Hemolytic anemias

Anemia resulting from hemolysis or breakdown of RBCs is called hemolytic anemia. Due to hemolysis RBCs are destroyed earlier than their normal life span of 120 days, causing reduced RBC count and anemia. Broadly, hemolytic anemia can be classified as either intravascular or extravascular. Intravascular hemolysis is the destruction of RBCs within the circulation , which releases hemoglobin into the plasma and causes hemoglobinuria. Whereas, in extravascular hemolysis, the RBCs are broken down by macrophages of the reticuloendothelial system in the spleen and liver. As a result, hemoglobin levels in the plasma do not rise significantly in extravascular hemolysis and there is no hemoglobinuria.

Causes of intravascular and extravascular hemolytic anemia

Intravascular hemolysis

• AIHA
• Drug induced autoimmune hemolytic anemia e.g. meropenem, iron dextran, ceftriaxone, piperacillin
• Mechanical trauma like artificial heart valves, calcified aortic valve
• G6PD deficiency
• TTP, HUS
• DIC, HELLP syndrome
• PNH
• Mismatched blood transfusion
• Toxins in malaria (blackwater fever), clostridial toxins, sepsis, snake venom

Extravascular hemolysis

• AIHA
• Hereditary spherocytosis
• Hereditary elliptocytosis
• Pyruvate kinase deficiency
• Sickle cell anemia
• Thalassemias

Normally, haptoglobin in the plasma binds to free hemoglobin. Levels of haptoglobin decrease or it may be totally absent in severe intravascular hemolysis. This happens because haptoglobin bound to Hb is cleared by the reticuloendothelial system at a rate that cannot be countered by increased synthesis of haptoglobin.

Comparison between intravascular and extravascular hemolytic anemia**

1. Auto-immune hemolytic anemia or AIHA: It is an autoimmune disorder that results in the lysis of RBCs by auto-antibodies. It may cause intravascular or extravascular hemolysis. AIHA can be primary (idiopathic) or secondary (acquired). Secondary causes are autoimmune diseases, such as lupus, Chronic lymphocytic leukemia, non-Hodgkin’s lymphoma and other blood cancers, Epstein-Barr virus, Cytomegalovirus, Mycoplasma pneumonia, Hepatitis viruses and HIV. Some drugs may also induce auto-antibody production against RBCs. Apart from symptoms from anemia itself, severe cases will show jaundice and splenomegaly. DAT and indirect antibody test will be positive in AIHA.

   Acquired autoimmune hemolytic anemia occurs in different forms, including warm antibody hemolytic anemia (WAIHA) and cold antibody hemolytic anemia (CAIHA). In WAIHA, the autoantibodies present in the serum react optimally with human RBCs at 37ºC, while CAIHA is mediated by cold autoantibodies exhibiting affinity for RBCs at temperatures below 37ºC. WAIHA is the more common form and is associated with IgG1 or IgG3 antibodies. It is seen in CLL, non-Hodgkin’s lymphoma, solid tumors, SLE, viral infections, drugs like cephalosporins and piperacillin; previous transfusions or transplantation.

   CAIHA caused by antibodies that optimally react at 4°C includes cases of cold agglutinin syndrome (CAS) and paroxysmal cold hemoglobinuria (PCH). CAS characteristically occurs in middle-aged or elderly persons, often with signs and symptoms exacerbated by cold such as livedo reticularis, Raynaud disease, acrocyanosis, and cutaneous necrosis. CAS is associated with IgM and may be primary, nonmalignant clonal B cell disorder or secondary to non-Hodgkin’s lymphoma, CLL, Mycoplasma pneumoniae or infectious mononucleosis (Epstein–Barr virus). The diagnosis of CAS is suggested by the autoagglutination of the patient’s blood at room temperature that is exacerbated at 4°C, and is reverted at 37°C. Patients with CAS present a positive DAT with anti-C3 or anti-C3d, but is negative with anti-IgG reagents.

   PCH is more common in children and follows a viral illness or vaccination. It presents with intravascular hemolysis with hemoglobinuria and jaundice associated with abdominal pain, cutaneous and mucosal pallor, fever, chills, and chronic headache, preceded by 1–2 weeks of a respiratory tract infection. Renal failure may occur. PCH is caused by a cold agglutinin called Donath-Landsteiner antibody that corresponds to a polyclonal IgG biphasic antibody that binds to RBCs at cold temperatures and produces complement mediated intravascular hemolysis at normal body temperature.

   Rarely, some patients may have mixed AIHA and show both warm and cold antibodies. Treatment of AIHA depends on severity and type. Corticosteroids, rituximab, avoiding cold exposure, cyclosporine, splenectomy and blood transfusions are used as therapy.

2. Drug-induced hemolytic anemia or DIHA: It is caused by drug-related antibodies that cause RBC breakdown. DIHA can be differentiated from AIHA by the fact that the disease goes into remission on discontinuation of the offending drug.

   Following mechanisms are seen:

   i) Drug binds to RBC membrane proteins inducing typically IgG antibody production against the complex. Such antibody coated RBCs are removed by macrophages. Classic example is Penicillin, which binds covalently to RBC proteins. Diagnosed by positive DAT and negative antibody elution tests. Indirect antiglobulin test may be positive or negative. Drug dependent antibodies seen.

   ii) Combination of membrane plus drug can create an immunogen, stimulating the production of IgM or IgG antibodies and often activate complement, leading to acute intravascular lysis and sometimes renal failure. Seen with ceftriaxone, piperacillin, NSAIDS, quinine, probenecid etc. Diagnosed by positive DAT and negative antibody elution tests. Indirect antiglobulin test may be positive or negative. Drug dependent antibodies seen.

   iii) Modification of the immune system so that RBC auto-antibodies are produced e.g. Methyl dopa, fludarabine. Drug independent antibodies seen. Diagnosed by positive DAT, indirect antiglobulin and antibody elution tests.

   iv) Some drugs modify the RBC membrane so that proteins such as albumin, IgG, C3, fibrinogen etc, become non-immunologically adsorbed onto the RBC membrane, activating an immune response. Drug independent antibodies seen. Diagnosed by positive DAT, indirect antiglobulin and antibody elution tests.

Common drugs causing DIHA

• Methyldopa
• Penicillin
• Cephalosporins like ceftriaxone, cefotetan
• Piperacillin
• NSAIDS
• Quinine
• Hydrochlorothiazide
• Rifampicin
• Mefloquine
• Fludarabine
• Probenecid
• Beta lactamase inhibitors

3. Paroxysmal nocturnal hemoglobinuria or PNH or Marchiafava-Micheli Syndrome:

   Paroxysmal nocturnal hemoglobinuria is a condition affecting RBCs, WBCs and platelets that is characterized by sudden, recurring episodes of intravascular hemolysis and hemoglobinuria which may be triggered by stressors such as infections or physical exertion. Acquired somatic mutations in PIGA gene of hematopoietic stem cells cause PNH. The PIGA gene codes for a protein called phosphatidylinositol glycan class A. This protein is involved in the production of GPI anchor, whose function is to “anchor” proteins to the cell membrane. Important among them are CD55 (decay accelerating factor or DAF) and CD59. Absence of these proteins makes the RBCs more susceptible to complement mediated lysis. In many cases, the hemoglobinuria is seen in the morning, upon passing brown or cola colored urine that has accumulated in the bladder during the night. Apart from above symptoms, abdominal vein thrombosis, hemorrhage, pancytopenia, thrombocytopenia. Budd-Chiari syndrome may be seen. Patients with PNH are prone to AML. It is a diagnosis of exclusion. Flow Cytometry can detect abnormal cells lacking GPI anchored proteins like CD55 and CD59. Eculizumab and Ravulizumab can be used for therapy. They act by blocking complement mediated lysis. Bone marrow transplantation is curative. Folate and/or iron supplementation may be needed. Ham’s test and sucrose hemolysis test can also be used to detect hemolysis in PNH.