IMMUNE HEMOLYSIS
IgG and/or IgM bind to red blood cell (rbc) surface antigen– initiate rbc destruction via Complement system and RESImmune Hemolytic Anaemia – Classification – autoimmune (AIHA), alloimmune, drug inducedAIHA –antibody directed against self rbcAIHA – incidence 1-3 cases/100,000 per yearAuto a/b usually reacted against antigen – exhibit reactivity against allogeneic rbc as wellPathogenesis;Degree of haemolysis depends on :1. Characteristic of antibody- quantityspecificity Typeability to fix Complementability to bind to tissue machrophage.
2. target antigen – densityexpressionage of ptIgG a poor activator for classical Complement pathway· Generally IgG sensitized rbc are eliminated by phagocyte of RES· RES also have receptor for C3b and iC3b – can potentiate extravascular hemolysis· IgM -- sensitized rbc associated with extravascular and intravascular hemolysis
REC have receptor for rbc bound C3b and iC3b resulting for Complement activationSpleen – site for IgG associated with extravascular hemolysisLiver (kupper cell) – site for Ig M associated extravascular
CLASSIFICATION OF AIHA
1.Warm AIHA Idiopathic Secondary (LPD, A/ Immune) 2. Cold AIHA Cold Agglutinin syndrome PCH 3. Mixed AIHA Idiopathic secondary 4. Drug induced Autoimmune type Drug adsorption type Neoantigen typeClassification according to Type reactivity of antibodyWarm react strongly near 37 cCold bind rbc strongly near 0-4 cLymphoproliferative disorder – about half of cases of secondary cold and warm AIHAIdiopathic more in female, peak at 4 and 5 decade.2 criteria to dx AIHA – serologic evidence and clinical or lab evidence
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)
CLINICAL MANIFESTATION
Warm AIHA; 48% - 70% of AIHA· incidence increase around 40 year· children peak incidence at first 4 years of life variable clinical presentation· fulminant hemolysis – jaundice, pallor, edema, dark urine, hepatosplenomrgaly· pregnancy – 5x risk of developing auto a/b.Lab Evaluation
Hb, HCT – Normal in pt with indolent hemolysis Low in pt with fulminant hemolysis.ReticulocytosisReticulocytopenia – early in disorder , secondary to autoimmune disorder ,inadequate BM response WBC – mild leucocytosisFBP – Polychromasis, macrocytosis, normoblast Erythrophagocytosis, microspherocytes –BM – erythriod hyperplasis,
Indirect Bilirubin – increaseLDH – IncreasedSerum haptoglobin – reducedPositive urine Hb and hemosiderinDirect AntihumanTest(DAT-coomb`s) – positive in 95% cases of WAIHA IgG (20-66%) IgG + C3 (24 – 63%) C3 ( 7-14%)
Treatment
1.Removal of the underlying causeIf Bm can compensate – monitorAnaemia develop – steroids – first line of tx. 70 – 80% improve within 3 months 2.Corticosteroid : 1mg/kg prednisone (3- 4 weeks / check-Hb. & retics.) then slow tapering if pt. respond . in chronic cases, use low dose 3.Splenectomy –fail steroid, second line of treatment Removal of primary site of extravascular hemolysis and site of antibody production. Response rate 60 – 75%. .4. Cytotoxic drug - fail steroid and splenectomy Response rate 40 – 60%5. Recently reported cases fail to steroid and chemo response to Rituximab ( anti CD 20)6.Plasmapheresis benefit in fulminant hemolysis 7. Intravenous immunoglobulin ( IVIG) 8. Danazol, vincristine *** PACKED CELL transfusion Limited to life threatening anaemia Least incompatible unit transfused Slow infusion * Donor rbs are destroyed at same rate as auto rbc * Exhibit specificity – Ag negative unit should be transfused *Transfusion may induce further auto antibody formation
COLD AGGLUTININ SYNDROME (CAS) :
16 – 32% of AIHAprimary – older, peak incidence 70 year, > femaleection secondary – lymphoproliferative and infcold enviroment may exacerbate the conditionpt may present with acryocyanosis, raynoud’s phenomenonCAUSES 1.Idiopathic. 2.Secondary : infection ( mycoplasma pneumonia, infectious mononucleosis) 3.Lymphoma.
Lab Evaluation
FBP – rbc clumping, polychromasia, anisopoikilocytosis, occassional spherocytesMCV – increaseHB, HCT – mildly reducedRetic – mildly increased Indirect bilirubin,LDH – increasedReduced serum haptoglobin with hemoglobinuria – in severe exacerbationDAT – positive for C3 and neg for IgGMajority of auto antibody are benignPathophysiology .
IgM auto antibody fix C1 – then initiate Complement cascadeWarmer Temperature ( at central circulation) – maximize C fixation and activation – facilitate hemolysisDissociate cold agglutinin and allow them to bind back to rbc and repeatt the cycleComplement cascade progress to MACROPHAGE—INTRAVASCULAR hemolysisRbc bound C3d – EXTRAVASCULAR hemolysis90% directed to I Ag and remaining to i. AgTreatment : Directed at the cause Infection : transient, self limited need supportive treatment (transfusion , avoid cold temp). Lymphoma : specific therapy. Idiopathic : common . No response to steroid & splenectomy(benefit patient with IgG Cold Agglutinin Syndrome). * Alkalating agent (CHLORAMBUCIL). * Plasmapheresis.
PAROXYSMAL COLD HEMOGLOBINURIA (PCH)
uncommon2-10% of AIHAacute cases predom in children, secondary to infectionclinically presented with constitutional symptoms, hemoglobinuria, cold urticaria and raynoud’s phenomenon.HCT,Hb – lowReticulocytopenia in acute phase later reticulocytosisFBP – agglutination, polychromasia, normoblast, Spherocytes, erythrophagocytosisIncreased Indirect bilirubin ,LDHReduced se HaptoglobinPositive urine HB, hemosiderinuriaARFPCH – caused by biphasic IgG auto antibody ( fix C at cold temperture and dissociate at higher temperture) ( Donath Landsteiner a/b)A/b are potent , small titre can cause hemolysisDAT – positive anti C3 , IgG positive if performed at cold temperature.
3 mechanism The hapten mechanism : drugs firmly bind protein on red cell surface. penicillin , high dose (SBE) can cause formation of Ag against pencillin which may or may not cause hemolyis. IgG lead to hemolysis subsides after discontinuation of the drug. IgM commonly occur with penicillin and do not cause hemolysis. Innocent bystander : Drugs form immue complex with Ab (IgM) then attach to red cell membrane and target the cell for immune destruction example sulpha., chloropropamide, phenacetin,
DRUG INDUCED HEMLYTIC ANAEMIA
Auto-antibody mechanism : drugs induce the formation of Ab. against the patient own red cell (IgG) producing true auto immune hemolytic anaemia for example methyldopa over 5 yrs lead to coomb's test found positive in 10-20% of cases, true hemolytic anaemia in few pts. Hemolysis improve after discontinuation of drug but coomb's test remain positive.
Membrane defects
Hereditary spherocytosisFunctions of red cell membrane
To separate the contents of the cell from the plasma. To maintain the characteristic shape of the red cell. To regulate intracellular cation conc. To act as the interface between the cell and its environment via membrane surface receptors.The most common of the inherited RBC membrane defects, affecting 1 in 5000 individuals. The disorder characterized by RBC that appear spherical on peripheral blood smear. Autosomal dominant disorder. 25% of pts have no family history.
Hereditary spherocytosis (HS)
It is caused by a defect in the proteins involved in the interactions between the membrane cytoskeleton and the lipid bilayer of the red cell. ( ankyrin, spectrin). The abnormal red cells lysis is caused by a defect in cytoskeleton : involves a partial deficiency of spectrin (RBC membrane protein). The spherical shape has two effects on RBC. 1.they become trapped in the splenic cord. 2.they have shortened life span. The abnormal membrane also demonstrate an increased permeability of sodium.
Pathophysiology
Clinical manifestation The typical HS pts is relatively asymptomatic with well compensated hemolysis and palpable spleen. The rare pts with severe HS may present early in childhood with life threatening hemolysis. HS has the usual features of chronic hemolysis : *symptoms of anaemia. *splenomegaly. *gall stones. *Aplastic crisis #Family members have mild form of the disorder or are carrier.
*Suggested by a family history of HS and characteristic finding on Peripheral Blood Smear i.e.* spherocytes. Two studies are used to confirm the diagnosis. 1. osmotic fragility test. A positive test shows hemolysis of the patients red cells at saline concentration near isotonic conditions compared to simple swelling in normal cells. 2. Autohemolysis test : Normal autohemolysis is about 1% while HS cells will display Hb release rate of 3% or more.
Diagnosis
Reticulocytosis is a feature of increased red cell production. New methylene blue is used to stain the reticulocytes
There is increased haemolysis in the patient in comparison with the control. Glucose has given partial correction.
Autohaemolysis test in HS
1.Asymptomatic pts with compensated hemolysis should receive supportive treatment with folate supplements. 2.Aplastic crisis may require short period of blood transfusion. 3.Splenectomy is curative in most pts with HS.
Treatment
1.performed only when necessary (pts who required blood transfusion) 2.should be avoided in pts younger than 10 years of age (associated risk (fatal pneumonia, sepsis) 3.may be performed with cholecystectomy to decrease the amount of hemolysis and the risk of gall stones development. 4.Proceeded by vaccination against pneumococcus, H. influenza, meningococcal spp.. 5.Followed by penicillin-V as 250 mg twice daily for the rest of life .
Guide line for performing splenectomy
1. The embden-Meyerhof Pathway : for glycolysis provides energy for : a. Na+, K+ and Ca+2 pumps in the cell membrane. b. phosphorylation of membrane components and synthesis of glutathione for protection from oxidative damage. c. reduction of NAD to NADH which is important in reducing met-hemoglobin to hemoglobin.
Red cell housekeeping enzymes :
maintains levels of NADH which is important in the reduction of oxidized glutathione (GSSH) to reduced glutathione (GSH). GSH is important in : a. destroying the oxidant hydrogen peroxide with glutathione peroxidase. b. maintaining sulphhydryl in Hb. and in various enzymes and within the cell membrane.
2.The pentose-phosphate shunt :
G6PD functions to reduce nicotinamide adenine dinucleotide phosphate (NADPH) while oxidizing glucose-6-phosphate. NADPH is needed for the production of reduced glutathione (GSH) which is important to defend the red cells against oxidant stress.
G6PD deficiency
More than 400 variants due to point mutations or deletions of the enzyme G6PD have been characterized which show less activity than normal. Worldwide over 400 million people are G6PD deficient in enzyme activity. Enzyme variants : Normal forms: Two forms of G6PD have normal activity, B form and A form or (A+) .The inheritance is sex-linked, affecting males, and carried by females. The main races affected are in West Africa, the Mediteranean, the Middle East, and South East Asia.
(A-) isoenzyme & Mediterranean isoenzyme. *Enzyme may exist at normal intracellular levels but have decrease activity. *May be unstable lead to increased break down in red cells.
Deficiency forms
Pathophysiology the daily destruction of RBC, Hb. is being constantly oxidized to met Hb. but a special protective mechanism that uses the E.M pathway reduces it back to Hb. This is important because
Met-Hemoglobin. dissociates into heme and globin and then precipitates as an insoluble mass (Heinz body)in the RBCs. These red cells with Heinz body are non deformable and have difficulty passing through the spleen. Splenic removal of Heinz bodies damages RBCs membrane, forming spherocytes and causing hemolysis.
Precipitating factors : 1.oxidant drugs : (antimalarial, antipyretic, sulfonamide and related group, nitrofurantion,) (moth ball) 2.fava beans(Favism). 3.Infection (resp. viruses, hepatitis, infectious mononucleosis , bacterial preumonia, and septicemia). 4.Stressful conditions : diabetic ketoacedosis & uraemia
Clinical manifestations 1. General features, patient with G6PD deficiency display signs and symptoms of IV hemolysis. The deficiency usually is episodic and occasionally is chronic. 2. patient with the common (A-)form of G6PD deficiency generally have mild hemolysis only when stressed. - *hemolysis is self-limited
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reticulocyte have high levels of the enzyme thus, compensating for the decreased activity. 3. patients with the rare Mediterranean form of G6PD deficiency : - severe hemolysis when exposed to oxidants or stressed by surgery or infection the hemolysis persist as long as the stress or oxidant persists.
Diagnosis 1. red cell enzyme levels measured by electrophoresis show very low activities in severe disease and low to normal activity in mild disease depending on when the test is performed. measurement should not be taken at the time of hemolysis and reticulocytosis 2. Heinz bodies can be demonstrated only during early phase of G6PD deficiency. characteristic of IV hemolysis.
Between crises blood count is normal .The enzyme deficiency is detected by One of a number of screening tests or By direct enzyme assay on red cells. During the crisis, the blood film may show contracted and fragmented cells, bite and blister cells. Enzyme assay may give a false normal level in the phase of acute haemolysis. There are feaures of intravascular haemolysis.
The blood film shows irregularly contracted cells [deep red arrows] and sometimes hemighosts [deep blue arrow] in which all the haemoglobin appears to have retracted to one side of the erythrocyte.
Treatment 1.primary therapy is to avoid oxidative agents. 2.recovery is rapid , but if the anaemia is severe transfusion of red cells with normal enzyme complement may be required. 3.Splenectomy is without value.
Pyruvate kinase(PK) deficiency *Congenital non-spherocytic hemolytic anaemia(CNSHA). *Autosomal recessive. *Characterized by decreased generation of ATP. *Impairement of glycolysis lead to increased 2,3 DPG level.
Clinical manifestation 1.-chronic hemolytic syndrome of variable severity. 2.severe deficient may be apparent in neonatal stage. 3.-may be a symptomatic. 4.-manifestation (reticulocytosis, marrow hyperplasia, splenomegaly, jaundice, gall stone).
Diagnosis : measuring PK enzyme level in red cells Treatment : 1.depend on severity of anaemia. folate supplements, 2.Splenectomy