Lecture 14 Haemolytic Anaemia PDF

HAEMOLYTIC ANAEMIA Dr David Donaldson  LEARNING OBJECTIVES 1. State the red cell enzyme pathways and the deficiencies which can occur. 2. Describe the basic structure of the red cell membrane. 3. Summarise defects of the red cell membrane both congenital and acquired. ...

HAEMOLYTIC ANAEMIA Dr David Donaldson  LEARNING OBJECTIVES 1. State the red cell enzyme pathways and the deficiencies which can occur. 2. Describe the basic structure of the red cell membrane. 3. Summarise defects of the red cell membrane both congenital and acquired.  DEFINITIONS Haemolytic anaemia – anaemia due to a shortened survival of circulating red blood cells (RBCs) due to their premature destruction. Haemolytic anaemia can be caused by intrinsic red blood cell (RBC) defects (haemoglobin defects, enzyme defects, or membrane defects) or extrinsic causes (autoimmune, vascular defects, toxins, or heat) Happens- circulation in Intravascular haemolysis results from direct destruction of circulating RBCs by extreme heat, toxins, infectious agents, complement, or shear forces. outside Extravascular haemolysis results from phagocytosis of intact (but abnormal) rculation RBCs by macrophages of the spleen and liver. U PATHOPHYSIOLOGY  BLOOD FILM CLUES Bite cells, blister cells, or Spherocytes or microspherocytes Schistocytes suggest a thrombotic irregularly shaped red blood suggest immune haemolysis microangiopathy cells (poikilocytosis) suggest oxidative haemolysis ↳ Librin Strandset i acrossthrough S ↳ due to oxidative stress AUTOIMMUNE HAEMOLYTIC ANAEMIA  AUTOIMMUNE HAEMOLYTIC ANAEMIA Autoimmune mediated destruction of RBCs by autoantibodies with various properties and target specificities Primary (no obvious trigger/cause) or Secondary (caused by underlying condition, eg: lymphoma, infection, drug) Can co-exist with autoimmune thrombocytopenia (Evan's Syndrome) Haemolysis is initiated when an autoantibody binds to the red cell membrane andDrecruits complement& Red cells either destroyed in circulation (intravascular haemolysis) or by macrophages in liver and/or spleen (extravascular haemolysis)  WARM ANTIBODY HAEMOLYTIC ANAEMIA most common Warm AIHA is due to an antibody that is active at normal body temperature Typically the antibody is an IgG Primary warm AIHA (previously called idiopathic) refers to warm AIHA with no underlying condition or medication that could be responsible. Secondary warm AIHA refers to warm AIHA in the setting of an underlying condition such as lymphoma Antibodies tend to be panagglutinins - antibodies that reacts with all reagent RBCs. Alloantibodies usually react to specific RBC antigens (eg, Kell, Kidd). Cold agglutinins often have specificity against i or I.  PATHOPHYSIOLOGY In warm AIHA, RBCs are mostly cleared extravascularly (in reticuloendothelial macrophages). Typically spleen in warm AIHA Macrophages in the spleen have Fc-gamma receptors that recognize and phagocytose the IgG heavy chain and a portion of the RBC membrane, producing a spherocyte Hepatic macrophages – Hepatic macrophages have receptors for complement C3 fragments and can phagocytose RBCs with complement on their surface.  ASSOCIATED CONDITIONS Approximately 50 to 60 percent of warm AIHA is associated with an underlying condition The underlying condition generally produces some combination of immune activation, immune deficiency, or immune dysregulation. Infection – Includes viral infections such as HIV, Epstein Barr virus (EBV), or hepatitis C virus (HCV). Autoimmune disorders – Includes autoimmune disorders including systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, or ulcerative colitis Lymphoproliferative disorders – Includes autoimmune lymphoproliferative syndrome (ALPS), chronic lymphocytic leukemia (CLL), lymphoma, and monoclonal gammopathies Immunodeficiency – states including inherited immunodeficiencies, hematopoietic stem cell transplant (HCT), solid organ transplant, and hypogammaglobulinemia  CLINICAL MANIFESTATIONS Anaemia-related symptoms (shortness of breath on exertion, fatigue, bounding pulse or palpitations) in at least 75% of individuals. Jaundice and/or dark urine and splenomegaly in approximately 30%. In some cases, splenomegaly is due to an underlying lymphoproliferative disorder. Chest pain in 7%. Immune thrombocytopenia can co-exist in 7% (Evan’s Syndrome) Haemoglobinuria suggests intravascular haemolysis, which may occur in severe warm AIHA Thromboembolic events including deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, or myocardial infarction can complicate AIHA  Normal blood film INITIAL TESTING CBC - Anaemia is usually present but may be absent if haemolysis is mild. A typical haemoglobin at presentation is between 7 and 10 g/dL, although haemoglobin 64) at 4 oC, typical findings by the DAT, and the absence of an underlying clinical disease (eg: Lymphoma) Clonal lymphocyte population may be detected however - Primary CAD-associated lymphoproliferative disorder The typical DAT pattern is a positive monospecific test for C3d only. Serum IgMK paraprotein is found in about 90% of patients C3b-coated RBCs are phagocytosed by macrophages in the reticuloendothelial system (ie, extravascular haemolysis), predominantly Kupffer cells in the liver Secondary CAD (also known as Cold Agglutinin Syndrome) associated with underlying condition (eg lymphoma or infection). Kupffer Cells  COLD AIHA - PATHOPHYSIOLOGY Most cold agglutinins are IgM very large IgM antibodies are pentameric, which places antigen-binding sites sufficiently far apart to allow them to bridge the distance between RBCs. Most cold agglutinins are directed against "I" or "i" antigens of the I blood group The big I and little i antigens are ubiquitous (present on all cell membranes) Little I is progressively converted to big I during infancy, and most children become big I positive by the age of two years. The function of the big I and little i antigens is unknown!  COLD AIHA - PATHOPHYSIOLOGY The titer is the number of dilutions after which the antibody can still cause agglutination; it reflects antibody concentration and avidity. Generally, a titer ≥64 is considered clinically significant. Thermal amplitude – The temperature range at which the antibodies are active is referred to as the thermal range or thermal amplitude. The typical optimum temperature for antigen binding to cold agglutinins is 3 to 4°C (refrigerator temperature), but pathogenic cold agglutinins often have a thermal amplitude of 28 to 30°C or more and will be active at temperatures that occur in acral areas of the body (fingers, nose, toes typically) Antibodies with a lower thermal amplitude will cause agglutination in vitro but are unlikely to cause clinical disease.  COLD AIHA - PATHOPHYSIOLOGY Haemolysis in CAD is primarily extravascular and mediated by complement. The IgM cold agglutinin binds to its cognate antigen (usually "I" or "i") on the surface of RBCs in sites of the body where the temperature is low enough to be in the thermal range of the antibody The bound IgM recruits components of the classical pathway of complement C3b-coated RBCs are phagocytosed by macrophages in the reticuloendothelial system (ie, extravascular hemolysis), predominantly Kupffer cells in the liver Phagocytes tend to engulf the entire cell rather than a portion of the cell membrane, as occurs in phagocytosis mediated by IgG. This may explain the absence of spherocytosis in CAD On the remaining circulating RBCs (ie, those that are not phagocytosed), IgM dissociates upon warming, but C3b remains attached. Surface C3b undergoes cleavage to C3d, which can be detected by the direct antiglobulin (Coombs) test  COLD AIHA – CLINIC AL FEATURES Not all individuals with demonstrable cold Acrocyanosis agglutinins have a cold agglutinin-related disease. Anaemia (median haemoglobin, 95 g/L) – 90 percent Haemolytic markers (high LDH and bilirubin, low haptoglobin) – approximately 90 percent each Cold-induced symptoms (mostly acrocyanosis) – 52 percent More severe cold-induced symptoms such as Raynaud phenomenon or ulceration are seen in a smaller proportion. Livido reticularis (a blanchable, patchy, reticulated vascular pattern on the skin with a red-blue or Raynaud’s Livedo violaceous color) Phenomenon Reticularis  SECONDARY CAD Lymphomas – most commonly Lymphoplasmacytic lymphoma, marginal zone lymphoma and small lymphocytic lymphoma Infections – particular Mycoplasma pneumonia or EBV (infectious mononucleosis) It usually occurs approximately two weeks after onset of the primary infection, diminishes as the infection begins to resolve, and is gone within two to three months Autoimmune disorders – SLE, rheumatoid arthritis  COLD ANTIBODY HAEMOLYTIC ANAEMIA - TREATMENT Not all individuals with cold agglutinins have clinical manifestations, and asymptomatic patients have not been shown to benefit from therapy. Symptomatic anaemia, significant fatigue, or bothersome circulatory symptoms are indications for treatment. Treatment aims at minimising symptoms, maintaining an acceptable haemoglobin level, and, if required, addressing underlying disorders  PAROXYSMAL COLD HAEMOGLOBINURIA Paroxysmal cold haemoglobinuria (PCH) is an uncommon autoimmune haemolytic anaemia in which autoantibodies to red blood cells bind to the cells in cold temperatures and fix complement, which can cause intravascular haemolysis upon warming. PCH antibodies bind to the RBC P antigen and are polyclonal and IgG (AKA Donath-Landsteiner antibody) Most common presentation is following a viral or bacterial infection in children Can also rarely be associated with syphilis and autoimmune/lymphoprilerative disorders Acute attacks are treated with rest, pain medication if needed, and cold avoidance Some individuals with severe haemolysis may require other interventions such as transfusions, hydration, or steroids. DRUG INDUCED HAEMOLYTIC ANAEMIA  DRUG INDUCED HAEMOLYTIC ANAEMIA Immune mechanisms – antibiotics, diclofenac, platinum based chemotherapy Oxidative mechanisms – primaquine and dapsone. Patients with G6PD deficiency are prone to this. Methaemaglobinaemia – antibiotics (eg septrin), some anaesthetics, nitrites (including poppers!). Treatment with methylene blue can also cause haemolysis in patients with G6PD deficiency Thrombotic microangiopathy There are many other drugs associated with haemolytic anaemia, always important to refer to patients medication history and cross-check! INHERITED HAEMOLYTIC ANAEMIAS THE RED CELL MEMBRANE The red blood cell membrane consists of a phospholipd bilayer that is anchored to the 2-dimensional elastic network of skeletal proteins (mainly spectrin) through transmembrane proteins  HEREDITARY SPHEROCYTOSIS HS is the most common cause of haemolytic anaemia due to a red cell membrane defect. HS can be caused by variants in genes that encode RBC membrane/cytoskeletal proteins. Most HS variants act in an autosomal dominant fashion (ie, a single variant allele is sufficient to confer disease) Spectrin – Erythrocyte spectrin is composed of alpha, beta heterodimers; the proteins are encoded by the SPTA1 and SPTB genes, respectively. Ankyrin – Erythrocyte ankyrin is encoded by the ANK1 gene. Band 3 (the anion exchanger AE1) – This anion channel is encoded by the solute carrier family 4 anion exchanger (SLC4A1) gene. Band 4.2 (previously called pallidin) – Band 4.2 is encoded by the EPB42 gene. Spherocytes are prone to haemolysis. The mechanisms include reduced deformability, which impairs passage through constricted regions of the microcirculation, and phagocytosis by splenic macrophages, which occurs in response to splenic trapping  HEREDITARY SPHEROCYTOSIS – CLINICAL PRESENTATION HS can present at any age and with any severity, with case reports describing a range of presentations, from hydrops fetalis in utero through diagnosis in the ninth decade of life The majority of affected individuals have mild or moderate haemolysis or haemolytic anaemia and a known family history, making diagnosis and treatment relatively straightforward. Individuals with significant severe haemolysis may develop additional complications such as jaundice/hyperbilirubinemia, folate deficiency, or splenomegaly Severe HS (5 percent of cases) – Haemoglobin 6 to 8 g/dL; reticulocytes >10%; bilirubin > 51 micromol/L  HEREDITARY SPHEROCYTOSIS - EVALUATION direct lobulin - antigfest The diagnosis of HS should be suspected in an individual with Coombs-negative (ie, non-immune) haemolytic anaemia and spherocytes on the peripheral blood smear, especially if there is a relevant family history. EMA binding is the most widely used confirmatory test EMA (eosin-5-maleimide) is an eosin-based fluorescent dye that binds to RBC membrane proteins, especially band 3 and Rh-related proteins The mean fluorescence of EMA-labeled RBCs from individuals with HS is lower than controls, and this reduction in fluorescence can be detected in a flow cytometry-based assay Advantages of EMA binding include its high sensitivity and specificity; rapid turnaround time (approximately two hours); and need for only a minimal amount of blood (a few microliters), which is especially advantageous for testing neonates HEREDITARY SPHEROCYTOSIS - EVALUATION  HEREDITARY SPHEROCYTOSIS – TREATMENT As with most inherited haemolytic anaemias, treatment is directed at preventing or minimising complications of chronic haemolysis and anaemia. Folic acid supplementation is appropriate for those with moderate to severe haemolysis and/or during pregnancy Red cell transfusion is often required in severely affected infants and may be needed during certain times in other settings (eg, aplastic crisis, pregnancy). For those with relatively severe haemolysis and symptoms, splenectomy is effective at improving anaemia. Ideally, this is delayed until the individual is older than six years to reduce the likelihood of sepsis due to absent splenic function. ↳ as you are removing a large component of immune system Simultaneous cholecystectomy can be performed if gallstones are also present (increased risk in chronic haemolytic conditions). Family testing, prenatal testing, and/or genetic counselling may be useful in family members of affected children, children of affected parents, and individuals of childbearing age.  HEREDITARY ELLIPTOCYTOSIS + PYROPOIKILOCYTOSIS Hereditary elliptocytosis (HE) is a heterogeneous group of disorders characterised by the presence of elongated, elliptically-shaped RBCs on the peripheral blood film. Haemolytic anaemia in these disorders ranges from absent to life-threatening. HE and related disorders are caused by pathogenic variants in the following genes: SPTA1, coding for α-spectrin (alpha-spectrin) SPTB, coding for β-spectrin (beta-spectrin) EPB41, coding for protein 4.1R GYPC, coding for glycophorin HE is transmitted in an autosomal dominant pattern; affected individuals are heterozygous for a pathogenic variant in EPB41 or an HE-causing variant in SPTA1, SPTB  HEREDITARY ELLIPTOCYTOSIS + PYROPOIKILOCYTOSIS Hereditary pyropoikilocytosis (HPP) – Homozygous or compound heterozygous individuals often have severe, symptomatic haemolytic anaemia, with RBC morphology characterised by anisopoikilocytosis, with elliptocytes and fragmented RBCs, a morphology resembling that seen in patients with thermal burns, giving to this disease the name HPP. HE/HPP are most common in individuals of African, Mediterranean, or Southeast Asian descent, paralleling the distribution of malaria Studies have shown resistance to invasion by Plasmodium falciparum parasites  he clinical presentation of the HE syndromes is highly variable, ranging from clinically silent/asymptomatic, in which elliptocytosis is an incidental finding on the blood smear, to severe hemolytic anemia.  HE/HPP DIAGNOSIS Osmotic gradient ektacytometry (OGE) measures the osmotic fragility, deformability, and hydration status of the RBC population. OGE is effective in confirming the diagnosis when RBC morphology does not provide a clear diagnosis. OGE is also helpful in distinguishing HE from HS Results of EMA binding are variable (and therefore less helpful) Typical EMA in HPP  HE/HPP MANAGEMENT Most individuals with HE are asymptomatic and require no specific therapy or follow-up care. Routine monitoring is not necessary. Individuals with intermittent episodes of haemolysis require closer medical attention. Individuals with HE who have low-grade, compensated haemolysis may not require extensive evaluations or treatment once the baseline laboratory values are established and other concomitant causes of anaemia such as iron deficiency or vitamin B12 or folate deficiency have been eliminated. Splenectomy can be considered in those with more severe chronic haemolysis Reproductive counselling — It is appropriate to perform reproductive counselling in couples at risk for having a severely affected child, as follows: Heterozygosity of both parents for a common HE variant, which places the child at risk for homozygosity HPP in one or both parents Consanguinity Previous child with clinically significant elliptocytosis with haemolysis and/or anaemia  SOUTH EAST ASIAN OVALOCYTOSIS Most frequently seen in individuals from parts of Southeast Asia, including Malaysia, New Guinea, Indonesia, and the Philippines. The causative deletion of 27 base pairs in SLC4A1 causes abnormal folding of band 3. SAO RBCs have a characteristic RBC morphology often described as stomatocytic elliptocytosis. Haemolysis and anaemia are usually absent after three years of age Confers some resistance to malaria RED CELL ENZYME DEFECTS  PYRUVATE KINASE DEFICIENCY Pyruvate kinase (PK) deficiency is an inherited (autosomal recessive) RBC enzyme disorder that causes chronic haemolysis Estimated prevalence of 3:1,000,000 to 1:20,000 Diagnosis of PKD is based on the presence of chronic haemolytic anaemia, reduced PK enzyme activity, and molecular characterisation of the pathogenic mutations. Haemolysis in PK deficiency is mainly extravascular  G6PD DEFICIENCY G6PD deficiency is the most common enzymatic disorder of red blood cells affecting 400 to 500 million people worldwide G6PD protects red blood cells against oxidant injury. G6PD catalyses the initial step in the hexose monophosphate shunt, oxidizing glucose-6-phosphate to 6-phosphogluconolactone and reducing nicotinamide adenine dinucleotide phosphate (NADP) to NADPH Almost all haemolytic episodes related to altered HMP shunt and glutathione metabolism are due to G6PD deficiency The gene for G6PD is located on the X chromosome G6PD deficiency is expressed in males carrying a variant gene, while heterozygous females are usually clinically unaffected Females can be affected by the skewed X inactivation that occurs with aging  G6PD DEFICIENCY - PATHOPHYSIOLOGY G6PD-deficient erythrocytes that are exposed to oxidants (eg, drugs, infection) become depleted of GSH; this change is followed by oxidation of other sulfhydryl- containing proteins with the following consequences Oxidation of the sulfhydryl groups on hemoglobin leads to the formation of methemoglobin and then denatured globin or sulfhemoglobin, which form insoluble masses that attach to the red cell membrane (called Heinz bodies) ↳ need specific stain to see This oxidative denaturation of haemoglobin leads to its cross bonding; as a result, haemoglobin is no longer free to flow in the cytosol, producing the puddling of haemoglobin and bite or hemi-blister cells in the peripheral smear The net effect is that the deficient red cells become rigid and non-deformable, making them susceptible to destruction by reticuloendothelial macrophages in the marrow, spleen and liver. Although this type of haemolysis is predominantly extravascular, intravascular haemolysis also occurs, leading to haemoglobinemia and haemoglobinuria Examples of a bite cell (thick arrow) and blister cells (arrows)  The majority of individuals are asymptomatic and do not have haemolysis in the steady state At two to four days after drug ingestion, there is the sudden onset of jaundice, pallor, and dark urine, with an abrupt fall in the hemoglobin concentration by 3 to 4 g/dL. There may be abdominal pain and/or back pain. Once patients are diagnosed and are able to reduce oxidant stress exposures through medication avoidance, the frequency of haemolysis may decline dramatically. The acute haemolytic process ends after approximately one week, with ultimate reversal of the anaemia, MICROANGIOPATHIC HAEMOLYTIC ANAEMIA  MICROANGIOPATHIC HAEMOLYTIC ANAEMIA MAHA is non-immune haemolysis (i.e, Coombs- negative haemolysis) resulting from intravascular red blood cell fragmentation that produces schistocytes on the peripheral blood smear Abnormalities in the microvasculature, including small arterioles and capillaries, are frequently involved Thrombotic microangiopathy describes microangiopathic haemolytic anaemia, The film shows multiple helmet cells thrombocytopenia and microthrombi leading to (arrows) and other fragmented red ischemic tissue injury cells (small arrowhead); microspherocytes are also seen (large arrowheads)  THROMBOTIC THROMBOCYTOPENIC PURPURA TTP is caused by severely deficient activity of the ADAMTS13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motif, member 13) protease ADAMTS13 is a plasma protease that was initially defined by its function as a von Willebrand factor (VWF)-cleaving protease This normal cleavage to smaller sized multimers prevents ultra large multimers from accumulating, especially in areas of high shear stress (eg, small arterioles and capillaries) When protease activity is reduced, ultra large VWF multimers accumulate on the endothelial surface, where platelets attach and accumulate The major cause of severe ADAMTS13 deficiency is an autoantibody; inherited gene mutations account for a small additional number of cases  TTP – CLINICAL FEATURES ADAMTS13 activity testing is important for the diagnosis, but this testing cannot be used in isolation, nor should therapy be delayed when appropriate while waiting for results.  TTP - MANAGEMENT TTP is a medical emergency requiring time-critical transfer to a dedicated treatment centre. From referral of a suspected diagnosis of TTP and transfer, Plasma exchange (PEX) should be initiated within four to eight hours. Platelet transfusion should be avoided (Don’t feed the clots!) Caplacizumab should be initiated on confirmation of acute iTTP and for up to 30 days following completion of PEX. In patients who remain severely ADAMTS13 deficient (150 × 10 9 /L. Monoclonal anti-CD20 therapy should be initiated within 3 days of acute iTTP admission (the purpose is to turn off antibody production by B lymphocytes)  CASE 1 5 year old female is referred to clinic with pallor and jaundice. Her dad is there and tells you that his spleen was removed when he was 14. What investigations would you do? CBC, Bilirubin, Reticulocytes, Blood film ↳ suspectron Blood film shows abnormal small, round red cells with no central pallor What diagnosis do you suspect? Hereditary Spherocytosis – How would you treat this patient? Transfusion support if required, folic acid supplementation and consideration of splenectomy  CASE 2 67 year old with known history of chronic lymphocytic leukaemia Presents to Emergency department with severe shortness of breath on exertion, chest pain and jaundice HB 54, Bilirubin 112, Reticulocyte count 290 (high). LDH 546. What investigations would you request? Blood film – Spherocytes +++ Direct Coomb’s test – Positive for IGG only. Haptoglobin – not reportable What is the diagnosis? Secondary Warm Autoimmune Haemolytic Anaemia What treatment would you start? Steroids, other immunosuppression, think about treating underlying CLL. Transfuse if severe symptoms. Folic Acid