1. MLAB 1415- Hematology Keri Brophy-Martinez
Hemolytic Anemia: Immune Anemias

2. Classification of Immune Hemolytic Anemia’s
Autoimmune hemolytic anemia
Drug-induced hemolytic anemia
Alloimmune hemolytic anemia

3. Role of Complement
Group of serum proteins that interact with each other to bring about complement-dependent, cell-mediated lysis.
Can be activated by two pathways:  classical and alternate.
Classical pathway activated by antigen-antibody complexes.
Alternate pathway triggered by certain microorganisms, polysaccharides, liposaccharides, and cells or particles even in absence of antibody.  Still results in cell lysis.
WILL LEARN ABOUT COMPLEMENT IN IMMUNOLOGY.  DO NOT MEMORIZE PATHWAYS NOW!

4. Mechanisms of Hemolysis
Intravascular
Results from activation of the classic complement pathway via immunoglobulin IgG or IgM antibodies. IgM more efficient activator of complement
Antibodies bind to antigens on red cells and activate complement resulting in lysis of the cell.
Extravascular
Most common with immune-mediated hemolysis
Usually involves IgG
Antibody coated or complement coated red cells are fully or partially phagocytized by cells in the reticuloendothelial system (RES), particularly in the spleen and liver. Partially phagocytized cells are seen as spherocytes on the peripheral smear.

5. Laboratory Findings in Intravascular Hemolysis
Hemoglobinemia
Hemoglobinuria
Haptoglobin
Other test results include elevated lactate dehydrogenase (LDH) levels – LDH is enzyme of red cell metabolism.
May or may not see reticulocytosis.

6. Laboratory Findings in Extravascular Hemolysis
Will see spherocytes on peripheral blood smear.
Serum bilirubin (indirect) may be elevated and urobilinogen concentration may be increased in both urine and stools.
Direct Antiglobulin Test (DAT) may be positive, as well as indirect antiglobulin test (antibody screening test).

7. Laboratory identification of sensitized red cells
Two major techniques
Agglutination in saline
Detects IgM class of antibodies
Antihuman globulin(AHG) or Coombs test(DAT)
Detects IgG class of antibodies and/or complement
Difference in the ability of IgG and IgM to cause agglutination in saline is based on the difference in size of the two antibodies in relation to the zeta potential

8. RBC Zeta Potential
Caused by a difference in the charge density of the inner and outer layers of the ionic cloud around rbcs when they are suspended in saline. The force keeps rbcs about 25nm apart in solution. So any antibody that causes agglutination must be > 25nm to span the gap between cells

9. RBC Zeta Potential
IgM can span 35nm to overcome forces, causing agglutination
IgG spans about 14nm, so it can not reach antigens on separate cells to cause agglutination. Therefore, another technique must be used.

10. Autoimmune Hemolytic Anemias

11. Autoimmune Hemolytic Anemia (AIHA)
Caused by an altered immune response resulting in production of antibody against the patient’s own red cells, with subsequent hemolysis
These autoantibodies are directed against high-incidence antigens
Mechanism of antibody formation unknown

12. Warm AIHA (WAIHA)
Most common form of AIHA ~70%
Reacts best at body temperature (37oC)
Can occur at any age, but incidence increases over the age of 40
Cases can be idiopathic or secondary to another disease
IgG coats red cells with or without complement fixation
Antibody usually directed against antigens of the Rh system

13. Laboratory findings of WAIHA
Common Findings
Positive DAT
Normocytic, normochromic anemia
Increased reticulocytes
Spherocytes
Presence of autoantibody in serum
Increased bilirubin
Decreased serum haptoglobin

14. Cold Agglutinin Disease (CAD)
Optimal thermal activity below 30oC
Cases can be idiopathic or secondary to another disease
Often seen secondary to Mycoplasma pneumoniae (anti-I) or infectious mononucleosis
Mechanism of action
IgM antibody attaches to erythrocyte in the cold and fixes complement
After warming, the antibody dissociates from the cell, but complement remains, causing lysis or initiating extravascular hemolysis
Side note: Almost all adults have cold antibodies in low quantity which cause no problems because they react in the temperature range of 4̊ to 22̊ C. The antibody is usually anti-I.

15. Laboratory findings of CAD
Common Findings
Positive DAT
Normocytic, normochromic anemia
False increase in MCV, MCH, MCHC
False decrease in RBC count
Increased reticulocytes
Spherocytes
Agglutinated RBCs, rouleaux, nRBCS
Cold agglutinin titer high at 4oc
Increased bilirubin
Decreased serum haptoglobin

16. Paroxysmal Cold Hemoglobinurea (PCH)
Cold hemolytic anemia which can be severe
Common in children under age 5
Seen following respiratory viral infections
Caused by a bi-phasic complement-fixing IgG antibody( Donath-Landsteiner)
Antibody reacts with RBCs in the capillaries at temperatures < 20oC and avidly binds early complement. Upon warming to 37oC , the antibody molecule disperses from the cell, but the membrane attack complement components are activated on the cell membrane causing lysis
Treatment is to avoid the cold

17. Laboratory findings of PCH
Common Findings
Decreased hgb
Hemoglobinemia
Hemoglobinuria
Spherocytes
Increased bilirubin
Decreased serum haptoglobin

18. Drug-Induced Hemolytic Anemias

19. Drug-Induced Hemolytic Anemia
Drugs that attach to the RBC membrane or alter it in some way
Methyldopa/ procainamide, penicillin, cephalosporins
Patient produces antibodies directed against a particular drug, its metabolites or the RBC coated with drug
Acquired
The drug itself does not cause the RBC injury, and not all people taking the drug are affected

20. Alloimmune Hemolytic Anemia

21. Alloimmune Hemolytic Anemia
A foreign antigen stimulates the host’s immune system to produce a corresponding antibody called an alloantibody
Two main types
Hemolytic transfusion reactions
Hemolytic Disease of the Fetus and Newborn(HDFN)

22. Types of Alloimmune Hemolytic Anemia
Hemolytic transfusion reactions
Acute transfusion reaction
Occurs within minutes- hours after transfusion
The wrong ABO type of blood is transfused. If a patient develops symptoms such as fever, shaking, chills, pain or burning at the site of the iv, the transfusion should be stopped immediately.
Delayed transfusion reaction
Occurs days – weeks after transfusion
Sensitization to non-ABO blood groups occurs during a primary transfusion. When re-exposed during a second transfusion, antibodies build up and a delayed reaction occurs. Symptoms are usually mild and non-specific.

23. Types of Alloimmune Hemolytic Anemia
Hemolytic Disease of the Fetus and Newborn (HDFN)
This is caused when the mother’s and baby’s blood groups are incompatible and there is an exchange of fetal-maternal blood bfore or during delivery.
The mother builds antibodies against the fetal red cells which then become coated with antibody and are destroyed in the baby’s RES.
Associated with ABO or Rh incompatibility.
It can be severe and treatment would consist of an exchange transfusion of the newborn.

24. Referenes
Harmening, D. M. (2009). Clinical Hematology and Fundamentals of hemostasis (5th ed.). Philadelphia, PA: F.A. Davis Company.
McKenzie, S. B. (2010). Clinical Laboratory Hematology (2nd ed.). Upper Saddle River, NJ: Pearson Education, Inc.
Rodak, B. F. (2002). Hematology Clinical Principles and Applications (3rd ed.). St. Louis: Saunders Elsevier.