesthesia in which pH and oxygen levels are sufficiently lowered RBCs carrying HbAS have normal shape.

49. Other hemoglobinopathies Hemoglobin C Described after sickle cell Anemia . Cause by amino acid substitution at position 6 (Glutamate to lysine) of β chains Like HbS , Hb C polymerizes at low oxygen tension but structure differs for HbS . Hb S polymers are long and thin, whereas the polymers in Hb C form a short, thick crystal within the RBCs Short HbC polymers do not alter RBC shape. less splenic sequestration and hemolysis of HbC RBCs vasoocclusive crisis does not occur

50. Hemoglobin-C- georgetown Aka. Hb C-Harlem. Caused two amino acid substitution of of β chains termied as Hb C-Harlem or Hb C-Georgetown) ; Position 6 (Glutamate to valine) Position 73: asparagine to aspartate Asymptomatic in the heterozygote state Patients with both Hb C-Harlem and HbS , clinical manifestation of HbSS disease.

51. Hemoglobin E Rare in Africa Cause by amino acid substitution at position 26 (glutamate to lysin) of of β chain. Hemoglobin polymerization does not occur. Amino acid substitution cause alternative splicing of mRNA, thus it is both a qualitative defect (defective β chain produced) and quantitative defect with a β -thalassemia phenotype (decreased β chain production)

52. Hemoglobin D A group of 16 β chain variants Named based on the location where they were identified e.g. Hb D-Punjab and Hb D-Los Angeles= position121 (glutamate → glutamine) No sickling of RBCs Heterozygote is asymptomatic Homozygote (Hb DD disease) is symptomatic Marked by mild hemolytic anemia and chronic nonprogressive splenomegaly.

53. Thalassemia associated with Hb structural variants Hemoglobin-S Thalasemia due to the coinheritance of two abnormal β -globin genes for Hb S and an α -thalassemia haplotype HbSS α + thalassemia is common since HbS and α + common in the population. It is a compound heterozygous condition that results from the inheritance of a β -thalassemia gene from one parent and an Hb S gene from the other Clinical manifestation depends on the type of β -thalassemia mutation inherited Hb S- β +-thalassemia produce variable amounts of normal β chain

54. Hemoglobin-S- β° - Thalasemia Distinguished from sickle cell anemia by the presence of microcytosis, splenomegaly, an elevated Hb A 2 level, and an Hb A level that is less than the Hb S level Interraction of S and thalassemia result to a more severe sickle cell trait which resemble sickle cell anemia Typically, there is mild hemolytic anemia with splenomegaly.

55. Hemoglobin C-Thalassemia produces moderately severe hemolysis , splenomegaly, hypochromia, microcytosis, and numerous target cells ( codocytes ). is minimal or no β chain production Codocyte are RBCs that appear as a shooting target.

56. Hemoglobin E-Thalassemia Prevalent in Southeast Asia and Eastern India Hb E is due to a change in the splice site of β -globin gene thus decreased production of Hb E. homozygous state (Hb EE) the clinical symptoms are similar to a mild β -thalassemia. Coinheritance of both HbE & thalassemia causes marked reduction of β chain production