Hemolysis and Hemolytic Anemias

Alice Ma, M.D.

Hematology UNC-CH February 12, 2004

Overview
Clinical features of hemolysis Laboratory features of hemolysis Congenital hemolytic anemias
Membrane defects Enzyme defects

Immune Hemolysis Non-Immune Hemolysis

Hemolytic AnemiasIntroduction
Definition: a group of disorders characterized by decreased red cell lifespan. Normal red cell lifespan is 120d. Remember that the patient may not always be anemic, depending on the degree of marrow compensation.

Hemolytic Anemias General Clinical Features

General Clinical Features

Splenomegaly is generally present Increased incidence of pigmented gallstones. Dark urine (tea-colored or red) Patients may have chronic ankle ulcers. Aplastic crises associated with Parvovirus B19, may occur Increased requirement for folate

Parvovirus B19
Non-encapsulated DNA virus. Infects and lyses RBC precursors in marrow, causing 7-10d cessation of erythropoiesis. Normal individuals have no significant hematologic effect, since RBCs have normal life span. In pts with hemolytic anemias, loss of red cell production causes H/H to plummet dramatically = Aplastic Crisis

Hemolytic Anemias Classification Schemes

Classification by sites of red cell destruction Classification as acquired vs congenital Classification by mechanism of red cell damage

Hemolytic Anemias Sites of Red Cell Destruction

Extravascular Hemolysis Macrophages in spleen, liver, and marrow remove damaged or antibody-coated red cells Red cells rupture within the vasculature, releasing free hemoglobin into the circulation

Intravascular Hemolysis

Hemolytic Anemias Laboratory Features

Laboratory Evidence for Presence of Hemolysis

Evidence for increased red cell production Evidence for increased red cell destruction

Evidence for increased red cell production

In the blood: Elevated reticulocyte count Circulating NRBCs may be present In the bone marrow: erythroid hyperplasia reduced M/E (myeloid/erythroid) ratio In the bone: Deforming changes in the skull and long bones (frontal bossing)

Polychromatophilic cell on WrightGiemsa staining

Reticulocytes (supravital staining)

Erythroid Hyperplasia

Expansion of the erythroid lineage. Arrow is pointing to red cell precursor. Myeloid:erythroid ratio is normally 3:1. The slide above has ratio of 1:10.

Frontal Bossing
Results from increased erythropoietic activity and marrow space expansion

Evidence for Increased Red Cell Destruction

Biochemical consequences of hemolysis in general Morphologic evidence of red cell damage Reduced red cell life-span
seen on peripheral smear Biochemical consequences of intravascular hemolysis

Measurement of red cell survival no longer routinely done.

Biochemical consequences of hemolysis in general

Elevated LDH levels Elevated unconjugated bilirubin

Only clinically significant if liver function is abnormal Only begins to rise when red cell life span is 50 days or less

Biochemical Consequences of Intravascular Hemolysis Reduced serum haptoglobin Hemoglobinemia Hemoglobinuria Hemosiderinuria

Hemolytic Anemias Classification by Etiology

Congenital
Defects in membrane skeleton proteins Defects in enzymes involved in energy production Hemoglobin defects Immune-mediated Non-immune-mediated

Acquired

Congenital Hemolytic AnemiasMembrane Defects

Hereditary spherocytosis is the most common defect leading to anemia, affecting 1/5000 Europeans Other
Hereditary elliptocytosis
Usually does not produce anemia Autosomal dominant, affecting 1/2500 Europeans Usually results from double recessive or homozygous defect

Hereditary pyropoikilocytosis

Hereditary Spherocytosis
Defect is in proteins of the membrane skeleton, usually spectrin or ankyrin Lipid microvesicles are pinched off in the spleen and other RE organs, causing decreased MCV and spherocytic change.

Hereditary Spherocytosis
Autosomal Dominant trait Patients can develop increased hemolysis and abdominal pain with even trivial infectious illnesses Diagnose by Osmotic Fragility Aplastic crises with Parvovirus B19 Treatment: Folate supplementation Splenectomy will cause hemolysis to abate, but spherocytes will persist.

Osmotic Fragility
Normal

Abnormal

Congenital Hemolytic Anemias Enzyme Defects

Red Cell Enzyme Defects

RBCs require constant energy to maintain biconcave disc shape and hemoglobin in reduced form. Without adequate energy, red cells lyse and/or deform. Energy from glucose is derived from metabolism via anaerobic glycolysis (Embden-Myerhof pathway). There is an alternate aerobic pathway (pentosephosphate shunt) which starts with G-6P. Rate-limiting enzyme in this pathway is G6PD

RBC Metabolism Simplified

Glucose Glucose-6-P G6PD Aerobic metabolism F-6-P Anaerobic glycolysis

Detoxification of metabolites of oxidative stress Elimination of methemoglobin

 Low G6PD activity results in low levels of NADPH and reduced glutathione, which are required to protect hemoglobin from oxidative damage. In the absence of adequate reducing ability (provided by G6PD), oxidizing agents convert hemoglobin to methemoglobin, then denature it, causing it to precipitate as Heinz bodies. The spleen pinches off the Heinz body and the overlying membrane, leaving a bite cell or blister cell

G6PD Deficiency Pathophysiology

G6PD
Two normal forms of enzyme. Most prevalent type is B. 20% of healthy Africans have type A. Deficiency is X-linked. In Africans with G6PD deficiency, mutant protein is A-, which is unstable and loses activity as the red cell ages. Mediterranean variant has baseline low activity. In US, 10-14% of AA men affected In NC, deficiency can be seen in women, since prevalence is high enough.

G6PD Deficiency Agents to avoid

Anti-malarials Sulfa drugs Dapsone Vitamin K Fava beans Naphtha compounds (mothballs)

Blister cell

 Typically, hemolysis is triggered by drugs or infections. Anemia is maximal 7-10 d after exposure. Individuals with A- begin to compensate for the anemia by making reticulocytes, despite continuation of drug. Immediately after a hemolytic episode, G6PD levels in pts with A- may be normal, since the mature cells have been lysed, and only younger cells with normal G6PD levels, are present

G6PD Deficiency Clinical Features

Congenital Hemolytic Anemias Hemoglobinopathies

Structural Hemoglobin Defects Hemoglobin S Hemoglobin C Thalassemias Alpha thalassemia Beta thalassemia

Thalassemia-like syndromes from decreased synthesis of an abnormal globin chain: Hb E, Hb LePore, Hb Constant Spring

Acquired Hemolytic Anemias Immune-Mediated Hemolysis

Pathophysiology
Red cells react with antibody, with or without complement fixation, leading to RBC destruction. IgG-coated RBCs interact with Fc receptors on macrophages, leading to complete or partial phagocytosis, with spherocyte formation (extravascular hemolysis). C3-coated red cells interact with C3 receptors on macrophages, with phagocytosis (extravascular hemolysis). Alternatively, complement cascade can be competed, with complement-mediated RBC lysis (intravascular hemolysis).

Immune hemolytic anemias Classification

Autoimmune Warm antibody-mediated Cold antibody-mediated Paroxysmal Cold Hemoglobinuria Drug-related hemolysis Hemolytic transfusion reactions Hemolytic disease of the newborn Paroxysmal Nocturnal Hemoglobinuria

Auto-Immune Hemolytic Anemias

Antibodies causing hemolysis can be broken down into 2 general categories: warm and cold. Warm antibodies react with RBCs best at 37 and typically do not agglutinate red cells Cold antibodies typically react best at <32 and do cause RBC agglutination The hallmark of this disease is a positive Coombs test

Coombs Test
The Direct Coombs = DAT (Direct Antiglobulin Test) - tests for IgG or C3 DIRECTLY ON THE RED CELLS. The Indirect Coombs - tests for IgG or C3 in the serum which react with generic normal red cells. This is also known as the antibody screen in blood-banking.

Direct Coombs Test

Patients RBCs coated with IgG or C3

Antibodies to human IgG/C3 (Coombs reagent)

Agglutination

Patient serum containing anti RBC Abs

Indirect Coombs Test

Normal RBCs and Coombs reagent

Agglutination

Normal control

Agglutinated red cells (positive test)

Note: the agglutination here takes place in the test tube--not in the body.

Warm-Antibody Hemolytic Anemias Pathophysiology

IgG antibodies directed against RBC membrane surface molecules are formed (Indirect Coombs test positive) IgG antibodies coat RBCs, with or without C3 complement (Direct Coombs positive) IgG-coated red cell membrane fragments engulfed by macrophages in RE system (usually spleen). Spherocytes form.

Spherocyte Formation

Progressive loss of membrane, loss of surface area, more spherocytic shape

Warm-Antibody Hemolytic Anemias Etiology

Can be either primary or secondary to another disorder, such as a hematologic malignancy or another autoimmune disease such as lupus Can be induced by drugs Can be associated with immune platelet destruction = Evans syndrome

Warm-Antibody Hemolytic Anemias Clinical Features

Splenomegaly, jaundice usually present. Depending on degree of anemia and rate of fall in hemoglobin, patients can have VERY symptomatic anemia Lab Dx reticulocytes, bili, LDH, positive Coombs test - both direct and indirect. SPHEROCYTES are seen on the peripheral smear.

Warm Autoimmune Hemolytic Anemia - spherocytes

Warm-Antibody Hemolytic Anemias Treatment Patients may require red cell transfusions, if they are symptomatic with their anemia However, immunosuppression is the mainstay of therapy.

Warm-Antibody Hemolytic Anemias Immunosuppressive Treatment

First line is corticosteroids If steroids fail to work, or if patient relapses after steroid taper, splenectomy may be necessary. IVIg can be used as adjunctive therapy. Immunosuppressives such as cyclophosphamide or azathioprine may be required as third-line therapy. Rituximab has been used successfully.

Drug-Induced Immune Hemolysis

Three general mechanisms Innocent bystander Hapten

Quinine, Quinidine, Isoniazide Penicillins, Cephalosporins Alpha-methyldopa, L-DOPA, Procainamide

True autoimmune

Cold Agglutinin Disease

Pathogenic antibodies are usually IgM, which bind to red cells in the cooler extremities, then fix complement. When red cells return to the warmer torso, IgM falls off. Complement-coated red cells can be lysed directly within the vessel (intravascular hemolysis). Alternatively, complement-coated red cells can be engulfed by complement receptors on macrophages within the liver (extravascular hemolysis)

Cold Agglutinin Disease

In the cold, IgM can lead to red cell agglutination Red cells clumps cannot pass through microvasculature, leading to cyanosis and ischemia in extremities. Smear shows RBC agglutination.

Digital ischemia from coldagglutinin disease

Cold Agglutinin Disease Clinical features

Can be associated with infection with either Mycoplasma or Mononucleosis, Can also be idiopathic or associated with a lymphoproliferative disease. Treatment is to keep patient (especially the extremities) warm. Blood and IV fluids should be warmed. Immunosuppression with oral chemotherapy may be required. Steroids and splenectomy are usually ineffective.

Paroxysmal Cold Hemoglobinuria

Pathogenic antibody is an IgG which only binds to red cells in the cold. It can fix complement in the warm. This antibody is called the DonathLandsteiner antibody. Formerly associated with syphilitic infection, now is mostly pediatric or idiopathic. Sample needs to be collected and kept warm. Sample is then chilled and rewarmed--want to see red cell lysis.

Paroxysmal Nocturnal Hemoglobinuria

An acquired disease in which an abnormal stem cell clone gives rise to abnormal RBCs, WBCs, and platelets all missing proteins which are attached to the cell surface by a GPI (glycero-phoshpatidylinositol) anchor. Among these proteins are CD55 and CD59, which protect red cells from complementmediated lysis. Diagnose by flow cytometry for CD55 and CD59 (at UNC, order as PNH Screen). Former diagnostic test was Hams test looking for abnormal sensitivity to addition of acidified serum. PNH cells lyse at a higher pH than normal red cells.

Paroxysmal Nocturnal Hemoglobinuria Clinical features Pancytopenia may be seen. Predisposition to thrombosis.

May develop during the course of aplastic anemia, either preceding aplasia or after recovery from aplasia. May develop into acute leukemia Hemolysis is predominantly nocturnal (when pH usually falls), usually mild.

Both arterial and venous, ae well as clots in odd places. Associated with development of BuddChiari syndrome.

Non-Immune Hemolytic Anemia Classification

Mechanical trauma to red cells
Microangiopathic Hemolytic Anemia Abnormalities in heart and large vessels March Hemoglobinuria

Infections Drugs, Chemicals, and Venoms

Microangiopathic Hemolytic Anemias

Shear damage to red cells as the result of endothelial cell activation/damage. Hallmark is presence of schistocytes on the peripheral smear. Can be caused by the following disorders:

TTP/HUS DIC

Malignant hypertension Ecclampsia, HELLP syndrome Vasculitis Other, including metastatic cancer, scleroderma renal crisis, solid organ transplant rejection

MAHA - Schistocytes

Macroangiopathic Hemolytic Anemias Abnormalities within heart and large vessels can cause shear damage to RBCs. Schistocytes are also seen here Prior to surgery: AS, ruptured sinus of valsalva, ruptured chordae, coarctation, aortic aneurysm and/or dissection. After surgery: complement activation in bypass or dialysis tubing, after patching operations, after valve replacement

March Hemoglobinuria
1861 - German soldier complained of dark urine after strenuous marches. 1964 - 2 track runners noted dark urine after races. Disappeared after softer shoe insoles used. Mechanical damage to red cells also reported after conga drum playing, head beating, and karate exercises.

Hemolysis from Infections

Malaria Babesia microti or divergens Bartonella bacilliformis
acquired from tick bite or from transfusion. Causes Oroya Fever--seen in South America hemolysis from extracellular parasite can be dramatic. Hemolysis can be so severe it produces a dissociation b/w hemoglobin and hematocrit

Clostridium welchii toxin

 Arsenic, especially arsine gas Lead - produces some shortening of RBC lifespan, but anemia mainly due to defect in heme synthesis. Copper - deliberate ingestion, accumulation of toxic amounts from dialysis fluid exposed to copper pipes, and Wilsons disease. Insect, spider (esp brown recluse), snake venoms Heat/burns

Hemolysis Associated with Chemical and Physical Agents