Red Blood Cell Disorders.psa

8/14/2019 Red Blood Cell Disorders.psa

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Red Blood Cell Disorders

PETER S. AZNAR, MD, FPSP


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3 Formed Elements of Blood

Red Blood Cells (RBC) Erythrocytes

White Blood Cells (WBC) Leukocytes

Platelets Thrombocytes


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Functions of the Three Formed Elements

RBC- carries oxygen

WBC- responsible for the immune system and

prevention of infection

Platelets- coagulation


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Rouleaux formation- artificial stacking of red blood

cells

Reticulocytes- immediate precursor of a maturered blood cell

Anisocytosis- variation in RBC size

Poikilocytosis- variation in RBC shape

Definition of Terms:


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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)



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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



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Order of Red Blood Cell Maturation


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Erythropoiesis

Basophilic erythroblast

Polychromatophilic

erythroblast

Orthochromatophilicerythroblast Reticulocyte

Mature RBC

Proerythroblast


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Life Span of RBC

90- 120 days


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Immediate Precursor of Red Blood Cell

Reticulocyte

Normal value: 0.5 to 1.5%


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1. Inadequate intake

3. Malabsorption

5. Diversion of iron during pregnancy

7. Blood loss

Causes of Iron Deficiency


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RBC Characteristics in Iron Deficiency

Anemia?

Microcytic small RBCs

Hypochromatic pale RBCs


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Ways to diagnose iron deficiency anemia?

2. Serum ferritin

will be decreased. Careful, itll also be low in

diseased/ill patients (an acute phase reactant)

4. Bone Marrow

staining

6. Treatment

iron


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Causes of Macrocytic Anemia

B12 deficiency

Folate deficiency


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Uses of B12 and Folate

DNA synthesis

B12 = co-factor

Folate = transfer single carbon groups


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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


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Folate Deficiency 3 major causes

Dietary

Malabsorption

Increased usage


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3 Ways to Diagnose Folate Deficiency

2. Morphology

macrocytic RBCs and hypersegmented

neutrophils

3. Serum folate

5. Red cell folate


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Megaloblastic AnemiaMegaloblastic Anemia

Possible Causes?Possible Causes?

B12 or folate deficiencyB12 or folate deficiency


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B12 Deficiency 3 major causes

2. Pernicious Anemia

4. Pancreatic Insufficiency

6. Malabsorption


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3 Ways to Diagnose- B12 Deficiency

Morphology

Serum B12

Neurologic findings

Demyelination of spinal cord, cerebral cortex


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Treating B12 & Folate Deficiencies

B12

IM B12 supplementation for life

Folate

Daily folate supplement (1mg/day)


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What do you see in the RBCs below?

How would we quantitate this?

Anisocytosis refers tored cells which varywidely in size.

The RDWmathematicallymeasures the rangeof red cell sizes.


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What do you see in the RBCs below?

What diseases might they be associated with?

Microcytosis refers tored cells that are small.

You can use thelymphocyte nucleus as a

visual reference, or youcan use the MCV

Associated with Iron deficiency

Thalassemias

Sideroblastic anemia


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What do you see in the top

slide?

Characterizes whatdiseases?

Macrocytosis refers to largered cells.

Associated with Elevated reticulocyte count

B12/folate deficiency

Liver disease Thyroid disease

Chemotherapy

Anti-retrovirals (AZT)


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Whats wrong with these RBCs?

Measured how? A likely cause?

Hypochromasia refersto red cells that havetoo little hemoglobin.

The area of central

pallor is more than 1/3the total red celldiameter.

This is measured by theMCH (mean cellularhemoglobin)

Iron deficiency


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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.


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What do you see here? Diseases?

Target cells look likebulls-eyes.

Associated with

Liver disease

Thalassemias

Hemoglobin C

After splenectomy


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Spherocytes have a lossof central pallor.

Can be seen in

Hereditary spherocytosis

Autoimmune hemolysis

If due to autoimmune

hemolysis, the cells are

smaller (i.e.microspherocytes)


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Schistocytes are red

cell fragments with

sharp edges.

They are a hallmarkofMicroangiopathic

Hemolytic Anemia

(MAHA)


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What do you see here?

Sickle Cells are seen

in sickle cell anemia.

Notice that this slide

has target cells aswell as a sickled cell.


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What RBCs are here?

How do you distinguish the

two? Associated disease?

Echinocytes, orburr cells,have small, regularprojections. Seen in renaldisease

Acanthocytes, orspurcells, have larger, irregularprojections, and are seenin liver disease.


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What do you see here?

What causes it?

Teardrop cells

Seen in myelophthisic

processes, or

diseases ofmarrowinfiltration.

Deformed as it tries to

squeeze out of the

bone marrow


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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.


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Rouleaux are linear arrangements of red cells typically

described as piles of coins on a plate

They are typically seen in disorders with increasedlevels of immunoglobulin, such as Multiple Myeloma orWaldenstroms macroglobulinemia.

Severe hypo-albuminemia can also lead to reouleuxformation


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Red cell agglutination occurs when the redcells are coated with IgM. IgM is largeenough to bridge two red cells and causeagglutination.

Unlike rouleaux, the red cell clumps are notorderly and linear.


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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, scleralicterus

Patients may have chronic ankle ulcers.

Aplastic crises associated with Parvovirus B19, may

occur

Increased requirement for folate


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Post-splenectomy blood findings


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

Red cell abnormalities target cells, acanthocytes, schistocytes, NRBCs


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Hemolytic Anemia


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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!)


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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)


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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


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Classification by Etiology

Congenital Defects in membrane skeleton proteins

Defects in enzymes involved in energyproduction

Hemoglobin defects

Acquired

Immune-mediated Non-immune-mediated


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Most common defect leading to anemia? Hereditary spherocytosis

Frequency? Affects 1/5000 Europeans

Transmission?

Autosomal dominant


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Pathophysiology?

Defect is in proteins of the membrane skeleton, usuallyspectrin 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)


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Functions of GP6D?Functions of GP6D? Detoxification of metabolites of oxidative stressDetoxification of metabolites of oxidative stress

Elimination of methemoglobinElimination of methemoglobin

Important Products of GP6D?Important Products of GP6D? NADPHNADPH

Reduced glutathioneReduced glutathione


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Diagnostic methemoglobin precipitate?Diagnostic methemoglobin precipitate? Heinz bodiesHeinz bodies

Causes the formation of bite/blister cellsCauses the formation of bite/blister cells

Epidemiology of GP6D Deficiency?Epidemiology of GP6D Deficiency? Type B is more prevalentType B is more prevalent

Type A is in 20% of healthy AfricansType A is in 20% of healthy Africans

In 10-14% of African American menIn 10-14% of African American men

Also prevalent in the MediterraneanAlso prevalent in the Mediterranean X-linkedX-linked


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G6PD Deficiency: Agents to avoid

For SKAND

Fava beans

Sulfa drugs

Vitamin K

Anti-malarials

Naphtha compounds (mothballs)

Dapsone

G6PD D fi i


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G6PD Deficiency

Blister cellBlister cell


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How will you diagnose an autoimmune

cause of hemolytic anemia?


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Coombs Test

The Direct Coombs DAT (Direct Antiglobulin Test) - tests for IgG or C3

DIRECTLY ON THE RED CELLS. Youre adding

patient RBCs!

The Indirect Coombs

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. Youre addingpatient serum!


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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 Coombs test - both direct and indirect.

SPHEROCYTES are seen on the peripheral

smear.


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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.

W A ib d H l i A i


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Treatment

Folate repletion

Transfusion determining factors: Heart failure, shock?

Inadequate reticulocyte count?

D I d d I H l i


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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


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Drug-Induced Immune Hemolysis

Three general mechanisms

True autoimmune

You dont need the drug in the body any more to get the

hemolysis

Alpha-methyldopa, L-DOPA, Procainamide


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Cold Agglutinin Disease

IgM antibodies bind to I antigens of RBCs when cold (fallsoff when warm)

Causes agglutination cyanosis & ischemia ofextremities

Direct Coombs test + for C3, but not IgM!

Has both intravascular and extravascular hemolyticcomponents


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Cold Agglutinin Disease

Primary, or associated w/ Mycoplasma, Mononucleosis, orlymphoproliferative disease

Treat by avoiding cold & folate repletion

Corticosteroid and splenectomies uneffective (big differencefrom warm antibody-mediated hemolysis)

N I H l ti A i


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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


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Infections Causing Hemolysis

Malaroa

Babesia microti

Clostridium welchii

Bartonella bacilliformis

Basic Structure of All Human Hemoglobin


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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 globin chain has 141 amino acids

All non- chains have 146 amino acids

There is considerable structural homology among the

non-alpha chains

N l H H l bi


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Normal Human Hemoglobins

Gower Hemoglobin (Embryonic) 22

Fetal Hemoglobin (HbF)

22

Major Adult Hemoglobin (HbA)

22

Minor Adult Hemoglobin (HbA2)

22


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Heme Synthesis

Begins with condensation of glycine & succinylCo-A-amino levulinic acid (-ALA).

The rate-limiting step in heme synthesis

Requires intra-mitochondrial enzyme ALA-synthase

-ALA travels to cytoplasm; converted toporphobilinogen (PBG), a monopyrrole.

Heme Synthesis


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Heme Synthesis

PBG converted from monopyrrole to biologically activeform 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)

f G G


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Location of the Globin Genes

Genes for the non- chains are located onChromosome 11. This is referred to as the -globin gene cluster

Chain genes are located on Chromosome 16

There is duplication of the genes for:

Globin Globin (Gand A) *

Gand Adiffer from one another only at position 136 wherethey have glycine & alanine respectively


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Location of the Globin Genes

Synthesis of the non- chains involves acoordinated switching that proceeds from

embryonic () to fetal () to adult () globinchains Yolk sac () liver/spleen () marrow ()


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Structure of the Hemoglobin Molecule

Each Hb is comprised of 4 subunits: 2 identical chains & 2 identical non- chains

Each chain is arranged in the form of an -helix with8 individual helical segments (labeled A - H)

Each globin molecule has both hydrophobic &

hydrophilic areas



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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 theiron residue from oxidation, thereby maintaining it in

the active, reduced form

Each iron atom in the center of the heme residue isheld in place and kept in the active, reduced Fe++

stateby two histidine residues

P ibl C f


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Possible Consequences of a

Hemoglobinopathy

No detectable effect

Instability of the hemoglobin moleculeInstability 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

U t bl H l bi thi


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Unstable Hemoglobinopathies

Most of the unstable hemoglobinopathies involve amutation in the region of the heme pocket

These mutations enable water to gain access to thisvery hydrophobic region of the molecule

The end result is heme instability, denaturation, andrelease of heme from its binding site

The demonstration ofHeinz Bodies in these red cellsis evidence of the presence of an unstablehemoglobin mutant

Hemoglobinopathy Altering Oxygen


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g p y g yg

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



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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

H l bi M Di


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Hemoglobin M Diseases

The Hemoglobin M disorders are seen when asubstitution 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:


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Inheritance, Appearance of Symptoms,

Diagnosis

The most common sickle cell disease (SCD) iscalled sickle cell anemia (HbSS)

However, there are a number of other SCD

genotypes - compound heterozygous states

The sickle mutation is inherited in an autosomalco-dominant fashion

Individuals with sickle cell trait (AS) have roughlyequal amounts of HbA & HbS and are generallyasymptomatic



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Inheritance, Appearance of Symptoms,

Diagnosis

Compound heterozygotes (e.g., SC or S-Thalassemia) generally express a significant sicklecell disease

We dx/ with electrophoresis:

- Hb C has a positive; HbS is neutral, HB A is

negative.- Movement: HbA > HbS > HbC


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Sickle Cell Anemia Pathophysiology

q

The presence of the abnormal (or sickle)hemoglobin (HbS) within the cells of theaffected individuals

q The decreased solubility & the tendency of

this abnormal hemoglobin to polymerize whenit assumes the deoxy conformation

q

In HbS, the negatively charged glutamic acidat 6 position is replaced by an unchargedvaline residue

Si kl C ll A i P th h i l


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Sickle Cell Anemia Pathophysiology

q

In deoxy conformation, the valine at 6

positionapproaches the phenylalanine at 85 position onadjacent HbS molecule.

Multiple critical contact points that enable thehemoglobin molecules to attach to one another

The polymer begins as a small nucleus of hemoglobin

molecules aligned polymer with a total of 7 anti-parallel pairs (or 14 individual hemoglobin chains)

SICKLE CELL DISEASE


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SICKLE CELL DISEASE

Clinical Features

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


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SICKLE CELL DISEASETherapeutic Approaches

Reactivate Fetal Hemoglobin Production usingReactivate Fetal Hemoglobin Production usingHydroxyureaHydroxyurea!!

Chemical inhibition of Hb S polymerization

Increase in intracellular hydration Altering RBC/Endothelial cell interactions

Bone marrow transplantation

Gene therapy


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Megaloblastic

Anemia

Red cells aremacrocytic

Hypersegmented

neutrophils can be

seen

Vitamin B12 or folate

deficiency

Sickle Cell Anemia


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Sickle Cell Anemia

Target Cell

Sickled Cell

Myeloproliferative Diseases


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Myeloproliferative Diseases

Includes:

Polycythemia vera

Essential Thrombocythemia

Myelofibrosis

Chronic Myelogenous Leukemia

P l th i


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Polycythemia vera

Most of cells in circulation are derived from asingle, neoplastic stem cell

Does not need Epo to produce more cells

Diagnosis based on low/absent levels of Epo

Polycythemia vera


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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


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Those common to ALL erythrocytosis Headache

Decreased mental acuity

Weakness

Pruritis after bathing

Hypermetabolic sxs

Erythromelalgia

Thrombosis

Hemorrhage



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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


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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


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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 1-

2% to 11%

Single injection may control hemoglobin and platelet

count for a year or more. Alkylating agents such as busulfan

P vera Treatment


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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-likesymptoms, fatigue, fever, myalgias, malaise

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Red Blood Cell Disorders.psa