Anemia – Part 8 – Hemolytic Anemias, Autoimmune Hemolytic Anemia, Causes and Lab Diagnosis

Sample

1. Make fresh blood smear.
2. Take blood in EDTA.
3. Bone marrow may be advised if needed.

Definition of hemolytic anemia

1. Hemolytic anemia results from the premature destruction of the peripheral blood RBCs.
   1. The normal removal of the RBCs:
   2. RBCs destruction occurs after a mean life span of 120 days when these are removed in the extravascular space by the MN phagocytic system, especially in the bone marrow and the spleen, and the liver.
2. Hemolytic anemias are defined as when there is increased destruction of the red blood cells.
   1. Because of the compensation from the bone marrow anemia appears, when the destruction of RBCs becomes several times more than this compensatory mechanism, it will lead to hemolytic anemia.
   2. Hemolytic Anemia  may be:
      1. Intrinsic where the RBCs are abnormal.
      2. Extrinsic when the external factors will destroy the RBCs.
3. Hemolytic anemias may be due to congenital or acquired defects. The site of hemolysis is:
   1. Intravascular hemolysis when the destruction of the RBCs is in the circulation (blood vessels).
   2. Extravascular hemolysis when the RBCs are destroyed by the mononuclear phagocytic system of the spleen, liver, and bone marrow.
4. All these anemias share common defects, which are shortened survival of the RBCs.
5. The outcome of the hemolysis:
   1. If the bone marrow hyperplasia compensates for the RBC loss, then no anemia will develop, called compensated hemolytic disease.
   2. The bone marrow becomes hyperplastic, and the increase in erythropoiesis increases 6 to 8 times the normal. Also, there is an increase in the volume of active marrow.
   3. Immature RBCs (reticulocytes) are released prematurely into the circulation and give rise to polychromasia.
      

      Hemolytic anemia leading to reticulocytosis and polychromasia

Anemias based on Coomb’s test:

Coomb’s positive hemolytic anemia	Coomb’s negative hemolytic anemia
Antibody-dependant hemolysis: Alloantibodies Autoantibodies Drugs-related antibodies	Non-antibody dependant hemolysis: Intrinsic RBCs abnormalities: Red blood cell membrane defects RBCs enzyme deficiency Hemoglobinopathies (Hb S, Hb C) Extrinsic RBCs mechanism: Burns DIC Vasculitis Intracellular infection (Malaria) Mechanical damage (Heart valve replacement) Toxins

The causes or etiology of RBC hemolysis may be:

1. Hereditary is due to intrinsic red cell defects.
2. Acquired are due to environmental changes.
   1. Hemolytic anemia can also be divided into:
      1. Anemias associated with intrinsic/intracorpuscular defects of RBCs.
         1. The life of the RBCs in patients and even in the recipient is shortened.
      2. Anemias associated with extrinsic/extracorpuscular defects of RBCs.
         1. In these cases, if transfuse normal RBCs into this group will be destroyed more rapidly.
3. Intravascular hemolysis when the destruction of the RBCs takes place in the blood vessels and hemoglobin is released.
   1. This is associated with physical trauma, like an artificial heart valve.
   2. Or this is associated with the complement system like paroxysmal nocturnal hemoglobinuria.
      1. Hemoglobin in the blood gives rise to hemoglobinuria.
   3. Causes are:
      1. Mismatched blood transfusion.
      2. G6PD deficiency associated with stress.
      3. March hemoglobinuria.
      4. Few autoimmune hemolytic anemias.
         

         Intravascular Hemolysis

4. Extravascular hemolysis when the destruction of the RBCs takes place in the mononuclear phagocytic system.
   1. The MNphagocytic system will trap and disrupt the abnormal cells.
   2. The spleen is very efficient in this respect and is followed by the liver.
      1. That is why there is splenomegaly in the hemolytic anemia (disease).
         

         Extravascular Hemolysis

5. Abnormal hemoglobin: The abnormalities of the hemoglobin function, and Heinz bodies formation, causes the RBCs to lose their elasticity. When such RBCs are going through the microcirculation, the RBC membrane cannot adjust to the small blood vessels, so these are trapped and destroyed.

Hereditary Causes of hemolytic anemia:

1. RBC cell membrane defect:
   1. Abnormality in the cell membrane cytoskeleton leads to spherocytosis and elliptocytosis.
   2. Abnormal lipid synthesis like an increase in the membrane lecithin.
2. RBC cell enzyme deficiency:
   1. G-6-PD deficiency.
   2. Pyruvate kinase deficiency.
   3. Hexokinase deficiency
   4. Glutathione synthetase deficiency
3. Disorders of hemoglobin synthesis:
   1. Hemoglobin synthesis defect like Thalassemia.
   2. Abnormal structure of the hemoglobin-like Sickle cell anemia, Hb E.
      

      RBC Sickle cell

      

      RBC Spherocyte

Acquired Causes of hemolytic anemia:

1. Antibody-mediated cytotoxic reaction (Type II) like ABO incompatibility.
   1. Hemolytic autoimmune anemia is seen with cold antibody and warm antibody.
   2. Drug-induced hemolytic anemia seen in Methyldopa, penicillin,
2. Microangiopathic hemolytic anemia is seen due to RBCs’ trauma in the cardiac prosthetic valve and fibrin deposition in the microvasculature.
3. Infections: Hemolytic anemia due to toxins like a malarial parasite, septicemia, Clostridium welchii, pneumococci, staphylococci, and lead poisoning.
   1. Hemolytic anemia due to snake venom and spiders’ bite.
   2. Hemolytic anemia due to lead poisoning and copper toxicity.
4. Hemolytic anemia due to extensive burns.
5. Hemolytic anemia in splenomegaly.
6. Paroxysmal nocturnal hemoglobinuria.
7. Anemia due to acquired RBCs membrane abnormality.
8. Secondary to liver and kidney diseases.
   

   Hemolytic anemia causes

   Causes of hemolytic anemia:

   Congenital causes of hemolytic anemia (inherited)

   Acquired causes of hemolytic anemia

   Hemoglobinopathies: Sickle cell anemia (Hb S) Hb C Thalassemia RBC Membrane defects: Hereditary spherocytosis Hereditary acanthocytosis Hereditary elliptocytosis Hereditary stomatocytosis Enzyme deficiency (metabolic defect): G6PD deficiency Pyruvate kinase deficiency

   Autoimmune: Cold antibody Infections like mycoplasma pneumonia Lymphoma Infectious mononucleosis Paroxysmal cold hemoglobinuria Warm antibody SLE Drugs (methyldopa) Lymphoma CLL Alloimmune: Hemolytic disease of the newborn Blood transfusion reaction Marrow transplantation rejection (allograft rejection) Non-immune: Paroxysmal nocturnal hemoglobinuria Chemical agents: Drugs Industrial chemicals Domestic substances Infectious agents: Malaria Clostridia Physical trauma: Burn Valve prosthesis Microangiopathic hemolytic anemia Secondary causes: Liver diseases Renal diseases Hemolytic transfusion reaction

Another classification of hemolytic anemia:

Intracorpuscular causes	Extracorpuscular causes
Hereditary: Enzyme defects G6PD deficiency Pyruvate kinase deficiency Defects in the RBC membrane Hereditary spherocytosis Hereditary elliptocytosis Hemoglobinopathies: Thalassemia syndrome Hb S, Hb C	Immune hemolytic anemia Chemicals and toxins Physical agents Infections Hypersplenism Microangiopathic hemolytic anemia Macroangiopathic hemolytic anemia
Acquired: paroxysmal nocturnal hemoglobinuria Infections from Clostridia and P.falciparum

Pathogenesis of drug-induced hemolytic anemias:

There are three different mechanisms in the case of drug-induced hemolytic anemia:

Clinical presentation of hemolytic anemia:

1. There is pallor of the mucous membrane, which can be judged from the tongue.
2. There is a fluctuating jaundice. In some patients, bilirubin will be very high and may need blood transfusion exchange.
3. Urine shows no bilirubin, but there is the presence of the urobilinogen.
4. Gallstones are common in these patients.
5. Patients with sickle cell disease may develop ulcers around the ankle.
6. Aplastic crises may be seen in these patients.
7. Rarely folate deficiency causes aplastic crises.

Autoimmune hemolytic anemia:

1. 1. Definition of Autoimmune hemolytic anemia:
      1. These are caused by the production of the antibody against self RBCs.
      2. These are characterized by the direct coomb’s test (direct antiglobulin test) and divided into:
         1. Warm antibody AIH reacts more strongly at 37 °C.
            1. The type of antibody is IgG.
            2. Coomb’s test is strongly positive.
            3. Mostly the cause is idiopathic.
            4. This is an autoimmune phenomenon and the best example is SLE, lymphoma, Hodgkin’s disease, and drugs (methyldopa).
            5. Signs and symptoms:
               1. 30% of the cases cause is not known.
               2. These anemias may occur at all ages and in both sexes.
               3. But more common in middle-aged females.
               4. There attack of relapse and remission with a short period of anemia and jaundice.
               5. This may progress to the chronic form.
               6. It is usually splenomegaly.
               7. Infection or folate deficiency may lead to a falling level in the hemoglobin.
            6. Diagnosis:
               1. There is a reticulocytosis.
               2. Bone marrow shows erythroid hyperplasia.
               3. Raised serum bilirubin level.
               4. Increased urinary urobilinogen.
               5. Decreased plasma haptoglobin.
               6. There is spherocytosis.
         2. Cold antibody AIH reacts more strongly at 4 °C.
            1. This reaction takes place usually below 37 °C.
            2. Mostly the antibody is IgM is cold agglutinin reacting at 4 °C are present in the plasma.
            3. Mechanism: At the low temperatures these antibodies can attach to RBCs and cause their agglutination in the cold periphery of the body like hands and feet.
               1. In addition, there is the activation of the complement and this will leads to intravascular hemolysis.
               2. After the infection by Mycoplasma, cytomegalovirus, and Epstein Barr virus. there is increased synthesis of polyclonal cold agglutinins.
               3. These cold cold agglutinins lead to mild to moderate transient hemolysis.
               4. The chronic cold haemagglutinins disease is usually seen in elderly people. This is due to the production of the monoclonal IgM cold agglutinins.
            4. Coomb’s test is positive but not strongly.
            5. This may take place from infections like infectious mononucleosis, Mycoplasma pneumoniae, and rarely viral infection.
            6. This condition may be from lymphoma and paroxysmal cold hemoglobinuria (due to IgG).
            7. Diagnosis:
               1. RBCs agglutinate at cold temperatures or at room temperature. Some time when you keep the sample at a cold temperature will show agglutination.
               2. Direct Coomb’s test is positive with complement.
            8. Treatment:
               1. Try to find the underlying cause.
               2. Keep the body parts warm and avoid cold exposure.
               3. Treatment with steroids, alkylating agents, and splenectomy does help these patients.
   2. Clinical signs and symptoms of autoimmune hemolytic anemias:
      1. This may be seen at any age and sex, and anemia is of varying severity.
      2. There may often be splenomegaly.
      3. Usually, there are attacks and remissions.
      4. It may start after some drug therapy like methyldopa.
      5. It may be associated with other diseases Like SLE and ITP.
         1. In SLE, the cells are coated with immunoglobulins and complements.

Treatment of autoimmune hemolytic anemia:

1. 1. 1. Remove the cause like drugs (methyldopa).
      2. The first treatment line is corticosteroids, and prednisolone, starting as 60 mg/day in adults, and then it is tapered down.
      3. In the case of complement associated AIH, the response is poor to steroids and even splenectomy.
      4. Immunosuppression can be tried when other measures fail. It can give Azathioprine, cyclophosphamide, chlorambucil, mycophenolate, and ciclosporin.
      5. Monoclonal antibodies, anti-CD20, and anti-CD52, have been tried successfully in a few cases.
      6. Folic acid is given in severe cases.
      7. Blood transfusion is given in the case of anemia.
      8. In the case of ITP, immunoglobulin can be given.

 Hemolytic anemia, lab changes:

1. The lab findings can be divided into three broad categories:
   1. Evidence for increased RBCs breakdown:
      1. Increased serum bilirubin and mainly unconjugated bilirubin (indirect bilirubin).
      2. Excessive urobilinogen is excreted in the urine (this is due to the breakdown of the bilirubin in the intestine).
      3. Fecal stercobilinogen is also increased in the stool.
      4. There is an absence of serum haptoglobin due to saturation with hemoglobin, and the MN phagocytic system removes it.
      5. Raised level of lactate dehydrogenase (LDH).
         

         Hemolytic anemia and haptoglobin

2. Increased production of RBCs (Erythroid hyperplasia):
   1. Increased reticulocyte count (reticulocytosis). Normal range is 0.5% to 2%.
      1. They leave the bone marrow and goes to peripheral blood, where they get maturation, which is more than the normal of one day. This will leads to a false increase in the reticulocyte count.
   2. There is bone marrow erythroid hyperplasia with changed M: E (normal = 2:1 to 12: 1) is decreased to 1:1, or there is a reversal.
3. Evidence of damaged RBCs:
   1. Morphologically will see elliptocytes, fragmented RBCs, and microspherocytes.
   2. Osmotic fragility is increased.
   3. RBC survival is shortened, and it can be studied by labeling the RBCs with ⁵¹Cr. It also can identify the site of destruction.
   4. Separate the patients into groups:
      1. Coombs test positive group, e.g., immune hemolytic anemia.
      2. Coombs negative group, e.g., non-immune hemolytic anemia.
4. Hb is reduced and may be mild to moderate, decreased from 6 to 10 G/dl.
5. Reticulocytes are increased from 5 to 20 % (Reticulocytosis).
6. MCV is normal or slightly increased,
7. MCHC is increased.
8. Bone marrow shows erythroid hyperplasia.
   1. Normal myeloid: the erythroid ratio of 2:1 to 12:1 is reduced to 1:1 or much more reduced.
9. Coombs test direct is negative, which differentiates it from autoimmune hemolytic anemia.
10. The peripheral blood smear shows poikilocytosis and polychromatophilia due to reticulocytes.
    1. There are elliptocytes and fragmented RBCs.
    2. There are normochromic and normocytic anemia pictures.
11. RDW is increased due to anisocytosis and poikilocytosis. Unlike other normochromic and normocytic anemias.
    1. Increased reticulocytes lead to increased MCV but not like megaloblastic anemias.
12. Serum bilirubin is raised.
    1. Urine urobilinogen is positive.
    2. Stercobilinogen is increased
13. Serum haptoglobin is absent.

Lab findings in hemolytic anemia:

Parameters	Hemolytic anemia
Hb	Decreased  6 to 10 g/dL
Reticulocytes	Increased 5 to 20%
MCV	A normal or mild increase
MCHC	Increased
Bone marrow	Erythroid hyperplasia
Coombs test	Direct is positive in acquired AIH.
RDW	Increased

See more details in Anemia part 1.

---