Hemolytic anemia

Fazel Elahi (MD) Oncologist &Hematologist Production of Erythrocytes: Erythropoiesis Erythrocytes (RBCs) Components of Whole Blood

Plasma (55% of whole blood)

Buffy coat: leukocyctes and platelets (<1% of whole blood) Formed elements Erythrocytes 1 Withdraw blood 2 Centrifuge (45% of whole blood) and place in tube Erythropoietin Mechanism

Start Normal blood oxygen levels Stimulus: Hypoxia due to decreased RBC count,

decreased availability of O2 to blood, or increased Increases tissue demands for O2 O2-carrying ability of blood

Reduces O2 levels in blood

Erythropoietin Kidney (and liver to a Enhanced stimulates red smaller extent) releases erythropoiesis bone marrow erythropoietin increases RBC count Structure of Hemoglobin Hemolysis:

 Any condition characterized by a significantly decreased erythrocyte life span RBC destrution

 Hemolysis  Ineffective erythropoesis  hematoma

Haemolysis may be predominantly extravascular (i.e. phagocytic uptake) or intravascular (i.e. in the blood stream

Definition A haemolytic anaemia is an anaemia resulting from an increased rate of red cell destruction. This results in a shortening of the red cell life span  Erythropoiesis in the bone marrow can expand by up to 6-fold to compensate for accelerated red cell destruction, but anaemia results when red cell destruction exceeds erythropoiesis Increased erythropoiesis leads to a classification

 Intavascular or Extravascular

 Inheritance or Aquired

 Extrinsic or Intrinsic Causes

 INTRACORPUSCULAR HEMOLYSIS Membrane Abnormalities Metabolic Abnormalities Hemoglobinopathies  EXTRACORPUSCULAR HEMOLYSIS Nonimmune Immune Classification

The haemolytic anaemias can be classified as inherited or acquired Inherited haemolytic anaemias can be subclassified into

I. Haemoglobin defects II. Red cell membrane defects III. Red cell enzyme defects HEMOLYTIC ANEMIA Membrane Defects

 Microskeletal defects Hereditary  Membrane permeability defects Hereditary stomatocytosis  Increased sensitivity to complement Paroxysmal nocturnal hemoglobinuria Hereditary hemolyisis

 Hemoglobinopathy (alfa,beta thal, S,S beta ,C ,SC…..)  Membran disorders ( HS ,HE, HPP, STOMATOCYTOSIS …)  Enzyme defect: 1-Glycolytic pathway 2-Pentose phosphate pathway 3-Nucleotid metabolism INHERITED HAEMOLYTIC ANAEMIAS

 HAEMOGLOBIN DEFECTS  Thalassaemia  Sickle cell anaemia  Other haemoglobin defects

 MEMBRANE DEFECTS   Other membrane defects

 ENZYME DEFECTS  G6PD deficiency  Other enzyme defects  All Hereditary etiology of hemolysis have intrinsic defect

 Only nonhereditary intrinsic defect (PNH)

In some of intrinsic defect hemolysis one extrinsic factor cause hemolysis or aggrevated hemolysis (G6PD) Acquired haemolytic anaemias

immune or non-immune

The entire classification encompasses a very large number of different haemolytic disorders ACQUIRED HAEMOLYTIC ANAEMIAS

IMMUNE Auto-immune haemolytic anaemia Allo-immune haemolysis

NON-IMMUNE Microangiopathic haemolytic anaemias Infections Drugs and toxins Acquired membrane disorders Mechanical and physical agents Aquired

 Immunohemolytic  Microangiopathic  Traumatic  Infectious agent  Chemical agent  Physical agent  Spur cell anemia  PNH  hypophosphATEMIA  Vit .E deficency EXTRACORPUSCULAR HEMOLYSIS Nonimmune

 Mechanical  Infectious  Chemical  Thermal  Osmotic Microangiopathic Hemolytic Anemia Causes

 Vascular abnormalities  Thrombotic thrombocytopenic purpura  Renal lesions Malignant hypertension Glomerulonephritis Preeclampsia Transplant rejection Vasculitis Polyarteritis nodosa Rocky mountain spotted fever Wegener’s granulomatosis Microangiopathic Hemolytic Anemia

Vascular abnormalities

 AV Fistula  Cavernous hemangioma Intravascular coagulation predominant Abruptio placentae Disseminated intravascular coagulation Hemolysis

 Acute

 Chronic

 Acute on chronic Clinical finding

Acute haemolysis presents with :

Fatigue, Pallor, Jaundice, Fever, Chills, Low back pain, Splenomegaly and Congestive cardiac failure

Haemoglobinuria results if the haemolysis is predominantly intravascular Clinical finding

Chronic haemolysis : (e.g. thalassemia, sickle cell anaemia etc)

Present with pallor, jaundice, splenomegaly and congestive cardiac failure; but in addition there may be gallstones, bony deformities and pathological fractures due to marrow erythroid expansion with thinning of the bone cortex Causes of Intravascular Haemolysis

Infection : Malaria, clostridium perfringen G6PD deficiency Severe auto-immune haemolytic anaemia Paroxysmal nocturnal haemoglobinuria Paroxysmal cold haemoglobinuria  Transfusion : ABO mismatched blood, infected blood Burns Cardiac prosthesis Intravascular haemolysis

Elevated plasma haemoglobin (haemoglobinaemia) urinary haemoglobin (haemoglobinuria) serum LDH urinary haemosiderin

Elevated unconjugated bilirubin and reduced haptoglobins are found in both intra- and extravascular haemolysis Chronic intravascular haemolysis

Results in iron deficiency due to the loss of haemoglobin, haemosiderin and ferritin in the urine Extravascular haemolysis

Red cells are destroyed by phagocytosis by macrophages in the spleen, liver and bone marrow

This uptake system is often referred as the reticulo-endothelial system (R.E.S). In this situation, free haemoglobin is not liberated into the bloodstream Detecting reasons of hemolysis

 Duration and severity  Jundice (acholuric )  Splenomegaly  Cholelithiasis (black stone)  Leg ulcer  Skeletal abnormality  Fever , shaking chills  Pain ( limb, back,abdominal,headache )  Malaise ,vomiting ,hypotention  Shock ,oliguria, aneuria  History ( famalial , personal , drugs )  Physical exam  lab test : 1- Sign of accelerated RBC destruction 2- Sign of accelerated erythropoesis Normal blood film:  There is a slight degree of (variation in cell size). The width of central pallor is less than one- third of the diameter of the cell. Laboratory finding

Peripheral blood film

Increased red cell destruction e.g. jaundice, unconjugated hyperbilirubinaemia, elevated serum lactate dehydrogenase (LDH), reduced haptoglobins

Increased erythropoiesis e.g. reticulocytosis, polychromasia, radiological changes of erythroid hyperplasia in chronic haemolysis The biochemical profile is characteristic of a pre- hepatic jaundice with unconjugated hyperbilirubinaemia and elevated LDH, but normal hepatocellular enzymes

There are a relatively small number of causes of intravascular haemolysis Therefore, tests for intravascular haemolysis are useful because the differential diagnosis for haemolysis can be considerably simplified if it can be shown that predominantly intravascular haemolysis is taking place RBC MORPHOLOGY HAEMOLYTIC ANAEMIA

Spherocytes hereditary spherocytosis, immune haemolysis, sepsis

Fragmentation DIC, artificial heart valves, malignancy, TTP, HUS Agglutination immune haemolysis

Target cells liver disease, thalassaemia, haemoglobin C Sickle cells S/S, S/C, S/Thal Blister cells G6PD deficiency liver disease, pancreatitis, abetalipoproteinaemia Prickle cells pyruvate kinase deficiency post-splenectomy blood film ,nucleated red cells, Howell- Jolly bodies, acanthocytes, target cells, mild to moderate leukocytosis and thrombocytosis

This film is from a splenectomised patient who was in chronic renal failure, and therefore also shows burr cells.  Reticulocytes are larger than mature red cells and stain purplish with Romanowsky stains (e.g. May Grunwald Giemsa). Reticulocytosis leads to a rise in the MCV (mean cell volume). The reticulocyte count is a simple test that provides a rough guide to the rate of red cell production

The reticulocyte count does not neccessarily reflect true erythroid activity because of premature or delayed release of reticulocytes from the marrow and variable rates of reticulocyte maturation in response to different conditions Reticulocyte count

(reference range 0. 2 - 2.0%)

Number of reticulocytes per litre

(10-100 x 109/L) Reticulocyte count: methylene blue stain BMA Erythroid Hyperplasia (haemoglobinaemia): Reddish plasma in severe intravascular haemolysis Urinary haemosiderin May persist for several weeks after an acute haemolytic episode, and may therefore be useful in detecting a bout of recent haemolysis Urinary haemosiderin: Perl's iron stain of urine cellular debris Sign of RBC destruction

 Uncongugated bilirubin increased  Urobilinogen “ “ “  LDH LDH2>LDH1 “ “ “  Haptoglobin deceased  GHb glycosylated hemoglobin “  RBC life span (t ½ Cr ) “  Carbon menoxide increased  Free haemoglobin binds with high affinity to a serum glycoprotein, haptoglobin and the complex is cleared by the reticulo-endothelial system (R.E.S.), resulting in a low or absent serum haptoglobin.

Albumin combines with haem to form methaemalbumin which gives a brown colour to the plasma. Methaemalbumin can be biochemically detected by Schumm's test

 (LDH) is released from red cells during haemolysis and this contributes to a rise in serum LDH, which is a useful marker for intravascular haemolysis.

 Massive deposition of free haemoglobin in the renal tubules can cause acute oliguric renal shutdown, a medical emergency, as seen in the haemolytic transfusion reaction. Intravascular hemolysis

 HEMOGLOBINEMIA  HEMOGLOBINURIA  HEMISIDRINURIA  METHEMOGLOBINEMIA  METHEMALBUMINEMIA  HEMOPEXIN decreased  Urine hemosiderrin Spacific Lab test

 Antiglobulin (coomb”s ) test  Osmotic fragility test  Autohemolysis  Heat stability test (denaturation procedure )  formation  Isopropanol precipitation test  Sickling test  Retic with incubation (H.inclusion)  Identifying Enzyme deficency  Hemoglobin & globin chain study  PNH Test HAM& sucrose lysis test ,flucytometry  Glutathion stability test  Ascorbate - cyanid test Sign of accelerated erythropoesis

 Retic  NRBC (erythroblastosis)  Polychromatophilia   Basophilic stipling  Leukocytosis (PMN rise), leukopenia  Thrombocytosis ,thrombocytopenia, large Plt  Erythrophagocytosis  Autoagglutination  BM erythroid hyperplasia , megaloblastic change  Effective erythropoesis (PITR ,EITR )  Macrocyte  Tin-macrocyte ,leptocyte ,wafer cell  Target cell .  Spherocyte , microspherocyte  ovalocyte  Stomatocyte I,II,III, spertostomatocyte spheroechinocyte IV (dense crenated spher ,prickle cell )  .spur cell  .Burr cell,Crenated cell,berry cell type I,II,III  ,helmet ,fragmented RBC  Sickle cell ,Drepantocyte, boat shape RBC  Keratocyte ,Horn cell  Tear drop ,tennis racket, dacrocyte   Heinz body ,howell jolly body  Agglutination ,heophagocytosis  Cabot Ring Reticulocyte Manual Count by Supravital Stain: Normal Count Reticulocytes: Elevated Count Hemolytic crisis

Aplastic crisis

Megaloblastic crisis Compensated hemolytic state

A state of hemolysis in which the resulting increased erythrocyte production is able to keep up with accelerated RBC destruction, thus averting any anemia Hemolytic anemia

A state of hemolysis in which increased erythrocyte production is insufficient to keep up with accelerated RBC destruction, thus producing anemia

This anemia is characterized as normochromic/normocytic, except when sufficient outpouring of the larger reticulocytes produces a resulting elevation of the MCV Diagnosis of hemolytic anemia is performed in four steps Step -1

1- Establish that anemia exists Step - 2 Marrow response  Hemolysis is demonstration of an attempted marrow response to erythrocyte destruction

. Reticulocyte count . Absolute retic count (in cells/µL) . Reticulocyte production index (RPI) Step - 3

Look for erythrocyte detritus/their component catabolites, such as free hemoglobin, methemoglobin, methemalbumin, bilirubin, and urobilinogen, as well as the specific binding proteins for these catabolites, such as haptoglobin and hemopexin. Step - 4

The first distinction to make is to determine whether the hemolysis is (extravascular hemolysis) or(intravascular hemolysis)

Both types produce indirect hyperbilirubinemia, urobilinogen in stool and urine, decreased serum haptoglobin, and reticulocytosis intravascular hemolysis produces hemosiderin in the urine sediment, free hemoglobin in the serum (which may be grossly visible), and free denatured hemoglobin in the urine. helmet-shaped schizocytes (or ""), which can be seen on the routine peripheral blood film

Extravascular hemolytic anemias may produce spherocytes, Leptocyte (hypochromic microcytic red cell), Blood smear Poikilocyte (DIC), Blood smear, Tear Drop Cell, Blood smear Basophilic Stippling, Blood smear Pappenheimer's body, Blood smear Howell-Jolly's body, Blood smear Formation, Blood smear Malaria, Blood smear Thalassemia, Bone marrow Schizocytes

FREQUENCY

 Internationally: Hemolytic anemia represents approximately 5% of all anemias. RACE

. Sickle cell disorders are found primarily in Africans, African Americans, some Arabic peoples, and Aborigines in southern India

. Several variants of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency exist. The A variant generally is found in West Africans and African Americans. Approximately 10% of African Americans have at least 1 gene for this variant

. The Mediterranean variant occurs in individuals of Mediterranean descent and in some Asians SEX

 Most cases of hemolytic anemia are not specific to any gender.  Autoimmune hemolytic anemia (AIHA) is slightly more likely to occur in females than in males.  G-6-PD deficiency is a X-linked recessive disorder. Males usually are affected, and females are carriers AGE

 Hemolytic anemia can occur at any age.  Hereditary disorders usually are evident early in life  AIHA is more likely to occur in middle-aged and older individuals Peripheral smear and morphological examination

Identifies polychromasia, indicating RBC immaturity reticulocytosis

Demonstrates spherocytes, suggesting congenital spherocytosis or AIHA

Can identify schistocytes (fragmented red cells), suggesting TTP, HUS, or mechanical damage

Can help diagnose a concomitant underlying hematological malignancy associated with hemolysis (ie, CLL) Reticulocyte count

An increased reticulocyte count is a criterion for hemolysis but is not specific for hemolysis

An increase may be caused by blood loss or a bone marrow response to iron, vitamin B-12, or folate deficiencies

The reticulocyte count may be normal or low in patients with bone marrow suppression despite ongoing severe hemolysis Lactic acid dehydrogenase

 Serum LDH is a criterion for hemolysis. LDH is not specific because it is ubiquitous and can be released from the neoplastic cells of the liver or other damaged organs.

 Although an increase in LDH isozyme 1 and 2 is more specific for RBC destruction, these enzymes also are increased in patients with myocardial infarction. Serum haptoglobin

. A low serum haptoglobin is a criterion for moderate-to-severe hemolysis

. A decrease is more likely in intravascular hemolysis than in extravascular hemolysis, but it is an acute phase reactant

. The presence of concomitant infection, other reactive states, or chronic hemolysis may mask the diagnosis by raising haptoglobin levels Indirect bilirubin

 Unconjugated bilirubin is a criterion for hemolysis, but it is not specific because an elevated bilirubin also may indicate Gilbert disease

 With hemolysis, the level of indirect bilirubin usually is less than 4 mg/dL

 Higher levels of indirect bilirubin indicate compromised hepatic function or cholelithiasis and hemolysis  Changes in the LDH and serum haptoglobin levels are the most sensitive general tests because the indirect bilirubin is not always increased Imaging Studies:

 Use ultrasound to estimate spleen size. The physical examination occasionally does not detect significant splenomegaly.

 Chest radiograph is used to evaluate cardiopulmonary status Other Tests:

 ECG and other studies are used to evaluate cardiopulmonary status Prognosis:

 Depends upon the underlying cause for hemolysis

Patient Education:

 The patient must be able to identify symptoms and signs of hemolysis recurrence and seek prompt medical attention if they occur.  There are numerous fragmented RBC's seen here. Some of the irregular shapes appear as "helmet" cells. Such fragmented RBC's are known as "schistocytes" and they are indicative of a microangiopathic hemolytic anemia (MAHA) or other cause for intravascular hemolysis. This finding is typical for disseminated intravascular coagulopathy (DIC)  The CBC of a patient with microangiopathic hemolytic anemia (MAHA) demonstrates a markedly increased RDW (red cell distribution width) due to the marked variation in size and shape of the RBC population. Erythrocyte Inclusions with Wright’s Stain

Inclusion Composition Appearance Condition

Basophilic Precipitated Evenly dispersed Lead poisoning stippling ribosomes fine or coarse granules thalassemia other anemia Howell-Jolly Nuclear Dense, round Post splenectomy bodies fragment blue granule Pappenheimer Iron-containing Small blue granules Anemias bodies granules in clusters Organism Small blue inclusion Malaria Babesiosis Basophilic Stippling Howell-Jolly Body Malaria RBC Inclusions: Composite Erythrocyte Distribution Abnormalities

 Rouleaux formation Stacking of RBCs due to increased plasma proteins coating RBCs

 Agglutination Antibody-mediated clumping; temperature dependent Rouleaux Formation Agglutination Reaction Variations in RBC Size and Shape

 Anisocytosis Variations in size (e.g. microcytes)

Variations in shape (e.g. target cells)

 Hypochromia Increased central pallor due to decrease in hemoglobin Variations in RBC Size and Shape

 Anisocytosis Variations in size (e.g. microcytes)

 Poikilocytosis Variations in shape (e.g. target cells)

 Hypochromia Increased central pallor due to decrease in hemoglobin Hypochromic Microcytic RBC Normal Hypochromic microcytic Hypochromia without Anisocytosis: Thalassemia Trait Severe Hypochromia: Iron Deficiency Anemia Mixed Population: Treated Iron Deficiency Anemia Microcytic Hypochromia: Alpha Thalassemia (a-/--) Microcytic Hypochromia: Major Microcytic Hypochromia: Beta Thalassemia Major Macrocytic Anemia: Macro- Ovalocytes Shape Abnormalities of Erythrocytes Terminology Description Condition

Target cells Central hemoglobin; target-shaped Liver disease; thalassemia: Abnormal Hgb; iron deficiency Echinocyte Short spicules, equally-spaced Uremia, hypokalemia, artifact Acanthocyte Spiculated, irregular Liver disease (alcohol), Post-splenectomy Spherocyte Spherical, no central pallor HS, Immune hemolytic anemia Schistocyte Fragmented RBC, helmet cells MAHA, burns

Ovalocyte Oval/elliptical shaped Hereditary elliptocytosis, Megaloblastic anemia Sickle cell bipolar spiculated shape Hgb S-containing “banana” shaped hemoglobinopathy Teardrop cell single elongated extremity Myelophthistic changes Bite cells Irregular gap in membrane G6PD deficiency Target Cells

 Diagnostic possibilities  Liver disease  Hemoglobinopathy  Thalassemia  Iron deficiency  Post-splenectomy  Lipid disorders (Burr Cells) Acanthocytes (Spur Cells) Morphologic Changes in Liver Disease

Target Spur Cells Cells Hepatorenal Syndrome: Burr + Spur Cells Spherocytes Spherocytes: Autoimmune Hemolytic Anemia Spherocytes: Hereditary Spherocytosis Schistocytes: Microangiopathic Hemolytic Anemia : Hereditary Elliptocytosis Sickle Cell Anemia: Hgb SS Hemoglobin SC Disease Hemoglobin S-Beta Thalassemia Teardrop Cells Bite Cells Heinz Bodies