Red Blood Cell Disorders


3 Formed Elements of Blood
• Red Blood Cells (RBC) Erythrocytes • White Blood Cells (WBC) Leukocytes • Platelets Thrombocytes

Functions of the Three Formed Elements
• RBC- carries oxygen • WBC- responsible for the immune system and prevention of infection • Platelets- coagulation

Definition of Terms:
• Ferritin- storage iron • Transferrin- transport iron • Total Iron Binding Capacity (TIBC)- test which measures the extent to which iron-binding sites in the serum can be saturated • Erythropoiesis- is the process by which red blood cells (erythrocytes) are produced

Definition of Terms:
• Rouleaux formation- artificial stacking of red blood cells • Reticulocytes- immediate precursor of a mature red blood cell • Anisocytosis- variation in RBC size • Poikilocytosis- variation in RBC shape

Definition of Terms:
• Microcytosis- refers to red cells that are small • Macrocytosis- refers to red cells that are large • Mean Corpuscular Volume (MCV)• Mean Corpuscular Hemoglobin Concentration (MCHC)

Definition of Terms:
• Mean Cellular Hemoglobin (MCH) • Hypochromasia refers to red cells that have too little hemoglobin • Howell-Jolly bodies are peripheral, small, round, purple inclusions within red cells that represent nuclear remnants

Order of Red Blood Cell Maturation

Proerythroblast Basophilic erythroblast

Polychromatophilic erythroblast

Orthochromatophilic erythroblast


Mature RBC

Life Span of RBC

• 90- 120 days

Immediate Precursor of Red Blood Cell
• Reticulocyte – Normal value: 0.5 to 1.5%

Causes of Iron Deficiency
1. 3. 5. 7. Inadequate intake Malabsorption Diversion of iron during pregnancy Blood loss

RBC Characteristics in Iron Deficiency Anemia?
• Microcytic – small RBCs • Hypochromatic – pale RBCs

Ways to diagnose iron deficiency anemia?
2. Serum ferritin
– will be decreased. Careful, it’ll also be low in diseased/ill patients (an acute phase reactant)


Bone Marrow
– staining


– iron

Causes of Macrocytic Anemia
• B12 deficiency • Folate deficiency

Uses of B12 and Folate
• DNA synthesis
– B12 = co-factor – Folate = transfer single carbon groups

How do we get folate and B12?
• Folate
– In leafy green veggies, liver, yeast – Destroyed by cooking – Need 100-200 micrograms daily

• Vitamin B12
– In animal products – Unaffected by cooking – Need 1-2 micrograms daily

Folate Deficiency – 3 major causes

• Dietary • Malabsorption • Increased usage

3 Ways to Diagnose Folate Deficiency

2. Morphology
– macrocytic RBCs and neutrophils hypersegmented

3. Serum folate 5. Red cell folate

Megaloblastic Anemia Possible Causes? B12 or folate deficiency

B12 Deficiency – 3 major causes
2. Pernicious Anemia 4. Pancreatic Insufficiency 6. Malabsorption

3 Ways to Diagnose- B12 Deficiency

• • •

Morphology Serum B12 Neurologic findings
– Demyelination of spinal cord, cerebral cortex

Treating B12 & Folate Deficiencies
• B12
– IM B12 supplementation for life

• Folate
– Daily folate supplement (1mg/day)

What do you see in the RBCs below? How would we quantitate this?
• Anisocytosis refers to red cells which vary widely in size. • The RDW mathematically measures the range of red cell sizes.

What do you see in the RBCs below? What diseases might they be associated with?
• Microcytosis refers to red cells that are small. • You can use the lymphocyte nucleus as a visual reference, or you can use the MCV • Associated with
– Iron deficiency – Thalassemias – Sideroblastic anemia

What do you see in the top slide? Characterizes what diseases?
• Macrocytosis refers to large red cells. • Associated with
– – – – – – Elevated reticulocyte count B12/folate deficiency Liver disease Thyroid disease Chemotherapy Anti-retrovirals (AZT)

What’s wrong with these RBCs? Measured how? A likely cause?
• Hypochromasia refers to red cells that have too little hemoglobin. • The area of central pallor is more than 1/3 the total red cell diameter. • This is measured by the MCH (mean cellular hemoglobin) • Iron deficiency

What do you see on this slide?
• Poikilocytosis refers to red cells that vary widely in shape. • Remember that anisocytosis refers to red cells that vary widely in size.

What do you see here? Diseases?
• Target cells look like bulls-eyes. • Associated with
– – – – Liver disease Thalassemias Hemoglobin C After splenectomy

What do you see here? Diseases?
• Spherocytes have a loss of central pallor. • Can be seen in
– Hereditary spherocytosis – Autoimmune hemolysis

• If due to autoimmune hemolysis, the cells are smaller (i.e. microspherocytes)

What do you see here? Diseases?
• Schistocytes are red cell fragments with sharp edges. • They are a hallmark of Microangiopathic Hemolytic Anemia (MAHA)

What do you see here?
• Sickle Cells are seen in sickle cell anemia. • Notice that this slide has target cells as well as a sickled cell.

What RBCs are here? How do you distinguish the two? Associated disease?
• Echinocytes, or burr cells, have small, regular projections. Seen in renal disease • Acanthocytes, or spur cells, have larger, irregular projections, and are seen in liver disease.

What do you see here? What causes it?
• Teardrop cells • Seen in myelophthisic processes, or diseases of marrow infiltration. • Deformed as it tries to squeeze out of the bone marrow

• Howell-Jolly bodies
– are peripheral, small, round, purple inclusions within red cells that represent nuclear remnants.

• They are seen after splenectomy, or in cases of splenic hypofunction.

• Rouleaux are linear arrangements of red cells typically described as “piles of coins on a plate” • They are typically seen in disorders with increased levels of immunoglobulin, such as Multiple Myeloma or Waldenstrom’s macroglobulinemia. • Severe hypo-albuminemia can also lead to reouleux formation

• Red cell agglutination occurs when the red cells are coated with IgM. IgM is large enough to bridge two red cells and cause agglutination. • Unlike rouleaux, the red cell clumps are not orderly and linear.

General Clinical Features of Hemolytic Anemias
• Splenomegaly is generally present • Patients have an increased incidence of pigmented gallstones. • Dark urine (tea-colored or red), jaundice, scleral icterus • Patients may have chronic ankle ulcers. • Aplastic crises associated with Parvovirus B19, may occur • Increased requirement for folate

Post-splenectomy blood findings

• Howell-Jolly bodies
– small round blue DNA remnants in periphery of RBCs

• Red cell abnormalities
– target cells, acanthocytes, schistocytes, NRBCs

Hemolytic Anemia

Sites of Red Cell Destruction
• Extravascular Hemolysis
– Macrophages in spleen, liver, and marrow remove damaged or antibody-coated red cells

• Intravascular Hemolysis
– Red cells rupture within the vasculature, releasing free hemoglobin into the circulation (and the circulation does NOT like this!)

Evidence for Increased Red Cell Production
• In the blood: – Elevated reticulocyte count – May be associated with high MCV – Circulating NRBCs may be present • In the bone marrow: – erythroid hyperplasia – reduced M/E (myeloid/erythroid) ratio • In the bone: – Deforming changes in the skull and long bones (“frontal bossing”)

Evidence for Increased Red Cell Destruction
• Biochemical consequences of hemolysis in general
• • • • • • Elevated LDH Elevated unconjugated bilirubin  jaundice, scleral icterus Lower serum haptoglobin Hemoglobinemia Hemoglobinuria Hemosiderinuria

• Morphologic evidence of red cell damage
• Schistocytes • Spherocytes • Bite/blister cells

• Reduced red cell life-span

Classification by Etiology

• Congenital
– Defects in membrane skeleton proteins – Defects in enzymes involved in energy production

– Hemoglobin defects

• Acquired
– Immune-mediated – Non-immune-mediated

• Most common defect leading to anemia?
– Hereditary spherocytosis

• Frequency?
– Affects 1/5000 Europeans

• Transmission?
– Autosomal dominant

• Pathophysiology?
– Defect is in proteins of the membrane skeleton, usually spectrin or ankyrin – Lipid microvesicles are pinched off in the spleen and other RE organs, causing decreased MCV and spherocytic change.

• Diagnosing?
– Increased osmotic fragility

• Treatment?
– Supplemental folate – Splenectomy (but carefully consider timing in children)

• Functions of GP6D?
• • • • Detoxification of metabolites of oxidative stress Elimination of methemoglobin

• Important Products of GP6D?
NADPH Reduced glutathione

• Diagnostic methemoglobin precipitate?
• • • • • • • Heinz bodies Causes the formation of bite/blister cells

• Epidemiology of GP6D Deficiency?
Type B is more prevalent Type A is in 20% of healthy Africans In 10-14% of African American men Also prevalent in the Mediterranean X-linked

G6PD Deficiency: Agents to avoid
– – – – – – Fava beans Sulfa drugs Vitamin K Anti-malarials Naphtha compounds (mothballs) Dapsone

G6PD Deficiency

Blister cell

How will you diagnose an autoimmune cause of hemolytic anemia?

Coomb’s Test
• The Direct Coomb’s
– DAT (Direct Antiglobulin Test) - tests for IgG or C3 DIRECTLY ON THE RED CELLS. You’re adding patient RBCs!

• The Indirect Coomb’s
– tests for IgG or C3 in the serum which react with generic normal red cells. This is also known as the antibody screen in blood-banking. You’re adding patient serum!

Warm-Antibody Hemolytic Anemias Clinical Features
• Splenomegaly, jaundice usually present. • Depending on degree of anemia and rate of fall in hemoglobin, patients can have VERY symptomatic anemia • Lab Dx ↑reticulocytes, ↑ bili, ↑ LDH, – positive Coomb’s test - both direct and indirect. – SPHEROCYTES are seen on the peripheral smear.

Warm-Antibody Hemolytic Anemias Treatment
• Immunosuppressive Treatment
– First line is corticosteroids (i.e. prednisone). – If steroids fail to work, or if patient relapses after steroid taper, splenectomy may be necessary. – Immunosuppressives such as cyclophosphamide (Cytoxan) or azathioprine (Immuran) may be required as third-line therapy.

Warm-Antibody Hemolytic Anemias Treatment
• Folate repletion • Transfusion – determining factors:
– Heart failure, shock? – Inadequate reticulocyte count?

Drug-Induced Immune Hemolysis Three general mechanisms
• Innocent bystander
– the Ab was directed at the drug, but it cross reacted w/ RBCs – Drug must be present for hemolysis to occur – Quinine, Quinidine, Isoniazide

• Hapten
– Drug binding to RBC  Abs that react to this complex – Penicillins, Cephalosporins

Drug-Induced Immune Hemolysis Three general mechanisms

• True autoimmune
– You don’t need the drug in the body any more to get the hemolysis – Alpha-methyldopa, L-DOPA, Procainamide

Cold Agglutinin Disease
• IgM antibodies bind to I antigens of RBCs when cold (falls off when warm) • Causes agglutination  cyanosis & ischemia of extremities • Direct Coomb’s test + for C3, but not IgM! • Has both intravascular and extravascular hemolytic components

Cold Agglutinin Disease
• Primary, or associated w/ Mycoplasma, Mononucleosis, or lymphoproliferative disease • Treat by avoiding cold & folate repletion • Corticosteroid and splenectomies uneffective (big difference from warm antibody-mediated hemolysis)

Non-Immune Hemolytic Anemia Classification
• Mechanical trauma to red cells
– Microangiopathic Hemolytic Anemia – Abnormalities in heart and large vessels – March Hemoglobinuria

• Infections • Drugs, Chemicals, and Venoms

Chemical & Physical Agents Causing Hemolysis

“BAr CoIns”
– – – – Severe Burns Arsenic Copper Insect and spider bites

Infections Causing Hemolysis
• Malaroa • Babesia microti • Clostridium welchii • Bartonella bacilliformis

Basic Structure of All Human Hemoglobin
• Each hemoglobin molecule is composed of:
– 4 iron-containing, tetrapyrrole heme rings – 4 polypeptide globin chains
• • 2 alpha chains 2 non-alpha chains

• • •

Each α globin chain has 141 amino acids All non-α chains have 146 amino acids There is considerable structural homology among the non-alpha chains

Normal Human Hemoglobins
• Gower Hemoglobin (Embryonic)

• Fetal Hemoglobin (HbF)

• Major Adult Hemoglobin (HbA)

• Minor Adult Hemoglobin (HbA2)

Heme Synthesis
• Begins with condensation of glycine & succinyl Co-A  δ-amino levulinic acid (δ-ALA). – The rate-limiting step in heme synthesis – Requires intra-mitochondrial enzyme ALAsynthase ∀ δ-ALA travels to cytoplasm; converted to porphobilinogen (PBG), a monopyrrole.

Heme Synthesis
• PBG converted from monopyrrole to biologically active form protoporphyrin IX, a tetrapyrrole. Iron inserted into tetrapyrrole ring n the mitochondria Heme synthesis stimulated by iron & repressed when iron is inadequate (e.g., iron deficiency)

• •

Location of the Globin Genes
• Genes for the non-α chains are located on Chromosome 11. This is referred to as the βglobin gene cluster ∀ α Chain genes are located on Chromosome 16 • There is duplication of the genes for:
∀ α Globin ∀ γ Globin (Gγ and Aγ) *
• γ and Aγ differ from one another only at position 136 where they have glycine & alanine respectively

Location of the Globin Genes
• Synthesis of the non-α chains involves a coordinated switching that proceeds from embryonic (ε) to fetal (γ) to adult (β) globin chains
• Yolk sac (ε)  liver/spleen (γ)  marrow (β)

Structure of the Hemoglobin Molecule
• Each Hb is comprised of 4 subunits: 2 identical α chains & 2 identical non-α chains Each chain is arranged in the form of an α-helix with 8 individual helical segments (labeled A - H) Each globin molecule has both hydrophobic & hydrophilic areas

Structure of the Hemoglobin Molecule
• The iron-containing heme ring is buried within a very hydrophobic region of the globin that is called the “Heme Pocket” The hydrophobic nature of this region protects the iron residue from oxidation, thereby maintaining it in the active, reduced form Each iron atom in the center of the heme residue is held in place and kept in the active, reduced Fe++ state by two histidine residues

Possible Consequences of a Hemoglobinopathy
• No detectable effect • Instability of the hemoglobin molecule • An increase or a decrease in oxygen affinity • Inability to maintain the heme iron in its active, reduced state (methemoglobinemia) • Decreased solubility of the hemoglobin molecule

Unstable Hemoglobinopathies
• • • • Most of the unstable hemoglobinopathies involve a mutation in the region of the heme pocket These mutations enable water to gain access to this very hydrophobic region of the molecule The end result is heme instability, denaturation, and release of heme from its binding site The demonstration of Heinz Bodies in these red cells is evidence of the presence of an unstable hemoglobin mutant

Hemoglobinopathy Altering Oxygen Affinity
• Increased Oxygen Affinity
– Stabilization of the Oxy conformation increases the oxygen affinity of the hemoglobin molecule The presence of such an effect can be confirmed by demonstrating a left shift in the Oxygen Saturation Curve Individuals with an increase in oxygen affinity typically exhibit erythrocytosis

Hemoglobinopathy Altering Oxygen Affinity
• Decreased Oxygen Affinity
– Stabilization of the Deoxy conformation produces a decrease in the the oxygen affinity of the hemoglobin molecule – The presence of such an effect can be confirmed by demonstrating a right shift in the Oxygen Saturation Curve – Individuals with a decrease in oxygen affinity are typically somewhat anemic

Hemoglobin M Diseases
• The Hemoglobin M disorders are seen when a substitution has occurred at the locus of either the proximal or distal histidine Typically, this involves a his tyr substitution which then forms an iron-phenolate complex Hemoglobin with its iron in the oxidized Fe+++ state is incapable of binding oxygen This form of hemoglobin (called Methemoglobin) has a brownish appearance Patients with Hemoglobin M disease are typically cyanotic

• • • •

The Sickle Cell Diseases: Inheritance, Appearance of Symptoms, Diagnosis
• The most common sickle cell disease (SCD) is called sickle cell anemia (HbSS) • However, there are a number of other SCD genotypes - compound heterozygous states • The sickle mutation is inherited in an autosomal co-dominant fashion • Individuals with sickle cell trait (AS) have roughly equal amounts of HbA & HbS and are generally asymptomatic

The Sickle Cell Diseases: Inheritance, Appearance of Symptoms, Diagnosis
• Compound heterozygotes (e.g., SC or S-β Thalassemia) generally express a significant sickle cell disease • We dx/ with electrophoresis: - Hb C has a positive; HbS is neutral, HB A is negative. - Movement: HbA > HbS > HbC

Sickle Cell Anemia Pathophysiology

The presence of the abnormal (or sickle) hemoglobin (HbS) within the cells of the affected individuals The decreased solubility & the tendency of this abnormal hemoglobin to polymerize when it assumes the deoxy conformation In HbS, the negatively charged glutamic acid at β6 position is replaced by an uncharged valine residue



Sickle Cell Anemia Pathophysiology

In deoxy conformation, the valine at β6 position approaches the phenylalanine at β 85 position on adjacent HbS molecule. Multiple critical contact points that enable hemoglobin molecules to attach to one another the

The polymer begins as a small nucleus of hemoglobin molecules  aligned polymer with a total of 7 antiparallel pairs (or 14 individual hemoglobin chains)

– – – – – – – – – Painful vaso-occlusive crisis Strokes Retinopathy Acute chest syndrome Pulmonary hypertension Sickle cell nephropathy Biliary tract disease Leg ulcers Avascular necrosis of the large joints

SICKLE CELL DISEASE Therapeutic Approaches
• Reactivate Fetal Hemoglobin Production using Hydroxyurea! • Chemical inhibition of Hb S polymerization • Increase in intracellular hydration • Altering RBC/Endothelial cell interactions • Bone marrow transplantation • Gene therapy

Megaloblastic Anemia
• Red cells are macrocytic • Hypersegmented neutrophils can be seen • Vitamin B12 or folate deficiency

Sickle Cell Anemia

Target Cell

Sickled Cell

Myeloproliferative Diseases

• Includes:
• • • • Polycythemia vera Essential Thrombocythemia Myelofibrosis Chronic Myelogenous Leukemia

Polycythemia vera
• Most of cells in circulation are derived from a single, neoplastic stem cell • Does not need Epo to produce more cells • Diagnosis based on low/absent levels of Epo

Polycythemia vera
Natural History – 4 phases: 2. Latent phase - asymptomatic 3. Proliferative phase -pts may have sxs of:
– – – Hypermetabolism Hyperviscosity Thrombosis

• •

Spent phase - ↓ red cell mass, anemia, leukopenia, secondary myelofibrosis, increasing HSM. 20% of pts Secondary AML
– 1-2% of pts treated with phlebotomy alone

Symptoms of Polycythemia Vera
• Those common to ALL erythrocytosis
– Headache – Decreased mental acuity – Weakness

• • • • •

Pruritis after bathing Hypermetabolic sxs Erythromelalgia Thrombosis Hemorrhage

Symptoms of Polycythemia Vera
• PE findings
– – – – Facial plethora Splenomegaly Hepatomegaly Retinal vein distension

• Lab findings
– BASOPHILIA – Low EPO levels – Increased Hbg/HCT, WBCs, platelets, uric acid, B12, leukocyte alkaline phosphatase score

P vera - Treatment
• Phlebotomy – Draw 500 cc blood 1-2x/wk to target Hct 45%; maintain BP w/ saline – Generally, the best initial treatment for P vera – rapid onset – Downsides:
• Increased risk of thrombosis • No effect on progression to spent phase • May be insufficient to control disease

P vera - Treatment
• Myelosuppressive agents – Hydroxyurea • can be used in conjunction with phlebotomy • May increase the risk of leukemic transformation from 1-2% to 4-5% – 32P – kills some of the proliferating cells! • increase the risk of leukemic transformation from 12% to 11% • Single injection may control hemoglobin and platelet count for a year or more. – Alkylating agents such as busulfan

P vera - Treatment
• Interferon alpha – Benefits • No myelosuppression • No increase in progression to AML • No increase in thrombosis risk – Drawbacks • Must be given by injection up to daily • Side effects may be intolerable in many pts: flu-like symptoms, fatigue, fever, myalgias, malaise