ANEMIA

OBJECTIVES
At the end of this chapter the student will be able to:
 Define anemia

 Discuss classification of anemia

 Elaborate the morphological classification of anemia

 List the causes of the different categories of anemia

 Discuss the causes and clinical significance of anemias

 Discuss the laboratory findings for each category of anemia

 Correlate laboratory findings within each category of anemia

 Perform basic laboratory tests for the diagnosis of anemia

 QC, sources of error,

CHAPTER OUTLINE
1.1. Definition of terms
1.1.1. anemia (global epidemiology
1.1.2. anemia (anemia situation in Ethiopia)
1.2. Classification of anemias
1.2.1. Hematologic Response to Anemia
1.2.2. Signs of Accelerated Bone Marrow Erythropoiesis
1.2.3. Physiologic Response to Anemia
1.2.4. Methods of classification
1.2.5. Anemia Diagnosis/Cause
1.2.6. Lab Investigation of Anemia

1.3. Types of anemia

. 1.3.1 microcytic hypochromic anemia
1.3.2. macrocytic normocytic anemias
1.3.3. normocytic anemias
1.3.4. normocytic anemias due to hemoglobinopathies
INTRODUCTION
Definition of Anemia
 Anemia is a decrease in the RBC count, Hgb and/or HCT
values as compared to normal reference range for age and
sex
– ‘True’ anemia:
– decreased RBC mass and normal plasma volume
– Pseudo or dilutional anemia:
– normal RBC mass and increased plasma volume
– An increase in plasma volume can occur in

Pregnancy, volume overload (IVs) congestive heart

failure
– Low Hgb and HCT values
DEFINITION OF ANEMIA CONT’D
ANEMIA DEFINITION CONT’D
 clinically the diagnosis of anemia is made by:
 patient history,
 physical exam,
 signs and symptoms,
 and laboratory findings
 This definition emphasizes the differences one should expect
when evaluating probable anemia in the different age groups.
 Anemia must also relate to the level of hemoglobin the
individual normally possesses.
 If an adult male usually maintains a hemoglobin level of 16g/dl, and
over a period of days is noted to have decreased to 14g/dl, this must
be considered significant even though both values are within the
normal range for an adult male.
DEFINITION OF ANEMIA CONT’D
 Functionally anemia is defined as tissue hypoxia (inability of the body
to supply tissue with adequate oxygen for proper metabolic function)
HEMATOLOGIC RESPONSE TO ANEMIA
 Tissue hypoxia causes increased renal release of
erythropoietin (EPO) to accelerate bone marrow
erythropoiesis

 The normal bone marrow can increase its activity 7-8 times
normal
 Marrow becomes hypercellular
SIGNS OF ACCELERATED BONE MARROW
ERYTHROPOIESIS
 The marrow becomes hypercellular due to a marked increase in
RBC precursors (called erythroid hyperplasia) and the M:E
ratio falls.
 Nucleated RBCs may be released into the blood circulation
along with the outpouring of reticulocytes
 NRBC number tends to correlate with the severity of anemia
 Increased polychromasia on the Wright's- stained blood smear is
seen due to increased number of circulating Retics.
SIGNS OF ACCELERATED BONE MARROW
ERYTHROPOIESIS
NRBC

Polychromasia

Wright’s stained blood smear

 If demand exceeds maximal bone marrow activity, RBC
production may occur in extramedullary sites, liver, spleen
(hepatosplenomegaly).
1.1.3. PHYSIOLOGIC RESPONSE TO ANEMIA
 Ability to adapt to anemia depends on:
 Age and underlying disease
 Cardio/pulmonary function
 Rate at which anemia develops (BM can compensate easier if the
onset of anemia is slow),
 Underlying disease

 Symptoms of hypoxia: decreased oxygen delivery to the

tissues/organs causes:
 fatigue,faintness, weakness, dizziness, headaches, dyspnea, poor
exercise tolerance, leg cramps.
 Pallor
SYMPTOMS CONT’D

 General physical findings:

 Pallor,
rapid pulse, neurologic
problems (see table)
PHYSICAL SIGNS OF ANEMIA
1.2. METHODS OF ANEMIA CLASSIFICATION
 Several schemes of classifying anemias exist
1. Morphologic
 Basedon RBC morphology
 Anemia is divided into three groups mainly on the basis of the MCV
(RBC indices)
2. Pathophysiologic
 Anemia is divided using three main causes/mechanisms
I. Impaired erythrocyte formation (Aplastic anemia,
IDA, sideroblastic anemia, anemia of chronic
diseases, megaloblastic anemia)
 Retic count is low

 The bone marrow fails to respond appropriately due to

disease or lack of essential supplies

METHODS OF CLASSIFICATION CONT’D
III. Increased blood loss (Acute, Chronic)
 Retic count is typically high
 Anemia results when red cell loss exceeds the bone marrow’s
capacity to increase its activity

IV. Increased destruction of RBCs (hemolytic anemias)

 Retic count is typically high
 Anemia results when red cell destruction exceeds the bone
marrow’s capacity to increase its activity
MORPHOLOGIC CATEGORIES OF ANEMIA
 Originally proposed by Wintrobe, a morphologic classification
is based on how the cells appear on a stained smear and should
correspond with the red cell indices.
 Normocytic Normochromic (NCNC): anemia due to decrease in the
number of erythrocytes
 e.g. aplastic anemia, or acute blood loss
 Microcytichypochromic
 Macrocytic Normochromic,
MORPHOLOGIC CATEGORIES OF ANEMIA
CONT’D

1. Microcytic Hypochromic, anemia

 low MCHC
 low MCV
 These include:

1. iron deficiency anemia

2. sideroblastic anemia
3. Thalassemias
4. anemia of chronic disease (rare cases)
MORPHOLOGIC CATEGORIES OF ANEMIA
CONT’D

2. Macrocytic Normochromic anemia

 normalMCHC
 high MCV

 These include:
1. vitamin B12 deficiency
2. folate deficiency
MORPHOLOGIC CATEGORIES OF ANEMIA
CONT’D

3. Normocytic Normochromic (NCNC) anemia

 normal MCV
 normal MCHC

 These include:
1. anemia of acute hemorrhage
2. aplastic anemias (those characterized by disappearance of RBC
precursors from the marrow)
3. anemias of chronic disease (ACD)
4. hemolytic anemias (those characterized by accelerated destruction of
RBC’s)
 3 MORPHOLOGIC CATEGORIES OF ANEMIA

1 2
1 Microcytic/hypochromic 2 Macrocytic/normochromic

N.B. The nucleus of a small

lymphocyte (shown by the
arrow) is used as a reference to
3 a normal red cell size
3 Normocytic/normochromic
CLASSIFICATION OF ANEMIA
1.2.1. MICROCYTIC HYPOCHROMIC
ANEMIAS
Upon completion of this lesson the student will be able to:
 Evaluate the laboratory findings associated with
microcytic/hypochromic anemias based on etiology, clinical
findings, blood and bone marrow findings, and tests used to
aid in diagnosis and treatment:
 Iron deficiency anemia
 Sideroblastic anemias (primary and secondary types)
 Anemia of chronic disease
 Alpha and beta thalassemias (major and minor types)
MICROCYTIC/HYPOCHROMIC ANEMIAS
 Iron deficiency anemia (IDA)
 Anemia of chronic disease (ACD)
 Sideroblastic anemias (primary and secondary types)
 Alpha and beta thalassemias (major and minor types
 MICROCYTIC/HYPOCHROMIC ANEMIAS
 Characterized by impaired hemoglobin synthesis

Sideroblastic IDA
anemia ACD

Thalassemia ( or)
Abbott Manual
PATHOPHYSIOLOGY
Decrease in Hb synthesis
 Deficiency in heme synthesis
 Iron deficiency (IDA)

 Iron is trapped within macrophages and is unavailable for heme

synthesis (ACD)
 Failure of protoporpyirin synthesis (Sideroblastic anemia)

- abnormal metabolism within the RBC

itself. In this case, iron is sequestered in the developing
RBC mitochondria and is unavailable for heme synthesis
(e.g. sideroblastic anemia)
 Deficiency in globin chain synthesis ( or  Thalassemias)
MICROCYTIC/HYPOCHROMIC ANEMIAS

Normoblastic RBC maturation  normocytic red cells

RBC maturation in microcytic anemias

Abbott Manual
IRON DEFICIENCY ANEMIA (IDA)
 Is a condition in which the total body iron content is decreased
below a normal level
 This results in a reduced red blood cell and hemoglobin
production
 Causes:
 Nutritional deficiency
 Malabsorption (insufficient or defective absorption)
 Inefficient transport, storage or utilization of iron
 Increased need
 Chronic blood loss (GI bleeding, ulcer, heavy menstruation, etc)
IRON DEFICIENCY ANEMIA (IDA)
 Sequence of iron depletion
 Storeage 1st; Serum Iron depletion 2nd; cell morphology 3rd
DAILY IRON CYCLE (FIG)
IRON ABSORPTION, TRANSPORT AND
STORAGE
 Iron absorbed from duodenum and jejunum in the GIT, moves
via circulation to the bone marrow,where it is incorporated with
protoporphyrin in mitochondria of the erythroid precursor to
make Heme
 Three proteins are important for transporting and storage of iron
 Transferrin: transports iron from the plasma to the erythroblasts in the
marrow for erythropoiesis
 Transferrin will bind to Transferrin receptor on the
erythroblast membrane
 Excess iron is stored as Ferritin and hemosiderin in the
reticuloendothelial system

LAB INVESTIGATION OF MICROCYTIC
HYPOCHROMIC ANEMIA
 Iron tests
►Used to differentiate microcytic hypochromis anemias or detect
iron overload (hemochromatosis)
 Iron circulates bound to the transport protein transferrin

Transferrin is normally ~33% saturated with iron



 Iron tests include serum iron, Total Iron Binding Capacity

(TIBC), serum ferritin
 Serum iron level
 measures the amount of iron bound to transferrin
 Does not include the free form of iron
LAB INVESTIGATION CONT’D
 Total Iron Binding Capacity (TIBC)
 Isan indirect measure of the amount of transferrin protein in the
serum
 Inversely proportional to the serum iron level

 If serum iron is decreased, total iron binding capacity of

transferrin increased (transferrin has more empty space to
carry iron)
LAB INVESTIGATION CONT’D
 Serum ferritin
 indirectly reflects storage iron in tissues
 found in trace amount in plasma
 It is in equilibrium with the body stores
 Variation in the quantity of iron in the storing compartment is
reflected by plasma ferritin concentration
e.g.Plasma ferritin is decreases in IDA
Plasma ferritin increases in ACD

Limitation: During infection or inflammation Serum Ferritin increases

like other acute phase proteins, and then it is not an accurate indicator
in such situations.
BONE MARROW IRON (TISSUE IRON)
 Tissue biopsy of bone marrow
 Prussian blue stain

 Type of iron is hemosiderin

LAB INVESTIGATION OF ANEMIA CONT’D

 Transferrin levels are regulated by iron availability

↑ synthesis in iron deficient states…↓ serum iron, ↑ TIBC
 ↓ synthesis in iron overload states…↑ serum iron, ↓ TIBC
 ↓ synthesis in inflammatory states…↓ serum iron, ↓ TIBC
LAB INVESTIGATION OF ANEMIA CONT’D
IRON DEFICIENCY ANEMIA

Blood smear

 Lab findings
 Low RBC, Hgb, Hct
 Low MCV, MCH, MCHC
 Normal WBC and PLT
IRON DEFICIENCY ANEMIA
 RBC morphology
 Hypochromia
 Microcytosis
 Anisocytosis
 Poikilocytosis
 Pencil cells (cigar cells)
 Target cells
Blood smear
 no RBC inclusions
 Iron parameters
 Low serum iron,
 High TIBC,
 Low serum ferritin
 IRON DEFICIENCY

Ovalocytes - Pencil forms

No RBC inclusions

Wright’s stained blood smear

IRON DEFICIENCY ANEMIA
 Bone marrow (not usually performed)
 No stainable iron
 Increased NRBC
 Erythroid hyperplasia with decreased M:E ratio

Bone marrow 10x

No stainable iron (-) Prussian

blue iron stain
 IDA CONT’D
 Clinical signs and symptoms
 Pica – cravings for ice, dirt, laundry starch, clay
 Tongue atrophy/glossitis - raw and sore.
 Spoon‑shaped nails (koilonychia), brittle nails and hair.
 Numbness and tingling.
 IDA CONT’D

 Treatment
 Identify the underlying cause
 Oral iron is given; see increased Retic count post-therapy.
 May see dimorphism following treatment
 a dual red cell population with older microcytic red cells
along with the newly produced normocytic red cells.
SIDEROBLASTIC ANEMIA (SA)
 This group of anemias are characterized by defective protoporphyrin
synthesis (blocks) resulting in iron loading and a hypochromic anemia due
to deficient hemoglobin synthesis.
TERMS:
 Siderocytes are mature RBCs in the blood containing iron
granules called Pappenheimer bodies....abnormal.
 Sideroblasts are immature nucleated RBCs in the bone marrow
containing small amounts of iron in the cytoplasm....normal.
 Sideroblastic anemia is characterized by the accumulation of
iron in the mitochondria of immature nucleated RBCs in the
bone marrow; the iron forms a ring around the nucleus  these
are called ringed sideroblasts....abnormal.
 The iron accumulation in the mitochondria is the result of
blocks in the protoporphyrin pathway.
SA CONT’D
Lab findings:
 Microcytic/hypochromic red cells, low MCV and MCHC;
variable anemia, low retic.
 RBC inclusions: Basophilic stippling and Pappenheimer bodies
(siderocytes). (May see target cells).
 High serum iron and high serum ferritin (stores); low TIBC and
high % saturation.
 *Decreased transferrin synthesis occurs in iron overload states.
 Bone marrow: ringed syderoblasts (Hall mark of
Sideroblastic Anemia)
 SIDEROBLASTIC ANEMIA (SA)

Blood Bone marrow Bone marrow

Ringed Sideroblast
Pappenheimer bodies Sideroblast

RBC with iron NRBC with iron NRBC with ring of iron
Wright’s stain Prussian blue stain Prussian 47
blue stain
SIDEROBLASTIC ANEMIA (SA)
Blood Blood

Basophilic stippling/stippled RBCs

Pappenheimer bodies
Wright’s stain

Blood
Pappenheimer bodies
Prussian blue iron stain
 SIDEROBLASTIC ANEMIA (SA)

BONE MARROW FINDINGS (IF DONE):

1. *Ringed sideroblasts demonstrated with Prussian
blue stain.
2. Increased stainable iron in macrophages.
Bone marrow 100x Bone marrow 10x

Ringed Sideroblasts Increased stainable iron

Prussian blue iron stain Prussian blue iron stain
TYPES OF SIDEROBLASTIC ANEMIA (SA)
 Blocks in the synthesis of protoporphyrin may be
 primary and irreversible (idiopathic = cause unknown) or
 secondary and reversible
TYPES OF SIDEROBLASTIC ANEMIA CONT’D
 Blocks in the protoporphyrin pathway can be primary or
secondary.
 Primary ‑ cause of protoporphyrin blocks are unknown (can't
identify blocks) and are not reversible....called Idiopathic or
primary Sideroblastic anemia.
1. Elderly, responds to no treatment. Requires transfusion support
if severe anemia.
2. Characterized by a dimorphic red cell population - micro/hypo
red cells with
3. normocytic and/or macrocytic red cells....MCV is variable and
RDW is high.
4. Primary type of sideroblastic anemia is one of myelodysplastic
syndromes called Refractory Anemia with Ringed
Sideroblasts; may terminate in leukemia
SECONDARY TYPES OF SA
 Alcohol inhibits vitamin B6/pyridoxine
 Anti-tuberculosis drugs inhibit vitamin B6

 Lead causes multiple blocks

 Inhaled or ingested
 Abnormal lead level Stippled RBCs – Lead poisoning
 Neurologic problems
 Lead line (gums)
 Chelation therapy

Wright’s stained blood smear

ANEMIA OF CHRONIC DISEASE (ACD)
 Anemia of chronic disease (ACD) – inability to use iron and decreased
response to EPO
 Complex etiology, history important
 Associated conditions
 Persistentinfection (HIV), chronic inflammatory or collagen disorders (RA,
SLE), malignant disease
 Associated with systemic diseases, including
 chronicinflammatory conditions such as Arthritis
 Chronic infections such as TB
 Blood findings
 Microcytic
or normocytic; severity parallels disease
 Very common anemia, #2
 Low serum iron, low TIBC, normal or high serum ferritin
 Treat underlying disorder if possible
 Iron harmful, EPO may help
 ACD PATHOGENESIS
 Lactoferrin is an iron biding protein in the granules of neutrophils
 Its avidity for iron is greater than transferrin

 During infection or Inflammation, neutrophil-lactoferrin released

into plasma, Scavenges available iron
 Bind to macrophage and liver cells (because they have receptor for
lactoferrin)
 Though iron is present in abundance in bone marrow macrophages
its release to developing erythrocyte is slowed
 Cytokines: produced by macrophages during inflammation and
contribute to ACD by inhibiting erythropoiesis
 Inadequate erythropoiesis
LAB DIAGNOSIS
 Early stage: normocytic normochromic
 Late stage: hypochromic microcytic,

 Leukocytosis

 Abundant storage of iron in macrophage (Prusian blue)

THALASSEMIAS
 Inherited decrease in alpha or beta globin chain synthesis
needed for Hgb A; quantitative defect
 All have microcytic/hypochromic RBCs and target cells
 Genetic mutations classified by:
↓ beta chains = beta thalassemia…Greek/Italian
 ↓ alpha chains = alpha thalassemia…Asian

Beta

Alpha

Target cells/Codocytes
56
THALASSEMIAS

 Normal globin chain production is balanced

 Severity ranges from lethal, to severe transfusion-dependency,

to no clinical abnormalities
 Major types - severe, no alpha or no beta chains made
 Minor/trait types – mild, slight ↓ in normal hgb types
THALASSEMIAS

 Impaired alpha or beta

globin synthesis results in an
unbalanced number of
chains produced that leads
to:
 RBC destruction in beta
thalassemia major
 Production of compensatory
hgb types in beta thals
 Formation of unstable or non-
functional hgb types in alpha
thals
BETA THAL MAJOR (HOMOZYGOUS)
 Both beta genes abnormal
 Marked decrease/absence of beta chains leads to alpha chain
excess…no Hgb A is produced
 Rigid RBCs with Heinz bodies destroyed in bone marrow and blood
(ineffective erythropoiesis)

Heinz bodies Excess

alpha chains Supravital
stain
BETA THAL MAJOR (HOMOZYGOUS)
 Clinical findings
 Lab findings
 Severeanemia, target cells, nucleated red cells
 RBC inclusions
 No hemoglobin A; compensatory Hgb F

Target cell

HJB NRBC

Stippled NRBC

Wright’s stained blood smear

BETA THAL MAJOR
(HOMOZYGOUS)
Blood smear Howell-Jolly
body

Target cells
NRBC Pap bodies

Target Transfused
cell RBC Blood smear

Transfused RBC
 Treatment
 Transfusion
 Splenectomy
 Iron chelation
Hypercellular Bone Marrow (10x)
 BETA THAL MINOR (HETEROZYGOUS)
 One abnormal beta gene
 Slight decreased rate of beta
chain production
 Blood picture can look similar
to iron deficiency Stippled RBC

 Lab findings
 Mildanemia, target cells, no
nucRBCs, stippled RBCs
 No Heinz bodies Target cell
 Normal iron tests
Ovalocytes
 Compensates with
Wright’s stained blood smear
Hgb A2
ALPHA THAL MAJOR/HOMOZYGOUS
 Deletion of all 4 alpha genes results in complete absence of
alpha chain production
 No normal hemoglobin types made
 Known as Barts Hydrops Fetalis
 Die of hypoxia….Bart’s hgb
ALPHA THAL INTERMEDIA = HGB H
DISEASE
 Three alpha genes deleted
 Moderate decrease in alpha chains leads to beta chain excess…
unstable Hgb H
 Moderate anemia

Heinz bodies Excess

beta chains Supravital
stain

Target cells

Wright’s stain blood smear

ALPHA THAL MINOR (HETEROZYGOUS)
 One or two alpha genes deleted (group)
 Slightdecrease in alpha chain production
 Mild or no anemia, few target cells
 Essentially normal electrophoresis; many undiagnosed
BETA THALASSEMIAS
ALPHA THALASSEMIAS
DIFFERENTIAL DIAGNOSIS OF MICROCYTIC
ANEMIA

HGB Synthesis Defects

1.3.2. MACROCYTIC NORMOCYTIC
ANEMIAS

 Characterized by elevated MCV & MCH values; normal

MCHC
 Includes Megaloblastic and non-Megaloblastic anemia

Wright’s stained blood smear

LAB INVESTIGATION OF ANEMIA
MEGALOBLASTIC ANEMIA
 Macrocytosis due to a deficiency of vitamin B12 or folic acid
that causes impaired nuclear maturation
 Vitamin B12 & folate are DNA coenzymes necessary for DNA
synthesis and normal nuclear maturation
 Results in megaloblastic maturation…nucleus lags behind the
cytoplasm (asynchrony)
 Both deficiencies cause enlarged fragile cells
 Many cells die in the marrow (ineffective)
 Show a similar blood picture and clinical findings

 Only vitamin B12 deficiency causes neurological symptoms…

required for myelin synthesis
MEGALOBLASTIC ANEMIA
Megaloblastic RBC maturation  macrocytic red
cells

Normoblastic RBC maturation  normocytic red

cells

RBC maturation in microcytic anemias…IDA

Abbott Manual
MEGALOBLASTIC ANEMIA
 Lab findings
 Mild to severe anemia, macrocytic ovalocytes, teardrops,
schistocytes, NRBCs, RBC inclusions
 Hypersegmented neutrophils, giant platelets

Howell-Jolly body

Teardrop
Schistocyte

Blood NRBC Blood

Macrocytic Ovalocytes
MEGALOBLASTIC ANEMIA

Stippled RBC &

Cabot Ring

Giant Platelet
Pap bodies Hypersegmented neutrophil >5
lobes

 Bone marrow shows erythroid hyperplasia and megaloblastic

maturation
 Clinical findings
VITAMIN B12 (COBALAMIN) DEFICIENCY
 Dietary deficiency - rare
 Lack of intrinsic factor
 Pernicious anemia most common
cause
 PA is defined as the absence of IF
and/or the presence of antibodies to
IF or parietal cells; autoimmune
factors
 Low B12 level is followed by
antibody tests
 Total gastrectomy
 May develop B12 and iron
deficiency with macro and micro
red cells…a dual (dimorphic) RBC
population
 Competition
 Malabsorption
FOLATE (FOLIC ACID) DEFICIENCY

 Dietary deficiency – leading

cause, low stores
 Increased need Two RBC populations
 Pregnancy; may develop Dimorphism
concurrent iron deficiency and
a dimorphic population of red
cells with a falsely normal Macrocytic
MCV RBCs
 Malabsorption
 Anti-folate drugs
Microcytic
 Treatment for megaloblastic RBCs
anemia
NON-MEGALOBLASTIC ANEMIA
 Macrocytosis that is NOT due to vitamin B12 or folate
deficiency
 Accelerated erythropoiesis
 Regenerating marrow or marked retic response following recent
blood loss

NRBC

Polychromatophilic RBCs
Wright’s stain
NON-MEGALOBLASTIC ANEMIA

 Liver disease
 Complex & multiple problems
 Degree of anemia varies, round macrocytes

Target cells

Echinocytes
Acanthocytes

Stomatocytes, Alcoholic
DIFFERENTIAL DIAGNOSIS OF
MACROCYTIC ANEMIA

 Megaloblastic and
non-Megaloblastic
 Perform B12 and
folate levels
 Specific morphology

Blood smear
LAB INVESTIGATION OF ANEMIA
Typical peripheral blood values in patients with IDA
TEST MILD IDA MODERATE IDA SEVERE IDA

Hemoglobulin >11 g/dL < 10 g.dL < 10 g/dL

Or in normal range

Hematrocrit >36% < 30 % < 30%

Or in normal range

RBC count May be within May be within Decreased

normal range normal range

MCV May be within Microcytic; < 80 fL Microcytic; < 80 fL

normal range
MCH May be within < 27 pg < 27 pg
normal range
MCHC May be within Normochromic to Hypochromic
normal range hypochromic
1.3.3. NORMOCYTIC/NORMOCHROMIC
ANEMIAS
 Due to increased RBC loss or decreased RBC production
 Majority are hemolytic caused by increased destruction
 Classification of hemolytic anemias
 Mode of transmission
 Hereditary versus acquired
 Type of defect
 Intrinsic, the red cell is abnormal
 Extrinsic, an external agent or trauma destroys red cell

 Site of destruction
 Extravascular or Intravascular
NORMOCYTIC/NORMOCHROMIC
ANEMIAS
 Compensated hemolytic disease
 RBC replacement = RBC destruction
 Anemia does not develop; marrow production keeps up with loss
 Uncompensated hemolytic disease
 RBC destruction > RBC replacement by marrow
 See anemia with high retic but too low to keep up with rate of RBC
loss
NORMOCYTIC/NORMOCHROMIC
HEMOLYTIC ANEMIAS DUE TO INTRINSIC
DEFECTS
• Hereditary hemolytic anemias
– Intrinsic defects of the RBC membrane, hemoglobin
molecule or enzymes decrease lifespan
• Severity depends on rate of hemolysis and the degree of
bone marrow compensatory response
– Increased retic count but loss > production
• Characterized by abnormal RBC destruction tests and
damaged/rigid red cells
TERMS USED IN DESCRIBING
NORMOCYTIC/NORMOCHROMIC
ANEMIAS
 Antigen
 Antibody

 Ig types

 Complement
TERMS USED IN DESCRIBING
NORMOCYTIC/NORMOCHROMIC
ANEMIAS
 C3 induced RBC lysis
 Phagocytosis
 Osmotic lysis

 Fragmentation

 Hemoglobin denaturation
HEMOLYTIC ANEMIA
NORMOCYTIC/NORMOCHROMIC
HEMOLYTIC ANEMIAS DUE TO EXTRINSIC
DEFECTS
 Acquired hemolytic anemias
 Extrinsic defects due to antibodies, trauma, infectious agents,
heat, drugs/chemicals decrease lifespan
 Severity depends on rate of hemolysis and the degree of
bone marrow compensatory response
 Increased retic count but loss > production
 Characterized by abnormal RBC destruction tests and
damaged/rigid red cells
DEFINITIONS FOR IMMUNE ANEMIAS
 Antigens and antibodies
 Antigen(Ag) on RBC membrane
 Antibody (Ab) in serum/plasma; 5 immunoglobulin types
 IgM – cold, room temp; large, can visually see
 IgG – warm, body temp; small, can’t see; need to do DAT

 DAT detects IgG antibody and/or complement that has coated

the red cell membrane in vivo
 Normally, antibodies are not formed against antigens
you possess
 Iso/alloantibody is formed against a foreign (non-self)
antigen after exposure
 Autoantibody is produced against host ‘self’ antigens
ANTIGEN/ANTIBODY REACTIONS

 Most Ag/Ab reactions

require complement
 End result of C3
 Activation is lysis
 If RBC = hemolysis

 Macrophages have IgG &

C3 receptors
WARM AND COLD AUTOANTIBODIES

Spherocytes

Agglutination
WARM AUTOIMMUNE HA (WAIHA)
 Altered immune response causes production of an IgG
autoantibody against ‘self’ RBC antigens
 Antibody attaches to RBC antigen  spherocytes
 Primary (idiopathic) or secondary to disease

Spherocytes & polychromasia

Blood
WARM AUTOIMMUNE HA (WAIHA)
 Lab findings
 Mod to severe anemia, spherocytes, high MCHC
 Erythrophagocytosis
 Looks similar to H spherocytosis but positive DAT
 Treatment
Ingestion of coated RBC
Blood Electron
Microscopy
RBC

Monocyte with ingested RBC

RBC
COLD AUTOIMMUNE HA (CAIHA)

 Altered immune response causes production of an IgM

autoantibody against ‘self’ RBC antigens
 Antibody/C3 attaches to RBC antigen  agglutination
 Primary (idiopathic) or secondary to disease

50x 100x

RBC Agglutination
COLD AUTOIMMUNE HA (CAIHA)
 Few clinical problems unless IgM antibody titers are
high (>1:1000)
 Symptoms of acrocyanosis/Raynaud’s phenomenon
 Lab findings
 Severity varies with seasons….avoid the cold
 IgM antibodies cause RBC agglutination
 Positive Direct Antiglobulin Test (detects complement)

 Problems obtaining valid RBC parameters

 Blood sample must be warmed prior to analysis
HEMOLYTIC TRANSFUSION REACTION
 Incompatible blood transfusion
 Recipient has antibodies to antigens on the donor red cells
received
 Donor cells are destroyed

 ABO worst
 Intravascular hemolysis that is complement-induced lysis…
immediate
 Can be life-threatening
NORMAL NEWBORN BLOOD PICTURE

 High RBC count, Hgb (~20 g/dL) and HCT

 Macrocytic RBCs (MCV ~110 fL); high #retics
 Up to 10 NRBCs/diff

NRBC
Blood
HEMOLYTIC DISEASE OF THE
NEWBORN

 Caused by maternal IgG antibodies directed against baby RBC

antigens
 Antibodies cross placenta and destroy fetal red cells
 HDN due to Rh incompatibility
 Rh negative mother forms Rh antibody after exposure
 HDN due to ABO incompatibility
 Mother’s ABO blood type is O; baby is type A or B

B, C
A, D
HEMOLYTIC ANEMIAS DUE TO TRAUMA
 Fragmentation syndromes…most common finding on smear
are schistocytes; anemia varies
 Types of trauma
 Mechanical…valves/cardiac
 Microangiopathic
(MAHA)…small vessels (DIC, HUS)
 March hemoglobinuria…contact

Schistocytes

Fibrin Strands

RBC

RBC fragmentation on fibrin strands

HEMOLYTIC ANEMIAS DUE TO INFECTIOUS
AGENTS, THERMAL BURNS

 Anemia varies, with severe hemolysis common

 Schistocytes and spherocytes on blood smear
 Parasitize RBC, elaborate lytic toxins or cause direct damage to red
cell membrane

Schistocytes & Spherocytes

HEMOLYTIC ANEMIAS DUE TO
INFECTIOUS AGENTS, THERMAL BURNS
Clostridia (autopsy
specimen)

P. vivax gamete

Malarial ring forms, P.

falciparum

P. falciparum ‘bananas’
LAB INVESTIGATION OF ANEMIA
NORMOCYTIC ANEMIAS DUE TO
MARROW FAILURE

 Impaired cell production and/or pancytopenia

 Normal or decreased retic count
 Not hemolytic, normal RBC destruction tests and damaged red cells
are not evident on blood smear
 Marrow replacement anemia
 Infiltration
or replacement of normal marrow cells
 Immature WBCs (neutrophils) & immature RBCs (nucleated RBCs)
escape from bone marrow
 Leukoerythroblastic blood picture
 May result in extramedullary production
APLASTIC ANEMIA

 Aplastic anemia
 Condition of blood pancytopenia caused
by bone marrow failure…decreased
production of all cell lines
 Due to damaged stem cells, damaged bone
marrow environment or suppression
 No extramedullary hematopoiesis
 Types of aplastic anemia
 Primary/idiopathic
 Secondary/acquired….chemicals drugs,
infections
 Congenital….Fanconi’s
APLASTIC ANEMIA
Normal RBCs
No Platelets
 Blood findings
 Pancytopenia
 Mod to severe anemia
 Hypocellular marrow
Blood
 Complications
 Bleeding 10X
& infection
 Treatment
 Support
 Steroids/Growth factors
 Transplant
Bone marrow, decreased #
precursor cells
NORMOCYTIC/NORMOCHROMIC
ANEMIAS
1 2

Normocytic RBCs & polychromasia Microcytic & hypochromic RBCs

Which blood picture is most consistent with a recent acute

hemorrhage?
1 – Normocytic red cells with a retic response, acute blood
loss
2 – Microcytic red cells, chronic blood loss; iron deficiency
develops
HEMOLYTIC ANEMIA
HEMOLYTIC ANEMIAS DUE TO MEMBRANE
DEFECTS

 Most common is Hereditary

Spherocytosis (HS)
 Membrane defect is
decreased spectrin and
increased permeability of
membrane to sodium ions
 Lab findings
 Anemia varies
 Few to many spherocytes
on
smear, high MCHC
Spherocytes
H SPHEROCYTOSIS
 Treatment for H Spherocytosis is splenectomy
 Any post-splenectomy picture
 Increased poik
 Increased inclusions
 Increased platelets

H Spherocytosis Post-splenectomy

HJBs
Polychromasia

Spherocyte
H OVALOCYTOSIS/ELLIPTOCYTOSIS

 Membrane defect is H Ovalocytosis

polarization of cholesterol or
hemoglobin at ends and
increased sodium
permeability
 Over 25% ovalocytes
 Most asymptomatic
 Mild anemia in 10-15%
Normocytic ovalocytes
HEREDITARY STOMATOCYTOSIS

 Membrane defect is abnormal

permeability to sodium and
potassium
 20-30% stomatocytes on
blood smear
 Group of disorders
 Mild to severe
hemolytic H Stomatocytosis
anemia
H ACANTHOCYTOSIS

 Defect is increased membrane cholesterol due to abnormal

plasma lipids
 Numerous acanthocytes on smear
 Mild anemia
 Also known as abetalipoproteinemia

H Acanthocytosis =
Abetalipoproteinemia
OSMOTIC FRAGILITY TEST (OF)
• Most commonly used to diagnose Hereditary Spherocytosis
– Red cells are placed in hypotonic solutions

Hypotonic
OSMOTIC FRAGILITY TEST (OF)
Spherocytes Target Cells

Decreased Surface:Volume Ratio “Easy Increased Surface:Volume Ratio

to Lyse” “Hard to Lyse”
OSMOTIC FRAGILITY TEST (OF)
HEMOLYTIC ANEMIAS DUE TO ENZYME
DEFECTS

 Inherited enzyme deficiencies that lead to premature RBC

death
HEMOLYTIC ANEMIAS DUE TO ENZYME
DEFECTS

 Pyruvate kinase deficiency

 Mostcommon enzyme deficiency in
EMB pathway PK Deficiency
 G-6-PD deficiency
 Mostcommon enzyme deficiency in
HMP pathway
 PK deficiency Echinocytes
 ↓ATP impairs cation pump
 Severe hemolytic anemia
 Echinocytes
G-6-PD DEFICIENCY
 X-linked inheritance pattern;
malarial belt
 Exposure to oxidants induce Heinz
body formation and RBC
destruction G-6-PD Deficiency
 Primaquine, sulfa drugs, fava beans,
infection
 Unable to protect hemoglobin due to
decreased NADPH
 No clinical problems unless exposed
to oxidants Normal RBCs if no
 Normal G-6-PD variant = 100% exposure to oxidant
enzyme activity
 Africanvariant
 Mediterranean variant
G-6-PD DEFICIENCY
 Blood findings after oxidant exposure:
 Mod to severe anemia
 Schistocytes, spherocytes due to pitting out of Heinz bodies by
spleen
 Enzyme assay

G-6-PD deficiency after G-6-PD deficiency

exposure to oxidant Hemolytic episode

Heinz bodies - denatured hgb Damaged RBCs

Supravital stain Wright’s stain
 Heinz bodies are not visible on a Wright's stained
smear.
 Intravascular rbc destructio occurs....hemoglobinuria.
 After hemolytic crisis, see a reticulocyte response by the
bone marrow.
HEMOLYTIC ANEMIA SUMMARY
1.3.4. NORMOCYTIC ANEMIAS DUE TO
HEMOGLOBINOPATHIES
 Inherited hemoglobin defect with production of structurally
abnormal globin chains; qualititative
 All have target cells
 Beta chain amino acid substitution = variant hgb
 Hgb S = valine substituted for glutamic acid @ 6th of ß
 Hgb C = lysine substituted for glutamic acid @ 6th of ß

Target cells/Codocytes
HEMOGLOBINOPATHIES
 Severity depends on inheritance of
homozygous or heterozygous state
 Homozygous = disease; both beta
chains abnormal
 Heterozygous = trait; one beta chain
is abnormal
Sickle cells
 Hemoglobin S disorders are most
common
 RBCs contain Hgb S  sickle when
oxygen is removed or low pH
 Rigid sickle cells block vessels
leading to organs, increases tissue Target cell
hypoxia Sickle Cell Disease
 Irreversibly sickled forms on blood
smear
HEMOGLOBIN S DISORDERS

 Hemoglobin S disease/Sickle cell

anemia/Hgb SS
 Twosickle cell genes inherited
 Symptomatic by 6 months of age
 Clinical findings Target cell
 Vascularocclusive disease; organ
damage & pain
 Lab findings Sickle cell
 Severe anemia
 Targets, sickle cells
 NRBCs, inclusions
HGB S Disease (Hgb SS)
 No Hgb A, >80% Hgb S, ↑ F
 Treatment
SICKLE CELL ANEMIA

Is the patient’s
increased rate
of RBC
production
keeping up
with RBC loss?

No, high retic

count but
destruction >
production
HEMOGLOBIN S DISORDERS

 Hemoglobin S trait/Sickle cell

trait/Hgb SA
 One sickle cell gene inherited
 Lab findings
Target cells only NO
 Asymptomatic, targets only Sickle cells
 No anemia or sickle cells
 ~60% Hgb A, ~40% Hgb S

 Potential problems if hypoxic

 Surgery, pregnancy…screen for Hgb HGB S Trait (Hgb SA)
S
 Methods for inducing sickling
SICKLE CELLS VS OVALOCYTES &
PENCILS
Sickle cells

Ovalocytes

Sickle Cells

Sickle cells

Pencil forms

Target cell
HEMOGLOBIN C DISORDERS

 Hemoglobin C disease/Hgb CC
 Two C genes inherited C crystals
 Lab findings
 Mild anemia
 Many target cells Target cell

 Intracellular C crystals
 No Hgb A, >90% Hgb C
HGB C Disease (Hgb CC)
 ▪May compensate with hemoglobin
F but < 7%.
HEMOGLOBIN C DISORDERS

 Hemoglobin C trait/Hgb CA HGB C Trait (Hgb CA)

 One C gene inherited
 Lab findings
 Asymptomatic, no anemia
 Targets, no C crystals
 ~60% Hgb A, ~40% Hgb C Target cells only NO
C crystals
HEMOGLOBIN SC DISEASE

 Hemoglobin SC disease/Hgb
SC HGB SC Disease (Hgb S & Hgb C)
 One sickle gene and one C gene
inherited
 Lab findings
 Intermediate in severity between
Hgb SS & SA SC Crystals
 Several target cells
 Many bizarre SC crystals Target cells
 No Hgb A, ~50% Hgb S, ~50%
Hgb C, ↑ F
SC CRYSTALS VS SCHISTOCYTES

Schistocytes

SC Crystals
Hemoglobinopathies Summary

Target cells
SUMMARY OF NORMOCYTIC ANEMIAS
 Large group, the majority are hemolytic
 Retic count
 Anemia with a low retic count and low WBC/PLT counts
suggests marrow failure
 Anemia with a high retic count and evidence of damaged
red cells suggests a hemolytic process
LAB INVESTIGATION OF ANEMIA
1.2.5. ANEMIA DIAGNOSIS/CAUSE
 The cause of anemia is identified with laboratory tests as
well as consideration of patient history and physical
signs/clinical symptoms
 Patient evaluation
 Age, sex, ethnic background, family history
 Nutritional and hydration status, pregnancy
 Exposure to toxins or drugs
 Disease history (eg inflammation)
 Treatment
 Patientsymptoms and anemia severity determine the need for
a blood transfusion
1.2.6. LAB INVESTIGATION OF ANEMIA
 Begins with CBC/FBC
parameters and blood smear
evaluation
 Detects mild (~10 g/dl Hgb)
to
 Severe anemia (<8 g/dl Hgb)
 RBC indices (MCV) are used
to classify anemia
 RBC morphology
abnormalities can be
diagnostic or suggest a
cause that guides further
testing
 WBC & PLT counts are
normal or increased in most
anemias but low in aplastic
anemia

Damaged RBCs
LAB INVESTIGATION OF ANEMIA
 Retic count (absolute)
 Measuresrate of RBC production by the bone marrow
►Helps differentiate normocytic anemias

Reticulocytes

Increased Polychromasia Reticulocytosis New

Wright’s stain Methylene blue (supravital)
LAB INVESTIGATION OF ANEMIA
 Iron tests
►Used to differentiate microcytic anemias or detect iron
overload
 Iron circulates bound to the transport protein transferrin
 Transferrin is normally ~33% saturated with iron
 Includes serum iron, TIBC, serum ferritin
 Serum iron level measures the amount of iron bound to
transferrin
 Total iron binding capacity is an indirect measure of the
amount of transferrin protein in the serum
 Serum ferritin indirectly reflects storage iron in tissues
LAB INVESTIGATION OF ANEMIA
 Transferrin levels are regulated by iron availability
↑ synthesis in iron deficient states…↓ serum iron, ↑ TIBC
 ↓ synthesis in iron overload states…↑ serum iron, ↓ TIBC
 ↓ synthesis in inflammatory states…↓ serum iron, ↓ TIBC
LAB INVESTIGATION OF ANEMIA
 Tests to detect increased RBC destruction
►Useful for haemolytic anemias
 Both normal and increased red cell removal occurs mainly in the
tissues….extravascular destruction
 Intravascular RBC destruction occurs in the blood with release of free
Hgb….haptoglobin binds free Hgb
LAB INVESTIGATION OF ANEMIA

 Presence of increased RBC destruction

 Increased serum bilirubin
 Increased plasma haemoglobin
 Decreased haptoglobin…depleted as it binds free hgb
 Urinalysis:
 Increased urine urobilinogen
 Urine haemoglobin…haemoglobinuria follows depletion of

haptoglobin
 haemosiderinuria

 Increased LD level…LD in red cells is released

LAB INVESTIGATION OF ANEMIA
 Hgb electrophoresis
 Detects and quantitates both normal and abnormal Hgb types
 ►Useful for thalassemias and haemoglobinopathies
 Electrophoresis may be preceded by a screen for the presence of
haemoglobin S
LAB INVESTIGATION OF ANEMIA

 Vitamin B12/Folate levels

►Used to identify the cause of macrocytic anemias
 Direct antiglobulin test (DAT)
 Detectsantibody and/or complement coated red cells
►Useful for immune haemolytic anemias
 Bone marrow exam
 Evaluates# & type of precursor cells; assess iron stores
►Restricted to anemias due to production defects
 Others as indicated
BONE MARROW EXAMINATION

Decreased # Cells
10x Increased # Cells

Hypocellular Marrow
Normocellular Marrow Hypercellular Marrow Aplastic anemia

50x 10x Increased Iron Prussian

Normal Precursor Cells blue stain
LAB INVESTIGATION OF ANEMIA
HEMOLYTIC ANEMIA
EVALUATION ACTIVITY ON :

Recurrent dizziness

 Her anemia is best classified as:

 Microcytic
 Macrocytic
 Normocytic
► Microcytic (& hypochromic)
ACTIVITY EVALUATION:
Recurrent dizziness

 Next step:
 Serum iron, TIBC and ferritin levels
 Vitamin B12 & folate levels
 Retic count and bilirubin level
► Iron tests