Hemoglobin Structure.
Hemoglobin transports oxygen to the tissues. Each RBC contains hemoglobin. A normal hemoglobin molecule consists of: Four globin chains (2 alpha, 2 beta). Each globin chain has an iron containing heme molecule. The iron in the heme molecule binds to oxygen.Genetics.
In the first 8 weeks of embryonic life the predominant forms of hemoglobin are:Hb Gower 1 (ζ2ε2).Hb Gower 2 (α2ε2).Hb Portland 1 (ζ2γ2).By the 12th week embryonic hemoglobin is replaced by Hb F (α2γ2) which represents 70 – 100% of hemoglobin in fetal life.Genetics (2).
Adult hemoglobin Hb A (α2β2) detectable from 16/40, replaces Hb F as predominant hemoglobin by 6/12 after birth, up to 30% of Hb in fetal life.Hemoglobin HbA2 (α2Δ2) is present in utero but only very minor in normal adults. In normal adults 96 – 98% of hemoglobin is HbA, Hb A2 (2 – 3%) and HbF (<1%) constitute a minor component of the total hemoglobin.α2Δ2 α2γ2
Geography.Commonest genetic defect world wide with an estimated 269 million carriers. 90 million carriers in South East Asia, 85 million in Sub Saraharan Africa, and 48 million in the West Pacific region. Distributed across South East Asia in a line stretching from Southern China down the Malaysian Peninsula to Indonesian islands.
Geography (2).
Also distributed across the Mediterranean, Middle East, and Indian Subcontinent. The distribution of the defect is thought to be due to partial protection for carriers from Plasmodium Falciparum Malaria.Electrophoresis
Electrophoresis is a means of separating hemoglobin's. It depends on the migration of the hemoglobin molecules dissolved in a buffer on, or in, a supporting medium when an electric current is passed through them.Principle of cellulose acetate
In an alkaline pH (8.2-8.6) Hb is a negatively charged molecule and will migrate toward the anode (+). The various Hbs moves at different rates depending on their net negative charge, which in turn is controlled by the composition (amino acids) of the Hb molecule (globin chain).
Principle of cellulose acetate
The red cell hemolysate (red blood cell membranes are destroyed to free the Hb molecules for testing) is placed in a cellulose acetate membrane, which is positioned in an electrophoresis tray with the inoculated hemolysate near the cathode (-).HEMOGLOBINOPATHIES
Definition : Hemoglobinopathies are inherited disorders in which Mutation in or near the globin genes alter the structure of amino acid sequences or the rate of synthesis of a particular globin chain. Hb-S B6 glu val Hb-C B6 glu lysDefective haemoglobin
Sickle cell anaemiaIt results from single base change in the DNA coding for the amino acid in the sixth position in the b-globin chain.This leads to an amino acid change from glutamic acid to valine HbS will be formed instead of the normal Hb.INTRODUCTION
Sickle Cell Anemia is a hereditary disease which is cause by a disorder in the blood, a mutation in the Hemoglobin Beta Gene which can be found in the chromosome 11. This disease causes the body to make abnormally shapes red blood cells. A normal red blood cell is shaped as a round donut while the abnormal red blood cell has a “ C “ form.Sickle cell anaemia
Hb S is insoluble and forms crystals when exposed to low oxygen tension. Deoxygenated sickle Hb polymerizes into long fibrils. The red cells sickle may block the different areas of the microcirculation or large vessels causing infarcts of various organs. It is widespread in Africa. Individuals with sickle-cell trait are relatively resistant to the lethal effects of falciparum malaria in early childhood.INTRODUCTION CONT’
Hemoglobin Beta Gene (HB-B) also known as Beta Globin is a protein that resides in the red blood cells. The HBB is 146 amino acids long and its molecular weight is 15,867 Daltons. The molecules of the hemoglobin are responsible to carry oxygen through the body. The HBB is found in part 15.5 of the chromosome 11.Pathophysiological effects of sickled cells
1.Extravascular hemolysis . 2. Loss of splenic function. 3.Anaemia. 4.Compromisation of the microcirculation.CLINICAL MANIFESTATION
1.complication from moderate to severe anaemia 2.slowed growth and development . 3.cardiac over load leads to CHF . 4.Bilirubin stones and cholecystitis . 5.Aplastic crisis.Sickle cell crisis.
1.Splenic crisis (splenic sequestration syndrome, auto splenectomy) 2.Infections. 3.CNS and ophthalmic events (CVA, proliferative retinopathy). 4.Acute chest syndrome (chromic pulmonary hypertension lead to cor-pulmonale). 5.GIT : diffuse abdominal Pain. 6.Genitourinary symptoms: - Painless haematuria. - hyposthenuia. - priapism. - hypogonadism.Sickle cell crisis Cont.
7.skeletal complication - hand-foot syndrome. acute arthritis. aseptic necrosis of the head of femur. osteomyelitis . 8.Skin changes lead to chronic non-healing ulcerDIAGNOSIS
Peripheral blood smears : sickled cells, target cells, Howell-Jolly bodies, normoblast, red cell fragment, increase platelet and occasionally leukocytosis. screening test : sickling test The presence of HbS can be demonstrated by exposing red cells to a reducing agent such as sodium dithionite; HbA gives a clear solution,whereas HbS polymerises to produce a turbid solution. Definitive diagnosis by Hemoglobin electrophoresis (Hb-S = 87% , Hb-F = 9.7%, Hb-A2 = 3.3%)Howell Jolly Body
ErythroblastHaemoglobin Electrophoresis (alkaline pH )
Hb C
Hb S
Moves in same position as Hb A2
HbA
Anode
Haemolysate applied
Cathode
δ δ α α α s s α α γ γ α Hb S
Hb A2Hb F
Genotype αααα βsβs δδ γγ Haemoglobins Produced :
Diagnosis: Hb SS Disease
Laboratory diagnosis of sickle cell anaemia made by presence of only Hb S, Hb A2, and Hb F on Hb electrophoresis with no Hb A, a positive sickling test and presence of sickle cells in blood film
Diagnosis of sickle cell anaemia Cont.
Haemoglobin Electrophoresis: Hb A 0 % Hb S 87.0 % Hb F 9.7 % Hb A2 3.3 % Both parents of the affected individual will have sickle-cell traitTREATMENT
Painful vaso-occlusive crisis 1.hydration 2.precepitating factors 3.oxygen therapy 4.analgesic 5.exchange transfusion *Antisickling agent (Hydroxyurea) increase Hb F reduce sickling. *Bone marrow transplantation(Allogeneic-BMT). * Gene therapy.TREATMENT-cont.
maintenance therapy and prevention 1.folic acid 1 mg/d orally. 2.pneumococcal vaccine. 3.pregnancy (increase crisis, abortion, stillbirth) folate,, exchange transfusion. Exchange transfusion will increase Hb-A= 60%. 4.general anesthesia;Careful hydration and oxygenation. *Angiographic contrast media causing sickling should be avoidedThe thalassemia syndromes
are inherited disorders arising from globin gene mutations that either reduce or totally abolish the synthesis of one or more globin chains. These imbalance in chain synthesis lead to formation of unstable Hb. or decrease Hb. lead to hypochromic microcytic anaemia . The thalassaemia named according to globin chain involved.THALASSEMIA
Pathophysiology of thalassemiaα- thalassemia, gene deletions are responsible for the decrease or absence of α- chains. Я- thalassemia : usually due to an mRNA abnormality. This mutation reduces or eliminates the production of Я-globin chainsClinical manifestations of thalassemia
Decreased B- globin production has two major consequences :total Hb. synthesis is reduced leading to microcytic anaemia, low level of Hb-A lead to increase Hb-A2,Hb-F. free α-chain accumulates in the RBC α-chain accumulate and precipitate in RBC lead to hemolysis, destruction of RBC in the BM.The extent to which Я-chain synthesis is suppressed determine the degree of anaemia(ineffective erythropoiesis)Extramedullary hematopoiesis and increased erythropoiesis in the BM lead to over all RBC production is increased due to accumalation of α chain. Pathogenesis cont.
δ δ α α α γ γ α
Hb A2Hb F
Diagnosis: β Thalassemia major
Genotype αααα - - δδ γγ Haemoglobins Produced
Laboratory diagnosis of β thalassemia major made by CBC, absence of Hb A, with increased Hb F. Some patients have small amounts of Hb A if some β globin chain is produced.
Decrease synthesis of a-chains, lead to precipitation of Hb-H (4-Я chains) or Hb- Bart’s (4 δ-chains)Classification of α -thalassaemia : 1-Hydrops fetalis : severe, all 4 α -genes are deleted lead to severely anaemic, edematous, stillborn infant. Hb-barts (4 δ -chains had very high oxygen affinity). α-THALASSAEMIAS
αThalassemia cont. 2- HbH disease : deletion of 3 α -genes lead to unstable Hb. result in precipitation and extra-vascular hemolysis.3-α -thalassaemia Trait : deletion of 2 α genes. 4-α-thalassaemia Carrier: deletion of 1 α gene, asymptomatic.
DIAGNOSIS OF α-THALA Positive family history with lab finding non specific findings : Blood smear show microcytic, hypochromic red cells, target cells, aniso-poikilocytosis, and decrease MCV. Heinz bodies are evident. Specific findings : definitive diagnosis is finding of HbH by Hb electrophoresis.
Normal Control
Abnormal ControlAbnormal Control
Patient Hb H
HbA
HbA2
Hb –H Diagnosis Haemoglobin Electrophoresis: Hb A 91.5 % Fast moving band 8.5% Hb A2 and Hb F decreased
Hb H Preparation
Hb H inclusions in RBCsTREATMENT OF α- AND Я- THALASSEMIA 1.Regular red cell transfusions : hypertransfusion program (keep the level of Hb>90g/L) 2.Neocytes transfusion (increase RBC survival, decrease frequency of transfusion, and decrease iron over load). 2-3 units every 4-6 weeks . 3.Leukocyte filter will lowers rate of transfusion reaction. 4.Folic acid supplementation (5 mg) to prevent aplastic crisis.
5.Iron chelation: *Desferioxamine (Desferal) either with each unit of transfused blood (2 g) or by slow subcutaneous daily infusions by pump ( (1-4g over 8-12 *(Exjade ) Deferasirox *Deferiprone 6.Splenectomy ;indication: mechanical difficulty,hypersplenism. 7.Bone Marrow Transplantation : prior to development of hepatomegaly, portal fibrosis & iron over load.
TREATMENT –cont.
PRENATAL DIAGNOSIS OF THALASSAEMIA
Guide parents and physicians in deciding whether to complete pregnancy.Both parent carriers. Fetal diagnosis:fetoscope to sample fetal venous blood show α/Я chain synthesis ratio.Amniocentesis or trophoblast (chorionic villus) biopsy for DAN analysis using DNA probes.IMMUNE HEMOLYSIS
Definition : red cell life span is shortened because abnormalities in the components of the immune system are specifically directed against the patients own erythrocytes. 1.Auto-immune hemolytic anaemia. 2.Transfusion related hemolysis. 3.Drug-related immune hemolysis.AUTO- IMMUNE HEMOLYTIC ANAEMIA
The auto antibodies can be activated by either heat or cold. Warm reactive auto immune hemolysis (37oC) Causes : 1- idiopathic 2- secondary : I. Drugs (Methyldopa) II. Connective tissue disease (SLE) III. Lymphoproliferative (CLL, HD, NHL)CLINICAL MANIFESTATION
Onset rapid lead to anaemia, tiredness, fatigue. Elderly pts. With atherosclerosis lead to chest pain. Splenomegaly and Jaundice, may be absent in acute phase. Abdominal pain and fever may also occur.Diagnosis
spherocytosis, reticulocytosis, increase LDH, decrease serum haptoglolbin, increase indirect bilirubin positive direct coomb’s test; Patient’s CELLS are tested for surface Ab’sAround 10% of all warm autoimmunehaemolytic anaemias are Coombs test-negative.
Treatment
1.Removal of the underlying cause 2.Corticosteroid : 1mg/kg prednisone (3-4 weeks / check-Hb. & retics.) then slow tapering if pt. respond . in chronic cases, use low dose therapy 3.Splenectomy : in case of steroid failure or decrease Hb. following cessation / reduction of steroid. 4.Blood transfusion.*cold-reactive auto immune hemolysis
Auto Antibodies usually are IgM. Occasionally IgG. Low temp make the antigen(Ag) more prominent on the membrane lead to antibodies reaction. Warm temp hiding the Ag below the membrane below the lipid component lead to prevention of Ag-Antibodies(Ag-Ab.) reactionCAUSES
1.Idiopathic. 2.Secondary: *Infection(mycoplasma pneumonia, infectious mononucleosis) *LymphomaPathogenesis and clinical effects
The Blood from warm body cavities come to the surface low blood temp.this lead to activation of the autoAbs and to agglutination resulting in impairing circulation and producing cyanosis, pallor and ischemic pain (Raynauds phenomenon, Acrocyanosis). IgM binds Ag temporarily, but it fixes complement. IgM dis-engages from red cell to a attach to another cell. The fixed complement remains and activate C5-C6 causing hemolysis . C3-sensitized RBC removed by kuppfer cells in the liver and this why hemolysis not respond to splenectomy.1.positive direct coomb's test2.IgM Ab’s present in high titer.Treatment : Directed at the cause 1.infection : transient, self limited 2.supportive treatment (transfusion , avoid cold temp). 3.Lymphoma : specific therapy. 4.Idiopathic :common(No response to steroid, splenectomy) I-alkalating agent(Chlorambucil) II-Rituximab(mabthera) III-plamapheresis. Diagnosis
Paroxysmal Nocturnal Hemoglobinuria (PNH)
GlycosylphosPhatidylInositol ACQUIRED, NOT INHERITED like all the previous hemolytic anemias were ACQUIRED mutations in phosphatidylinositol glycan A (PIGA)It is “P” and “N” only 25% of the time.Triad of intravascular hemolysis,pancytopenia and thrombosis. PNH is also associated with hypoplastic bone marrow failure, aplastic anaemia and myelodysplastic syndrome.