Hemolytic Anemia - Corpuscular Hemolytic Anemia - Extracorpuscular Hemolytic Anemia

Question 1

THE THALASSEMIAS

Answer 1

A group of disorders, each of which results from an inherited abnormality of globin
chains production.
Hemoglobin electrophoresis in normal adults:
 HbA1 = α2β2 = 97%
HbA2 = α2δ2 = 2%
 HbF = α2γ2 = 1%
At birth HbF is more then 90% and gradually, in the first year of life, is replaced with HbA ( adult type )

Question 2

Globin chains:

Answer 2

 α are encoded by two genes on chromosome 16

 β, δ, γ are encoded by genes on chromosome

Question 3

COMMON FEATURES OF THALASSEMIAS

Answer 3

microcytic, hypochromic anemia - due to impaired hemoglobin synthesis;
peripheral blood smear: anisocytosis (small cells), target cells,
signs of hemolysis: ↑ LDH1,2, ↑ indirect-reacting serum bilirubin;
hemolytic jaundice, splenomegaly
elevated levels of serum iron, elevated transferrin saturation
hemoglobin electrophoresis confirms the diagnosis

Question 4

1. ALPHA (Α) THALASSEMIA = QUANTITATIVE IMPAIRED SYNTHESIS OF Α GLOBIN CHAINS

Answer 4

The inability to form α globin chains leads to synthesis of two abnormal types of hemoglobin:
Hb Bart’s = γ4 and Hb H = β4.
Hb β4 and γ4 are soluble and do not precipitate in the marrow.
However, they are unstable and precipitate in older red cells. Hence, the anemia of α thalassemia is
hemolytic rather than dyserythropoietic.
In addition Hb H and Bart’s have a higher affinity for oxygen and it is more difficult to release the oxygen in the tissues - in fact they are useless as oxygen carriers.
In α thalassemia one to four genes may be affected by ( deletions or mutations ) leading to clinical syndromes of variable severity

Question 5

a) HYDROPS FETALIS WITH HB BART’S (HB BART’S = HB Γ 4 )

Answer 5

Absence of all four of the α genes on chromosome 16
Clinical picture: premature, pale, and bloated infant who, if not stillborn, has significant cardiorespiratory distress at birth.
Death usually occurs within 1 hour of birth and results from severe hypoxia, a consequence of the high oxygen affinity of Hb Bart’s.
The hemoglobin concentration varies from 4 to 10 g/dl.
The peripheral blood smear: anisopoikilocytosis, severe hypochromia, and nucleated RBCs.
Hb electrophoresis: Hb Bart’s, Hb F and Hb A1 are absent

Question 6

b) HEMOGLOBIN H DISEASE (HB H = Β4 )

Answer 6

Deletion of three of the four α-globin genes
clinical picture is that of thalassemia intermedia: affected infants appear well at birth but develop anemia and splenomegaly by 1 year of age; thereafter, jaundice and hepatosplenomegaly are prominent.
Approximately one third of these patients have skeletal changes associated with an expanded erythron.
Transfusion therapy is unnecessary, except during intercurrent illness.
Moderate microcytic hypochromic anemia: Hb= 7 to 10 g/dl
Reticulocytes: 5 and 10%.
Hb electrophoresis : at birth Hb Bart’s =20-40%, replaced gradually during the first months by Hb H = 5-40%

Question 7

c) ALPHA (Α) THALASSEMIA MINOR

Answer 7

Deletion of two of the four α-globin genes
It is asymptomatic and usually is detected during routine hematologic examination
Mild hypochromic, microcytic anemia Hb = 10 to 11 g/dl
Hb electrophoresis: in the newborn period detection of Hb Bart’s, Hb H usually undetectable in adults

Question 8

d) SILENT CARRIER STATE

Answer 8

Deletion of a single α globin gene
No clinical or laboratory tests changes:
In very rare cases at birth Hb Bart’s 1-2%
Gene mapping is the only reliable means for precise identification

Question 9

2. BETA (Β) THALASSEMIA = QUANTITATIVE IMPAIRED SYNTHESIS OF B GLOBIN CHAINS

Answer 9

The pregnancy is normal and the newborns are well at birth (the synthesis of HbF - α2γ2 is normal).
Anemia usually develops during the first few months of life due to total or partial impaired synthesis of β chains.
Hemoglobin electrophoresis confirms the diagnosis showing decreased level of Hb A1 and increased level of Hb A2 and Hb F.
Hb α4 is very unstable and precipitate in the marrow . Red cell precursors that contain Hb α4 are recognized by the local macrophage and are phagocytized
resulting in ineffective erythropoiesis.
Therefore Hb electrophoresis will not reveal Hb α4.

Question 10

a) Β THALASSEMIA MAJOR = ANEMIA COOLEY

Answer 10

Clinical picture develops gradually after the first few months

• increased hematopoiesis leads to bone marrow expansion, impaired growth & thinning of cortical: frontal bossing, long bone fractures,
• hepatosplenomegaly, jaundice
• secondary hemosiderosis: liver cirrhosis (hepatosplenomegaly), myocarditis, arrhythmia, congestive heart failure, growth retardation, hypogonadotropic hypogonadism

Question 11

LABORATORY TESTS OF THALASSEMIAS

Answer 11

Severe hypochromic, microcytic anemia (Hb = 2-6 g/dl, MCV↓↓↓, , MCHC↓↓↓ )
reticulocytosis = 5-15% - lower than expected
peripheral blood smear: anisocytosis (small cells), target cells↑↑↑
Hb electrophoresis:
- Hb A1 less than 10% (even absent)
- Hb F= 40-90%
- Hb A = 24-10%

Question 12

b) Β THALASSEMIA INTERMEDIA

Answer 12

Signs and symptoms of thalassemia intermedia are comparable to those of thalassemia major but are of lesser magnitude
Hb = 6-9 g/dl, MCV↓↓,, MCHC ↓↓ (hypochromic / microcytic)
reticulocytosis = 3-10%
peripheral blood smear: anisocytosis (small cells), target cells, ↑↑
Hb electrophoresis:
- Hb A1 = ↓ ↓
- HbF = 10%
- HbA2 = 3-7%

Question 13

c) Β THALASSEMIA MINOR

Answer 13

Usually is asymptomatic; detected during routine hematologic examination
 Hb = 10-12 g/dl, MCV, MCHC (hypochromic / microcytic)
 Reticulocytosis = 2-4%
 Peripheral blood smear: anisocytosis (small cells), target cells↑
 Hb electrophoresis: HbA ↓, HbF ↑,HbA2↑
β thalassemia minima – also called the “silent β thalassemia” - virtually no clinical or laboratory changes

Question 14

EXTRACORPUSCULAR HEMOLYTIC ANEMIAS

Answer 14

1. Autoimmune hemolytic anemias
2. Drug-related immune hemolytic anemias
3. Alloimmune hemolytic disease of the newborn
4. Microangiopathic and macroangiopathic hemolytic anemias
5. Hemolytic anemia due to infections with microorganisms – malaria
6. Hypersplenism

Question 15

1. AUTOIMMUNE HEMOLYTIC ANEMIAS (AHA)

Answer 15

Autoimmune hemolytic anemias (AHA) occur when a patient produces pathologic autoantibodies that attach to and destroy red blood cells, causing anemia.
AHA are classified according to the characteristic temperature activity of the antibodies:
- Warm-active antibodies have their greatest affinity at 37°C. (80% of all AHA)
- Cold-active antibodies display increasing affinity for the RBCs as the temperature approaches 0°C. (20%)

Question 16

a) AUTOIMMUNE HEMOLYTIC ANEMIAS CAUSED BY WARM-ACTIVE ANTIBODIES

Answer 16

Warm-active antibodies are typically of the IgG variety, they have the highest affinity
at 37°C (the major share of autoantibodies is commonly bound to the patient’s
circulating erythrocytes at this temperature)
50% of warm-antibody AHA have autoantibodies specific for epitopes on Rh
proteins. The autoantibodies of such patients do not react with Rh negative RBC.
Autoantibodies with such specificity have been designated as Rh related.
50% of warm-antibody AHA have IgG autoantibodies that are fully reactive with Rh null RBC.
The exact specificity of the autoantibodies for many of these patients is undefined

Question 17

CLASSIFICATION OF AUTOIMMUNE HEMOLYTIC ANEMIAS CAUSED BY WARM-ACTIVE ANTIBODIES

Answer 17

Primary or idiopathic warm-antibody AHA - no recognizable underlying disease is present.
Secondary warm-antibody AHA - as a manifestation or complication of an underlying disorder
 Lymphocytic malignancies, particularly chronic
lymphocytic leukemia (CLL) and lymphomas

 Systemic lupus erythematosus (SLE)

Question 18

PATHOGENESIS OF AUTOIMMUNE HEMOLYTIC ANEMIAS CAUSED BY WARM-ACTIVE ANTIBODIES

Answer 18

Erythrocyte autoantibodies in warm-antibody AHA are pathogenic.
In warm-antibody AHA, the patient’s RBC typically are coated with IgG
autoantibodies with or without complement proteins.
Autoantibody-coated RBC are trapped by macrophages in the spleen and, to a lesser
extent, by Kupffer cells in the liver.

The macrophage has surface receptors for the Fc region of IgG and surface receptors for opsonic fragments of C3 (C3b) and C4 (C4b).
When present together on the RBC surface, IgG and C3b appear to act cooperatively as opsonins to enhance trapping and phagocytosis.
Interaction of a trapped RBC with splenic macrophages may result in phagocytosis of
the entire cell or a type of partial phagocytosis that results in the formation of spherocytes (the noningested portion of the RBC assumes a spherical shape, the shape with the lowest ratio of surface area to volume). Spherical RBC are more rigid and less deformable than normal RBC and are fragmented further and/or destroyed in future passages through the spleen. Spherocytosis is a consistent and
diagnostically important hallmark of AHA, and the degree of spherocytosis correlates well with the severity of hemolysis.
Direct complement-mediated hemolysis with hemoglobinuria is unusual in warm
antibody AHA, despite the fact that many warm autoantibodies fix complement.

Question 19

CLINICAL FEATURES OF AUTOIMMUNE HEMOLYTIC ANEMIAS CAUSED BY WARM-ACTIVE ANTIBODIES

Answer 19

Symptoms are usually slow and insidious in onset over several months:

• symptoms of anemia (tachycardia, pallor and adinamia)
• jaundice and modest splenomegaly
• Occasionally in very severe cases, particularly those of acute onset, patients may present with:
• fever, pallor,
• jaundice, hepatosplenomegaly,
• hyperpnea, tachycardia, angina or heart failure

Question 20

LABORATORY TEST OF AUTOIMMUNE HEMOLYTIC ANEMIAS CAUSED BY WARM-ACTIVE ANTIBODIES

Answer 20

severe to mild normochromic normocytic (MCV, MHCH normal) anemia (Hb = 5-11 g/dl)
reticulocyte count is elevated
blood smear: spherocytes (if hereditary spherocytosis is excluded, this finding suggests an immune hemolytic process) and schizocytes (red blood cell fragments).
Total bilirubin is increased, up to 5 mg/dl, (>85% unconjugated bilirubin)
Urinary urobilinogen is increased regularly, Serum haptoglobin levels are low and LDH levels are elevated.
positive direct Coombs’ test
positive indirect Coomb

Question 21

DIRECT COOMBS TEST

DIRECT ANTIGLOBULIN TEST – DAT

Answer 21

detects autoantibody or complement bound to the patient’s RBC

Question 22

INDIRECT COOMBS TEST

INDIRECT ANTIGLOBULIN TEST – IAT

Answer 22

detects “free” autoantibody in the plasma of these patients

Question 23

b) AUTOIMMUNE HEMOLYTIC ANEMIA

CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 23

AHA caused by cold active autoantibodies are chronic autoimmune hemolytic anemia in which the autoantibody directly agglutinates RBCs at temperatures below body temperature, maximally at 0 to 5°C.
Fixation of complement to RBCs by cold agglutinins occurs at higher temperatures, but below 37°C.
Cold-active antibodies (cold agglutinins) are of the IgM type, fix complement, and may lead to immediate intravascular destruction of erythrocytes or their removal from the circulation by the liver.
Most cold agglutinins have specificity for oligosaccharide antigens (I or i) of the RBC.

Question 24

CLASSIFICATION OF AUTOIMMUNE HEMOLYTIC ANEMIA CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 24

Primary or idiopathic cold agglutinin mediated AHA - no recognizable underlying disease is present.

Secondary cold agglutinin mediated AHA - as a manifestation of an underlying disorder

• Mycoplasma pneumoniae infections or infectious mononucleosis - EBV
• malignant lymphoproliferative diseas

Question 25

PATHOGENESIS (I) OF AUTOIMMUNE HEMOLYTIC ANEMIA CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 25

Most cold agglutinins are unable to agglutinate RBC at temperatures above 30°C . The highest temperature at which these antibodies cause detectable agglutination is termed the thermal amplitude. This value may vary considerably from one patient to another. Patients with cold agglutinins of higher thermal amplitudes have a greater risk for cold-agglutinin disease.  The cold agglutinins bind host RBC and activate complement (process called complement fixation) Complement fixation by these antibodies occur optimally at 20 to 25°C and even higher temperatures. Cold agglutinins bind to RBC in superficial vessels of the extremities, where the temperature ranges between 28 and 31°C and cause RBC to aggregate, thereby impeding RBC flow and producing acrocyanosis

Question 26

PATHOGENESIS (II) OF AUTOIMMUNE HEMOLYTIC ANEMIA CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 26

In addition, the RBC-bound cold agglutinin may activate complement via the classical pathway. Once activated complement proteins are deposited onto the RBC surface, it is no longer necessary for the cold agglutinin to remain bound to the RBC for hemolysis to occur. Complement fixation may effect RBC injury by two major mechanisms: (1) direct lysis (requires propagation of the full C1-to-C9 sequence on the RBC membrane) => intravascular hemolysis leading to hemoglobinemia and hemoglobinuria (2) opsonization (C3b,C4b)for hepatic and splenic macrophages => extravascular hemolysis A RBC heavily coated with C3b may be removed from the circulation by macrophages either in the liver or, to a lesser extent, the spleen. The trapped RBC may be ingested entirely or released back into the circulation as a spherocyte after losing some of its plasma membrane.

Question 27

CLINICAL FEATURES OF AUTOIMMUNE HEMOLYTIC ANEMIA CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 27

Most patients with cold-agglutinin hemolytic anemia have chronic hemolytic anemia with or without jaundice. In others, the principal feature is episodic, acute hemolysis with hemoglobinuria induced by chilling. Combinations of these clinical features may occur. Acrocyanosis affecting the fingers, toes, nose, and ears are associated with sludging (agglutination) of RBC in the cutaneous microvasculature.

Question 28

LABORATORY TESTS OF AUTOIMMUNE HEMOLYTIC ANEMIA CAUSED BY COLD-ACTIVE ANTIBODIES

Answer 28

mild to moderate normochromic normocytic anemia (Hb =8-11 g/dl). reticulocyte count is elevated blood smear: spherocytosis (less marked than in typical cases of warm-antibody autoimmune hemolytic anemia).  RBC auto agglutination may be noted at room temperature and intensified by cooling the blood to 4°C Mild hyperbilirubinemia (>85% unconjugated bilirubin) Serum haptoglobin levels are low and LDH1,2 levels are elevated. Hemoglobinemia, hemoglobinuria appear in acute hemolytic episodes positive direct Coombs' test for complement, negative direct Coombs’ test for autoantibodies negative indirect Coombs’ test (Coombs tests are performed at 37°C) !!!!!! Cold agglutinins are distinguished by their ability to agglutinate saline-suspended human RBC at low temperature, maximally at 0 to 5°C (32 to 41°F). This reaction is reversible by warming.

Question 29

2. DRUG-RELATED IMMUNE HEMOLYTIC ANEMIA

Answer 29

a) Hapten or drug adsorption mechanism - penicillin very high doses, cephalosporin, tetracycline b) Ternary complex mechanism (drug- antibody-target cell complex)- quinine, quinidine, sulfonamides, rifampicin c) Autoantibody induction. – alpha methyldopa, levodopa

Question 30

d) HAPTEN OR DRUG ADSORPTION MECHANISM PENICILLIN VERY HIGH DOSES, CEPHALOSPORIN, TETRACYCLINE

Answer 30

- Induction of antidrug antibody is thought to require firm chemical coupling of the drug (as a hapten) to a protein carrier. - The drug binds firmly to red cell membrane proteins in vivo. - Antidrug antibody (usually IgG) binds to the protein-bound drug. - Red cells coated with penicillin and IgG antipenicillin antibody are destroyed by splenic macrophages.

Question 31

b) TERNARY COMPLEX MECHANISM (DRUG-ANTIBODY-TARGET CELL COMPLEX) QUININE, QUINIDINE, SULFONAMIDES, RIFAMPICIN

Answer 31

Drug binds loosely or in undetectable amounts to red cell membrane. However, in the presence of appropriate antidrug antibody, a stable trimolecular (ternary) complex is formed among drug, red cell membrane protein, and antibody. In general, the antibody-combining site (Fab) recognizes both drug and membrane protein components but binds only weakly to either drug or protein unless both are present in the reaction mixture Drugs can induce immune injury not only of red cells but also of platelets or granulocytes The process differs in several ways from the mechanism of hapten/drug adsorption. - Drugs in this group exhibit only weak direct binding to blood cell membranes. - A relatively small dose of drug is capable of triggering destruction of blood cells. - Cellular injury appears to be mediated chiefly by complement activation at the cell surface

Question 32

c) AUTOANTIBODY INDUCTION | ALPHAMETHYLDOPA, LEVODOPA

Answer 32

The mechanism by which a drug can induce formation of an autoantibody is unknown Drug-induced antibodies can bind avidly to red cell membrane proteins (usually Rh proteins) in the absence of the inducing drug and are indistinguishable from the anti– red cell autoantibodies of patients with autoimmune hemolytic anemia

Question 33

3. ALLOIMMUNE HEMOLYTIC DISEASE OF THE NEWBORN | Rh hemolytic disease of the newborn

Answer 33

Rh hemolytic disease of the newborn occurs as a result of immunization (during transplacental passage of fetal red cells) of the Rh negative mother to red cell of the Rh positive fetus. This sensitization results in the transplacental passage of maternal IgG antibodies anti antigen D that bind to the fetal red cells, causing hemolysis (extravascular anti antigen D that bind to the fetal red cells, causing hemolysis (extravascular noncomplement-mediated phagocytosis and lysis), and as a consequence of the hemolytic process, anemia, extramedullary hematopoiesis, and neonatal hyperbilirubinemia, sometimes with devastating morbidity for the fetus and newborn infant.  ABO hemolytic disease of the newborn is limited to mothers who are blood group type O and whose babies are group A or B. Although ABO incompatibility exists in 15 % of O group pregnancies, ABO hemolytic disease is estimated to occur only in about 3 percent of all births. Most anti-A and anti-B antibodies are of the IgM type and do not cross the placenta. A small number of group O women produce anti-A and anti-B antibodies of the IgG type that can cross the placenta.

Question 34

4. MICROANGIOPATHIC AND MACROANGIOPATHIC HEMOLYTIC ANEMIA

Answer 34

a) MICROANGIOPATHIC HEMOLYTIC ANEMIA b) MARCH HEMOGLOBINURIA c) TRAUMATIC CARDIAC HEMOLYTIC ANEMIA

Question 35

a) MICROANGIOPATHIC HEMOLYTIC ANEMIA

Answer 35

Clinical disorders characterized by the fragmentation of red cells as they pass through the platelet-fibrin mesh present in microthrombi which are deposited in capillaries and arterioles.  The formation of arteriolar microthrombi can be caused by a variety of mechanisms: - activation of the coagulation system as occurs in disseminated intravascular coagulation (DIC), - antineoplastic and immunosuppressive agents as well as radiation therapy and bacterial toxins may induce endothelial cell injury leading to the formation of microthrombi in the affected vessels. - formation of platelet aggregates induced by the release of very large von Willebrand factor multimers as in thrombotic thrombocytopenic purpura (TTP).

Question 36

b) MARCH HEMOGLOBINURIA

Answer 36

In marchers and runners, traumatic hemolysis (red cells were destroyed in the soles of the feet during running) may cause hemoglobinuria and anemia, Similar traumatic red cell destruction with hemoglobinuria has been reported after beating the head against a wall, hand-strengthening exercises in a practitioner of karate and playing the conga drums

Question 37

c) TRAUMATIC CARDIAC HEMOLYTIC ANEMIA

Answer 37

The abrasive effect on red cells of arteriosclerotic or stenotic cardiac valves is usually minimal, resulting at most in a mild, often compensated hemolytic anemia.  However, the introduction of artificial valves was initially associated with marked red cell destruction and the development of an overt hemolytic anemia. Recently, the design of the artificial valves and the use of more compatible plastics or biologic materials have greatly reduced their traumatic effects and minimized hemolysis. Actually, the potential thrombogenic effect of artificial valves far outstrips their destructive effect on red cells, and cardiac hemolytic anemia is now an almost nonexistent problem.

Question 38

5. HEMOLYTIC ANEMIAS RESULTING FROM DIRECT EFFECTS OF INFECTIOUS AGENTS

Answer 38

Malaria - febrile disease caused by four species of Plasmodia: vivax, falciparum, malariae and ovale which are capable of parasitizing erythrocytes. P. falciparum invades erythrocytes of all ages =>50% of RBC may be affected. Within the erythrocyte, the malarial organisms divide asexually, the cell ruptures, and the merozoites are released to invade other cells.

Question 39

6. HYPERSPLENISM

Answer 39

Hypersplenism occurs when the size of the spleen is increased by tissue components or by vascular engorgement. This augments its filtering function, and even normal blood cells experience a delayed transit and temporary sequestration. The trapped red cells are usually destroyed causing a hemolytic anemia. Splenectomy is called for if the hypersplenic cytopenias are severe enough to demand intervention or if the enlarged spleen causes pain and discomfort.