RBC Disorders

Glucose-6-phosphate dehydrogenase deficiency

The red cell  vulnerable to injury by endogenous and exogenous oxidants are normally inactivated by reduced glutathione (GSH). Oxidants such as hydrogen peroxide are stopped by GSH, which is converted to oxidized glutathione in the process 

•          X- linked recessive, males

•          Hemolysis after exposure to oxidant stress

•          Oxidant drugs: antimalarial, sulfonamide, nitrofurantoins

•          Infections: viral hepatitis, pneumonia, typhoid fever

•          Foods: Fava beans

•          Causes episodic intravascular and extravascular hemolysis, which involve the following sequence:

–        Exposure of G6PD deficient red cells to oxidants

–        Regeneration of GSH is impaired in G6PD-deficient cells, hydrogen peroxide attack globin chains, which have sulfhydryl groups that are susceptible to oxidation

–         Oxidized Hb denatures and precipitates, forming intracellular inclusions called Heinz bodies, which can damage the cell membrane sufficiently to cause

intravascular hemolysis

–        Less severely damaged à cell membranes are further damaged when splenic phagocytes attempt to "pluck out" the Heinz bodies, creating so-called bite cells. All of these changes predispose the red cells to becoming trapped in the splenic sinusoids and destroyed by the phagocytes (extravascular hemolysis).

•          Anemia, hemoglobinemia, hemoglobinuria 2 to 3 days following exposure to oxidants

•          Older red cells at risk for lysis

•          Self limited

Hereditary Spherocytosis (HS)

•          Intrinsic defects in the red cell membrane that render red cells spheroid, less deformable, and vulnerable to splenic sequestration and destruction.

•          Prevalence highest in northern Europe.

•          Autosomal dominant in 3/4^th of cases.

•          Mutation affecting ankyrin, band 3, spectrin or band 4.2

•          Mutation of red cell ankyrin most common cause

•          Reduced membrane stability leads to loss of membrane fragments

•          Spherocytes, being less flexible than normal RBCs, are destroyed in the spleen, often causing splenomegaly.

•          Spherocytes have reduced surface membrane area relative to the RBC volume. Spherocytes are osmotically fragile compared to normal RBCs.

Morphology

•          Spherocytes- small dark staining red cells without central pallor.

•          Not pathognomonic- also seen in autoimmune hemolytic anemia.

•          Reticulocytosis.

•          Marrow hyperplasia.

•          Hemosiderosis.

•          Mild jaundice.

•          Cholelithiasis (pigment stones- 40-50% of patients).

•          Moderate splenomegaly.

Laboratory Diagnosis

•          PBS

–        Spherocytes

•          Bone marrow

–        Increased erythropoiesis

•          Increased MCHC

•          Increased osmotic fragility

Clinical Course

•          Anemia

•          Splenomegaly

•          Jaundice

•          Aplastic crisis

•          Hemolytic crisis

•          Gall stones

Iron Deficiency Anemia

•          It is the most common form of nutritional deficiency

•          Prevalence higher in developing countries

Etiology

•          Iron deficiency results from

–        Dietary lack

–        Impaired absorption

–        Increased requirement

–        Chronic blood loss 

•          Dietary lack

•          Impaired absorption: sprue, intestinal steatorrhea , chronic diarrhea, gastrectomy

•          Increased requirement: growing infants and children, adolescents, pregnant

•          Chronic blood loss: peptic ulcer, gastric carcinoma, colonic carcinoma, hookworm or pinworm disease, urinary tract tumor, menorrhagia

Diagnosis

Symptoms

•          Weakness, loss of concentration

•          Headache and loss of appetite

•          Dyspnea and palpitations 

Examination

•          Pallor (nails, lips, conjunctiva)

•          Pulse: tachycardia

•          Cardiac examination: systolic murmurs

Investigations

•          Morphology

–        Complete blood picture shows a picture of  microcytic hypochromic anemia with some anisopoikilocytosis

–        Poikilocytosis in the form of small, elongated red cells (pencil cells)

•          Bone marrow examination

–        Mild to moderate increase in erythroid progenitors

–        Iron staining : disappearance of stainable iron 

•          Low RBC count and hemoglobin

•          Low hematocrit 

•          MCV, MCHC and MCH decreased

•          WBC and platelets are normal (platelets increase in hemorrhage)

•          Ferritin low

•          Serum iron is decreased

–        TIBC increased

•          Transferrin saturation <15%

Complication

•          Koilonychia

•          Alopecia

•          Atrophic changes in tongue and gastric mucosa

•          Intestinal malabsorption

•          Plummer-Vinson syndrome

–        Microcytic hypochromic anemia

–        Atrophic glossitis

–        Esophageal webs

Megaloblastic Anemia

Ø  Impaired DNA synthesis with distinctive morphologic changes in the blood and bone marrow

Ø  Erythroid precursors and red cells are abnormally large due to defective cell maturation and division

Ø  Characterized by macrocytic blood picture (larger red cell with MCV > 100 fl) and megaloblastic bone marrow

Ø  Two types:

l  Pernicious anemia (vitamin B12 deficiency)

l  Folate deficiency anemia

Ø  Common feature: impaired DNA synthesis

Pathogenesis

Ø  The morphologic hallmark of megaloblastic anemias is an enlargement of erythroid precursors (megaloblasts), which gives rise to abnormally large red cells (macrocytes)

Ø  Other myeloid lineages also affected

l  Giant metamyelocytes and hypersegmented neutrophils

Ø  impairment of DNA synthesis à delay in nuclear maturation and cell division

Ø   Synthesis of RNA and cytoplasmic elements proceeds at a normal rate

Ø   Nuclear-cytoplasmic asynchrony

Ø  Ineffective hematopoiesis: Apoptosis of megaloblasts in marrow

Ø   Granulocyte and platelet precursors are also affected à pancytopenia (anemia, thrombocytopenia, and granulocytopenia).

Morphology

Ø  Peripheral blood smear

Ø  Bone marrow

Ø  Alimentary tract

Ø  Central nervous system

Ø  Peripheral blood smear

l  Macro-ovalocytes

l  Hypersegmented neutrophils – before onset of anemia

Ø  Bone marrow

l  markedly hypercellular, due to increased numbers of megaloblasts; erythroid hyperplasia

l  Giant metamyelocyte

l  Large megakaryocyte with bizarre multilobed nuclei

l  Iron increased

Ø  Macrocytes/ hypersegmented neutrophils

Ø  Alimentary Tract: Shiny tongue- atrophic glossitis

Ø  Central Nervous System: Spinal cord

l  Degeneration of myelin in dorsal and lateral tracts

l  Loss of axons

 Folate (Folic Acid) Deficiency Anemia

Ø  After absorption, folate is transported in the blood mainly as a monoglutamate

Ø  Conversion from dihydrofolate to tetrahydrofolate by the enzyme dihydrofolate reductase

Ø   Tetrahydrofolate acts as an acceptor and donor of one-carbon units in a variety of steps involved in the synthesis of purines and thymidylate, the building blocks of DNA

Ø   Deficiency accounts for the inadequate DNA synthesis that is characteristic of megaloblastic anemia.

Etiology

Ø  Clinical features

l  Weakness, easy fatigability

l  Alimentary tract affected

l  Neurologic abnormalities do not occur

Ø  Diagnosis

l  Peripheral smear and bone marrow examination

l  The anemia of folate deficiency is best distinguished from that of vitamin B₁₂ deficiency by measuring serum and red cell folate and vitamin B₁₂ levels.

Vitamin B₁₂ (Cobalamine) Deficiency Anemia: Pernicious Anemia

Ø  The term pernicious anemia is used to describe vitamin B₁₂ deficiency resulting from inadequate gastric production or defective function of intrinsic factor

Ø  Etiology of B12 Deficiency

Ø  Decreased intake

l  Inadequate diet, vegetarianism 

Ø  Impaired absorption

l  Intrinsic factor deficiency

•          Pernicious anemia

•          Gastrectomy

l  Malabsorption

l  Diffuse intestinal disease- lymphoma, Ileal resection, ileitis

l  Competitive parasitic uptake

•          Fish tapeworm

l  Bacterial overgrowth in  diverticula of intestine

Ø  Increased requirement

l  Pregnancy, hyperthyroidism, disseminated cancer

Ø  long-standing malabsorption is the most common and important

Ø  Until proved otherwise, a deficiency of vitamin B₁₂ (in the western world) is caused by pernicious anemia

Anemia of Vit. B12 Deficiency

Pernicious anemia  is a specific form of megaloblastic anemia caused by atrophic gastritis and failure of intrinsic factor production that leads to vitamin B12 deficiency

Ø  Autoimmune destruction of gastric mucosa

Ø  Chronic atrophic gastritis

Ø  Loss of parietal cells , infiltration of lymphocytes and plasma cells

Ø  Old age: 5^th to 8^th decades

Ø  Tendency to form antibodies against multiple self antigens

Ø  Association with other autoimmune diseases: Hashimoto thyroiditis, Addison disease and type I DM.

Ø  Types of auto-antibodies

l  parietal canalicular antibodies, which bind to the mucosal parietal cells

l  blocking antibodies, which block the binding of vitamin B₁₂ to intrinsic factor; and

l   binding antibodies that react with intrinsic factor-B₁₂ complex and prevent it from binding to the ileal receptor

Ø  Autoreactive T-cell response initiates gastric mucosal injury, triggering formation of autoantibodies, which may excacerbate epithelial injury

Ø  The principal neurologic lesions associated with vitamin B₁₂ deficiency are demyelination of the posterior and lateral columns of the spinal cord

Diagnosis

1.       low serum vitamin B₁₂ levels

2.       normal or elevated serum folate levels

3.       serum antibodies to intrinsic factor

4.       moderate to severe megaloblastic anemia

5.       leukopenia with hypersegmented granulocytes

6.       a dramatic reticulocytic response (within 2-3 days) to parenteral administration of vitamin B₁₂

EXCLUDE VITAMIN B12 DEFICIENCY IN MEGALOBLASTIC ANEMIA BEFORE INITIATING THERAPY WITH FOLATE

Hemoglobinopathies

•          Hemoglobinopathies are a group of hereditary disorders that are defined by the presence of structurally abnormal hemoglobins

•          Sicke cell anemia (HbS)

•          Sickle cell trait (40% HbS, 60% HbA)

•          HbC

•          HbE

•          Thalassemia

Sickle cell Anemia

•          Hereditary hemoglobinopathy characterized by production of defective hemoglobins

•          Point mutation at the sixth position of Beta globin chain leading to substitution of a valine residue for a glutamic acid residue

•          Sickle hemoglobin (HbS)

•          Homozygous: all hemoglobin in red cell is HbS

•          Heterozygous: 40% HbS, remainder normal

•          America (blacks) and Africa

•          Protection against falciparum malaria

Malaria protection

•          Seen in common hemoglobinopathies (Thalassemia, HbS, HbC, HbE) and enzyme (G6PD) deficiency

•          Due to

–        Reduced parasite invasion/growth

–        Increased susceptibility to phagocytosis of infected RBC

                Pathogenesis

•          Deoxygenation

–        Aggregation and polymerization of HbS

–        Aggregated HbS molecules assemble into long needle like fibers within red cells, producing distorted sickle shape

–        Repeated episodes of sickling: membrane damage

–        eventually the cells accumulate calcium, lose potassium and water, and become irreversibly sickled.

–        Red cells abnormally sticky

•          infection, inflammation, dehydration, and acidosis trigger the sickling of reversibly sickled cells

Morphology

Ø  Anatomic alterations caused by chronic hemolysis, increased formation of bilirubin, small vessel stasis and thrombosis

Ø  Bone marrow hyperplastic

Ø  fatty changes in the heart, liver, and renal tubules

Ø  Prominent cheekbones and changes in the skull

Ø  Crew-cut in roentgenograms

Ø  Children- Spleenomegaly upto 500 gm

Ø  Histological examination- Marked congestion of red pulp

Ø  Autosplenectomy

Ø  Infarction- Leg ulcers

Ø  Vascular congestion, thrombosis, and infarction can affect any organ, including bones, liver, kidney, retina, brain, lung, and skin BONE MARROW

Ø  Gallstone- increased breakdown of hemoglobin

Ø  Hemosiderosis

Clinical Course

1. Severe anemia
2. Vaso-occlusive complications
3. Chronic hyperbilirubinemia
4. Infection with encapsulated organisms- pneumococci and Haemophilus influenzae—impaired splenic function and opsonisation

Ø  Septicemia and meningitis most common causes of death in children

Ø  Salmonella osteomyelitis

Ø  Reticulocytosis

Ø   hyperbilirubinaemia

Ø  Irreversibly sickled cells

Ø  Crises   

Ø  Vaso-occlusive crises (pain crises)- Hand-foot syndrome, acute chest syndrome,  seizures or stroke, ulcers. acute chest syndrome and stroke are the two leading causes of ischemia-related death

Ø  Sequestration crises- splenic enlargement,hypovolemia, shock

Ø  Aplastic crises- Parvovirus B19

Ø  Generalized impairment of growth and development

Ø  Organ damage affecting spleen, heart, kidneys and lungs

Ø  Damage to the renal medulla leads to hyposthenuria (inability to concentrate urine)

Diagnosis

Clinical findings :

Ø  Irreversibly sickled cells in peripheral blood smear

Ø  Sickling of cells by metabisulphite (oxygen consuming reagent)

Ø  Hemoglobin electrophoresis: HbS

Ø  Prenatal diagnosis by analysis of fetal DNA

Thalassemia

—  The thalassemias are a heterogeneous group of inherited disorders caused by mutations that decrease the rate of synthesis of α- or β-globin chains

—   Hematologic consequences are caused by:

o   Low intracellular hemoglobin (hypochromia)

o   Excess of unimpaired chain

Classification

—  b-thalassemia- deficient b -chain

o   Thalassemia major

o   Thalassemia intermedia

o   Thalassemia minor

—  a-thalassemia- deficient a-chain

o   Hydrops fetalis

o   HbH disease

o   a-thalassemia trait

o   Silent carrier

Beta Thalassemia

—  Beta thalassemia results from absence or decreased  production beta globin chains , coupled with unimpaired synthesis of alpha chains

—  Each erythrocyte precursor cell has a beta globin gene on each chromosome 11 that determines beta globin production.

—  Thus, there are a total of two beta globin genes. Beta thalassemia results from mutation of these genes.

Pathogenesis

—  Impaired beta globin synthesis results in anemia by two mechanisms:

—  Deficit in HbA produces under-hemoglobinized hypochromic microcytic red cells

—  Diminished survival of red cells and their precursors

o   Precipitation of  free  alpha chains within normoblast

o   Cell membrane damage

o   Ineffective erythropoiesis

o   Splenic sequestration and destruction due to cell membrane damage and reduced deformability

Clinical syndromes

—   classification based on severity of anemia, which in turn depends on the type of genetic defect (Bo or B+)  and the gene dosage (homozygous or heterozygous)

o    B Thalassemia major

o   B Thalassemia minor or B Thalassemia  trait

o   B Thalassemia intermedia

Beta Thalassemia Major

—  Inheritance of two abnormal beta globin genes

—  Homozygous

—  Severe transfusion dependent anemia

Beta Thalassemia Minor

—  Heterozygotes with one beta thalassemia gene and one normal gene

—  The result is mild anemia-or no anemia at all.

Beta Thalassemia Interemedia

—  Severe but doesnot require regular blood transfusion

Thalassemia major

—  Decreased MCV and MCH

—  Peripheral blood smear

o   Microcytosis and hypochromia

o   Marked anisopoikilocytosis

o   Target cells

o   fragmented red cells

o   Elevated reticulocyte count

—   Iron status

o   Serum iron and serum ferritin markedly increased

o   Total iron binding capacity reduced

—  Bilirubin increased mainly of unconjugated type

—  HbF increased; HbA2 levels normal, low or high

Morphology

—  Bone marrow

o   Hypercellularity with erythroid hyperplasia

o   Bone marrow iron increased

o   Expansion of  hematopoietically active marrow leading to skeletal deformities (facial bones)

o   Erosion of cortical bone and formation of new bone giving rise to crew- cut appearance on X-ray                             

—  Enlarged spleen

o   Mononuclear phagocytic cell hyperplasia

o   Extramedullary hematopoiesis

—  Hemosiderosis: Heart, liver, pancreas

Clinical course

—   Transfusion dependent

—  Untreated children: growth retardation and death

—  Enlarged and distorted cheekbones and other bony prominences

—  Hepatospleenomegaly

—  Cardiac disease due to iron overload

—  Survival upto third decade with transfusion and iron chelation

Thalassemia minor

—  More common

—  Asymptomatic or mild anemia

—  PBS: microcytic hypochromic cells, target cells

—  Hemoglobin electrophoresis: increase in HbA2 to 4% to 8% of total hemoglobin (normal: 2.5%)

—  Important to diagnose due to:

o   Differentiation from hypochromic microcytic anemia of iron deficiency

o   Genetic counseling

Alpha Thalassemia

—  Absence or decreased production of alpha globin chains.

—  Each erythrocyte precursor cell has two alpha globin genes on each chromosome 16 that determine alpha globin production.

—  Thus, there are a total of four alpha globin genes.

—  The types of alpha thalassemia result from deletion of one or more of these genes.

—  Silent carrier

o   Deletion of single a-globin gene

o   Not much reduction in a-globin chain synthesis

o   Individuals are completely asymptomatic

—  a-thalassemia trait

o   Deletion of two a-globin genes

o   Can be from the same chromosome or one from each chromosome

Hemoglobin H disease

o   Deletion of 3 a-globin genes

o   a-globin synthesis markedly reduced

o   Tetramere of beta-globin- Hb H

o   Hb-h has very high affinity to O2- tissue hypoxia

o   Hb H prone to oxidation, leading to formation of intracellular inclusions- removed by splenic macrophages

Hydrops fetalis

o   Most severe from

o   Deletion of all 4 globin genes

o   In fetus, excess g-globin chain form tetramere (Hb Barts)

o   Hb Barts has very high affinity to O2, delivers almost no O2

o   Survival in early development is due to formation of zeta x-globin with formation of functional Hb (x2g2)

o   Signs of fetal distress evident from 3^rd trimester

o   Severe tissue hypoxia leads to intrauterine death

o   Intrauterine transfusion can save such fetus

o   Fetus shows severe pallor, generalized edema, and massive hepatosplenomegaly similar to erythroblastosis fetalis