ANEMIA And

Its Laboratory Diagnosis

By: Ahmad Sh. Silmi

IUG, Hematologist

asilmi@jugaza.edu.ps

1
 Objectives
At the end of this chapter you will be able to:
• Define anemia
• Discuss the causes and clinical significance of
different categories of anemia
• Describe the classification of anemia
• Explain - Microcytic anemia
- Macrocytic anemia
- Normochromic normocytic anemia
• Discuss the laboratory findings for each category of
anemia
• Perform basic laboratory tests for the diagnosis of2
anemia
 Chapter Outline
3.1. Definition of anemias

3.2. Classification of anemias

3.2.1. Hematologic Response to Anemia
3.2.2. Signs of Accelerated Bone Marrow Erythropoiesis
3.2.3. Physiologic Response to Anemia
3.2.4. Methods of classification
3.2.5. Anemia Diagnosis/Cause
3.2.6. Lab Investigation of Anemia

3.3. Types of anemia

. 3.3.1 microcytic hypochromic anemia
3.3.2. macrocytic normocytic anemias
3.3.3. normocytic anemias
3.3.4. normocytic anemias due to hemoglobinopathies
3
 3.1. Introduction
3.1.1. 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 (Also determined by alteration in
plasma volume)
– ‘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 4
Definition of Anemia cont’d

5
3.1.1. Definition of Anemia cont’d…..

• 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.
6
 3.1.1. Definition of Anemia cont’d…..
• Various diseases and disorders are associated
with decreased hemoglobin levels. These
include:
– Nutritional deficiencies
– External or internal blood loss
– Increased destruction of RBCs
– Ineffective or decreased production of RBCs
– Abnormal hemoglobin synthesis
– Bone marrow suppression by toxins, chemicals, or
radiation & replacement by malignant cells
– Infection
7
3.1.1. 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)
– There is an abnormal hemoglobin with an
increased O2 affinity resulting in an anemia
with normal or raised hemoglobin levels,
hematocrit, or RBC count.

• Generally anemia is not a disease, but rather the

expression of an underlying disorder or disease.

8
3.1.1. Definition of Anemia cont’d…..
• Anemia may develop:
– When RBC loss or destruction exceeds
the maximal capacity of bone marrow
RBC production or
– When bone marrow production is
impaired

9
 3.1.2.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

10
 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. 11
• If demand exceeds maximal bone marrow activity,
RBC production may occur in extramedullary sites,
liver, spleen (hepatosplenomegaly).

12
3.1.3. 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

13
 3.1.4. Clinical features:
• Symptoms of hypoxia: decreased
oxygen delivery to the tissues/organs
causes:
– fatigue , faintness, weakness, dizziness,
headaches, dyspnea, poor exercise
tolerance, leg cramps.

14
 3.1.4.Clinical features cont’d….:

Signs of anemia
• general signs include pallor of mucous
membrane, which occur if the Hgb
concentration is less than 9g/dl,
• specific signs are associated with
particular types of anemia, for
example, jaundice in hemolytic
anemia, leg ulcer in sickle cell anemia

15
 3.1.5. Diagnosis of anemia

• Before making a diagnosis of anemia, one

must consider:
– Age
– Sex
– Geographic location
– Presence or absence of lung disease

16
3.1.5. Diagnosis of anemia cont’d……

• How does one make a clinical

diagnosis of anemia?
A. Patient history
–Dietary habits
–Medication
–Possible exposure to chemicals and/or
toxins
–Description and duration of symptoms

17
3.1.5. Diagnosis of anemia cont’d……
Patient history cont………..
–Tiredness
–Muscle fatigue and weakness
–Headache and vertigo (dizziness)
–Dyspnia (difficult or labored breathing)
from exertion
–G I problems
–Overt signs of blood loss such as
hematuria (blood in urine) or black stools

18
3.1.5. Diagnosis of anemia cont’d….
B. Physical exam
– General findings
• Hepato or splenomegaly
• Heart abnormalities
• Skin pallor
– Specific findings
• In vitamin B12 deficiency there may be signs of
malnutrition and neurological changes
• In iron deficiency there may be severe pallor, a
smooth tongue, and esophageal webs
• In hemolytic anemias there may be jaundice due to
the increased levels of bilirubin from increased RBC
destruction
19
 3.1.5. Diagnosis of anemia cont’d……

C. Lab investigations
1. A complete blood count, CBC
– RBC count
– Hematocrit (Hct) or packed cell volume
– Hemoglobin determination
– RBC indices calculation
– Reticulocyte count
2. Blood smear examination to evaluate:
– Poikilocytosis
– Leukocytes or Platelets abnormalities 20
3.1.5. Diagnosis of anemia cont’d…..

Lab investigation cont’d……

3. A bone marrow smear and biopsy to
observe:
– Maturation of RBC and WBC series
– Ratio of myeloid to erythroid series
– Abundance of iron stores (ringed sideroblasts)
– Presence or absence of granulomas or tumor
cells
– Red to yellow ratio
– Presence of megakaryocytes
21
 3.1.5. Diagnosis of anemia cont’d…..
4. Hemoglobin electrophoresis

22
 3.1.5. Diagnosis of anemia cont’d…..

Lab investigation cont’d……

5. Antiglobulin testing
6. Osmotic fragility test

23
 3.2. Methods of Anemia Classification
• Several schemes of classifying anemias exist
1. Morphologic
– Based on 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
24
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 25
Methods of Classification cont’d
Morphologic Categories of Anemia

1. Normocytic Normochromic anemia

(normal red cell indices)
• Blood loss anemia
• Hemolytic anemia
• Aplastic anemia
• Chronic diseaes
• Renal insufficciency
26
 Methods of Classification cont’d

Morphologic Categories of Anemia

• Microcytic hypochromic
( low red cell indices)
• Iron deficiency anemia
• Sideroblastic anemia
• Lead poisoning
• Thallassemia
• Chronic diseases
27
Methods of Classification cont’d
Morphologic Categories of Anemia
• Macrocytic Normochromic
( high MCV and MCH, normal MCHC)
• Megaloblastic anemia
• Liver disease
• Post splenectomy
• Hypothyroidism
• Stress erythropoiesis
28
 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
29
3 Normocytic/Normochromic
 3.2.2. Pathophysiological classification

1.
A.

B.

2.

30
1. Microcytic- Hypochromic
Anemia

31
 Microcytic- Hypochromic
Anemia
• Many RBCs smaller than
nucleus of normal
lymphocytes
• Increased central pallor.
• Includes
– Iron deficiency anemia
– Thalassemia
– Anemia of chronic disease
– Sideroblastic anemia
– Lead poisoning

32
The Cause of Microcytic-Hypochromic Anemia
Iron Protoporphyrin

• Iron deficiency
• Chronic inflammation • Sideroblastic anemia
or malignant (ACD)

Heme + Globin

• Thalassemia ( or )

Hemoglobin 33
Microcytic/Hypochromic
Anemias
Normoblastic RBC maturation  normocytic red cells
Normal RBC maturation is shown for comparison

RBC maturation in microcytic anemias

Abbott Manual

34
A. 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

• More than half of all anemias are due to iron

deficiency.

35
 Iron Deficiency Anemia (IDA)
• 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)
36
 DIETARY SOURCES OF IRON

Inorganic Iron eg lentils Organic iron eg beef

DAILY IRON REQUIREMENT 10-15mg/day (5-10% absorbed)

37
Daily Iron cycle (Fig)

38
 Estimated daily iron requirements
Units are mg/day

Adult men 0.5-1

Post menopausal female 0.5-1

Menstruating female 1-2

Pregnant female 1.5-3

Children 1.1

Female (age 12-15) 1.6-2.6

39
Iron absorption, Transport and storage

• Iron absorbed from duodenum and jejunum

in the GIT

• Moves via circulation to the bone marrow

• Incorporated with protoporpyirin in

mitochondria of the erythroid precursor to
make Heme

40
• There are three proteins important for
transporting and storage of iron:
–Transferrin,
–Transferrin receptors and
–Ferritin
Transport:
• Transferrin: transports iron from the
plasma to the erythroblasts in the marrow
for erythropoiesis
• The transferrin will bind to transferrin
receptor on the erythrocyte membrane
41
Storage
• Hgb contains about two third of the body iron

• At the end of their life, RBCs are broken down in

the macrophage of reticuloendothelial system and
then iron is released from Hgb enters plasma and
provided to transferrin.

• Some stored in reticuloendothelial cell as ferritin

soluble protein – iron complex) and hemosiderin
(37%) (degraded form of ferritin insoluble)

• iron is also found in muscles as myoglobin and in

other cells as iron containing enzymes
42
 The distribution of body iron

Amount of iron Male Female %

in average adult (g) (g) of total

Hb 2.4 1.7 65

ferritin & hemosiderin 1.0 0.3 30

Myoglobin 0.15 0.12 3.5

Heme enzyme 0.02 0.15 0.5

Transferrin-bound 0.004 0.003 0.1

iron

43
 Iron Deficiency Anemia (IDA)
• Sequence of iron depletion

When iron loss or use exceeds absorption, there is a

sequence of iron depletion in the body:

1. Storage iron decreases/ low serum ferritin; serum

iron & TIBC are normal, no anemia, normal red cells.

2. Serum iron decreases/TIBC increases (increased

transferrin); no anemia, normal red cells.

3. Anemia with microcytic/hypochromic red cells = IDA.

44
 CLINICAL FEATURES IRON DEFICIENCY
• Symptoms eg. fatigue, dizziness, headache
• Signs eg. pallor, Tongue atrophy/ glossitis -
raw and sore, angular cheilosis (Stomatitis)
Angular Cheilosis
or Stomatitis

Koilonychia Glossitis 45
• Clinical signs and symptoms
– Spoon-shaped nails (koilonychia), brittle
nails and hair.

46
 Lab Investigation of IDA

• Iron tests
►Used to differentiate microcytic
hypochromic anemia's 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 47
 Lab Investigation cont’d
• Serum iron level
– measures the amount of iron bound to transferrin
– Does not include the free form of iron

• Total Iron Binding Capacity (TIBC)

– Is an 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)
48
 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.

49
Bone marrow iron (Tissue iron)
• Tissue biopsy of bone marrow
• Prussian blue stain
• Type of iron is hemosiderin

50
ABSENTiron
Absent IRON STORES
stores IN BONE
in bone marrow
MARROW IN IRON DEFICIENCY

Normal control Iron deficiency 51

 Iron Deficiency Anemia

Blood smear

• Lab findings
– Low RBC, Hgb, Hct
– Low MCV, MCH, MCHC
– Normal WBC and PLT
52
 Iron Deficiency Anemia
• RBC morphology
– Hypochromia
– Microcytosis
– Anisocytosis
– Poikilocytosis
• Pencil cells (cigar cells) Blood smear
• Target cells
– no RBC inclusions
• Iron parameters
– Low serum iron,
– High TIBC,
53
– Low serum ferritin
 Iron Deficiency

Ovalocytes - Pencil forms

No RBC inclusions

Wright’s stained blood smear

54
 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.

55
 B. 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.

• Block(s) in protoporphyrin synthesis leads to

iron overload and microcytic/hypochromic
anemia
56
Sideroblastic Anemia (SA)

57
 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.

58
 SA
• Sideroblastic anemia is characterized by the
– Accumulation of iron in the mitochondria of
immature nucleated RBCs in the bone marrow;

– 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.
59
 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) 60
 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 61
blue stain61
 Sideroblastic Anemia (SA)
Blood Blood

Basophilic stippling/stippled RBCs

Pappenheimer bodies
Wright’s stain

Blood
Pappenheimer bodies
Prussian blue iron stain

62
 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 63
 Types of Sideroblastic anemia cont’d
• Primary - cause 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 64
 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
– Neurologic problems Stippled RBCs – Lead poisoning
– Lead line (gums)
– Chelation therapy
(EDTA).
Wright’s stained blood smear 65
66
 C. Anemia of chronic disease
• Anemia of chronic disease (ACD) – inability to use
iron and decreased response to EPO
– Very common anemia, #2

• Associated with systemic disease, including

chronic inflammatory conditions:
–Rheumatoid arthritis
–Chronic renal disease
–Thyroid disease
–Malignancies
–Tuberculosis
–Chronic fungal infections etc
67
 ACD pathogenesis
• Lactoferrin is an iron biding protein in the granules
of neutrophils
• Its avidity for iron is grater than transferrin
• During infection or Inflammation, neutrophil-
lactoferrin released into plasma and Scavenges
available iron
• Bind to macrophage and liver cells (because they
have receptor for lactoferrin

• Cytokines: Produced by macrophages during

inflammation and contribute to ACD by inhibiting
erythropoiesis 68
 Lab Diagnosis
• Blood findings
– Early stage: normocytic normochromic
– Late stage: hypochromic microcytic,
– Low serum iron, low TIBC, normal or
high serum ferritin
• Leukocytosis
• Abundant storage of iron in macrophage
(Prusian blue)
69
 D. 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
70 70
Haemoglobin Molecule
• Consists of 4 globin chains + 4 heme groups
• Normally, each individual inherits 2α, 1β, 1γ,
and 1δ gene from each parent.....so 4α, 2β, 2γ,
and 2δ genes are inherited.

Hgb A = 2α & 2β Hgb A2 = 2α & 2δ Hgb F = 2α & 2γ

97% 2% 1%
71
 Thalassemia
• 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

72
 Thalassemia
• Severity ranges from lethal, to severe
transfusion-dependency, to no clinical
abnormalities; severity depends on the
number and type of abnormal globin genes
inherited.
1.Major  severe anemia; no α (or β) chains
are produced, so cannot make normal
hemoglobin (s).
2.Minor/trait  mild anemia; slight decrease in
normal hemoglobin types made.
73
 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
74
 Beta Thal Major (Homozygous)
• Clinical findings
• Lab findings
– Severe anemia, target cells, nucleated red cells
– RBC inclusions
– No hemoglobin A; compensatory Hgb F
Target cell

HJB NRBC

Stippled NRBC

Wright’s stained blood smear 75

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) 76
 Beta Thal Minor (Heterozygous)
• One abnormal beta gene
– Slight decreased rate of
beta chain production
– Blood picture can look Stippled RBC

similar to iron deficiency

• Lab findings
– Mild anemia, target cells,
Target cell
no nRBCs, stippled RBCs
– No Heinz bodies
Ovalocytes
– Normal iron tests Wright’s stained blood smear

– Compensates with
Hgb A2
77
 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

78
 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

79
 Alpha Thal Minor (Heterozygous)
• One or two alpha genes deleted (group)
– Slight decrease in alpha chain production
– Mild or no anemia, few target cells
– Essentially normal electrophoresis; many
undiagnosed

80
Beta Thalassemias

81
Alpha Thalassemias

82
Differential Diagnosis of Microcytic Anemia

HGB Synthesis Defects

83
2. Macrocytic Normocytic
Anemias

84
Macrocytic Normocytic Anemias
Characteristics ??????

Wright’s stained blood smear 85

A. MEGALOBLASTIC ANEMIA
• Vitamin B12 deficiency
• Folate deficiency
• Abnormal metabolism of folate and vit B12

B. Non megaloblastic anemia

• Liver disease
• Alcoholism
• Post splenoctomy
• Neonatal macrocytosis
• Stress erythropoiesis
86
 A. 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 and leads unbalanced growth
called maturation 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
87
Megaloblastic Anemia
Megaloblastic RBC maturation  macrocytic red cells

Normoblastic RBC maturation  normocytic red cells

RBC maturation in microcytic anemias…IDA

88
 Lab Findings of Megaloblastic
Anemia
Mild to severe anemia,
– Increased MCV & MCH, normal MCHC
– Low RBC, HGB, WBC and PLT counts (fragile
cells) due to ineffective hematopoiesis.
– Low reticulocyte count
– Macrocytic ovalocytes and teardrops;
– Marked anisocytosis and poikilocytosis
– Schistocytes/microcytes - due to RBC breakage
upon leaving the BM
– Erythroid hyperplasia - low M:E ratio (1:1)
– Iron stores increased. 89
 Howell-Jolly body

Teardrop
Schistocyte

Blood NRBC Blood

Macrocytic Ovalocytes

Stippled RBC &

Cabot Ring

Giant Platelet
Pap bodies Hypersegmented Neutrophil >5
lobes
90
Vitamin B12 (Cobalamin) Deficiency

91
 Vitamin B12 deficiency
Occur as a result of one of the following
conditions
1. Nutritional Coballamin deficiency
• Strict vegetarianism
2. Abnormal intragasteric events ( i.e. inadequate
proteolysis of food Coballamin)
• atrophic gastritis.
3. Loss or atrophy of gastric mucosa ( deficient IF)
• total or partial gasterectomy
– May develop B12 and iron deficiency with macro and micro
red cells…a dual (dimorphic) RBC population
• pernicious anemia 92
 Cont…..
4. Abnormal events in the small bowel lumen
– Inadequate pancreatic protease
– Competing agents like fish tape worms (D.latum)
– Disorders of ileac mucosa
– Diminished or absent of IF – Coballamin
receptor
– Drug effects
– Metabolic disorders ( coballamin is not used by
cells)

93
 Folate (Folic acid) Deficiency:
• Deficient intake.
• Increased needs: pregnancy,
infant, rapid cellular
proliferation, and cirrhosis
• Malabsorption (congenital and
drug-induced) Two RBC populations
Dimorphism
• Inherited DNA Synthesis
Disorders: Deficient thiamine
Macrocytic
and factors (e.g. enzymes) RBCs
responsible for folate
metabolism. Microcytic
• Toxins and Drugs: RBCs 94
 1. Pernicious Anemia
• it is defined as anemia resulting from
defective secretion of IF associated with
autoimmune attack on the gastric mucosa
leading to atrophy of the stomach or Abs that
block IF action.

• Abs block the site of IF where vit B12 binds.

• The diagnosis is confirmed by low serum

B12 level and typically abnormal results of
schilling test 95
 Schilling test
• Used to diagnose pernicious anemia and
determine if IF is available.

• If absorbed a portion of oral dose of vit B12

(not used by the body) ---- excreted in
urine

• If not absorbed (malabsorption)…….. Not

detected in urine but pass out in feces 96
Stage I. To diagnose malabsorption
• give the patient a radio active cobalt labeled vit B12
orally and
• Non labeled vit B12 intramuscularly to saturate liver

• If IF is present in the stomach (no other disease) vit

B12 is absorbed and labeled vit B12 detected in
urine

• If absorption is impaired labeled vit B12 not

detected in urine instead in stool

• Diagnose malabsorption if it is not appear in urine

by proceeding stage II. 97
Stage II. Differentiate cause of
malabsorption

• Oral dose labeled vit B12 + IF

• Appear in urine…… pernicious anemia

(give IF)
• If not appear in urine it is other cause of
malabsorption
98
Stage III
• Performed to determine if the patient
suffer from mal absorption of the IF – B12
complex secondary to small intestinal
bacterial overgrowth.

• If this is the condition then tetracycline

should normalize vit B12 absorption.

99
 B. Non-Megaloblastic Anemia
• Macrocytosis that is NOT due to vitamin B12 or
folate deficiency
• Accelerated erythropoiesis
– Regenerating marrow or marked reticulocyte
response following recent blood loss

NRBC

Polychromatophilic RBCs Wright’s stain

100
 Non-Megaloblastic Anemia
• Liver disease and alcoholism
– Complex & multiple problems
– Degree of anemia varies, round macrocytes
– Target cells/acanthocytes - due to abnormal lipid
metabolism.
– Echinocytes are also commonly found on the
smear in liver disease.

Target cells

Echinocytes Stomatocytes, Alcoholic

Acanthocytes

101
 Differential Diagnosis of
Macrocytic Anemia

• Megaloblastic and
non-Megaloblastic
– Perform B12 and
folate levels
– Specific
morphology
Blood smear

102
3. Normocytic Normochromic
Anemia

103
 3. Normocytic Normochromic
Anemia
Is a condition in which the size & Hgb content
of RBCs is normal but the number of RBCs is
decreased.
• It includes
• Aplastic anemia
due to BM failure
• Blood loss anemia
• Hemolytic anemia
104
 A. Aplastic Anemia
– Condition of blood pancytopenia caused by
bone marrow failure…decreased production
of all cell lines and replacement of marrow
with fat.

– Due to damaged stem cells, damaged bone

marrow environment or suppression

– No extramedullary hematopoiesis
105
Types of aplastic anemia

–Primary/idiopathic = 50%

–Secondary/acquired….chemicals,
drugs, infections, radiation = 50%

–Congenital….Fanconi’s
• Aplasia plus dwarfism, skeletal
abnormalities, mental retardation,
abnormal skin pigmentation. 106
 Lab diagnosis of Aplastic Anemia
• Normochromic –Normocytic Normal RBCs
RBC (normal MCV & MCH) No Platelets

• Low reticulocyte count & Hgb

• Pancytopenia
• No abnormal cells
• Hypoplasia Bone marrow Blood

•Normal Serum iron, vitamin 10X

B12 and folate levels

Bone marrow, decreased #

precursor cells 107
 B. Hemolytic anemia
• Result from an increase in the rate of pre
mature red cell destruction.
• Compensated hemolytic disease
• Uncompensated hemolytic disease

• It leads to
– Erythropoietic hyperplasia
– BM produces red cells 6 to 8X the normal rate
– Marked reticulocytosis
108
 Hemolytic anemia

• Two main mechanisms for RBC

destruction in HA
–Intravascular hemolysis: in the
circulation
–Extravascular hemolysis: in RE
system (reticuloendothelial system)
109
 Extravascular hemolysis
Aged RBC 120 day Exstravascularly removed
Abnormal RBC by Macrophage (RES)
in BM, liver and spleen
During destruction RBC releases Hgb
Hgb Protoporphyrin

Unconjugated bilirubin
liver (glucuronic acid)
Iron Globin
reabsorbed conjugated bilirubin
Amino acid gut reabsorbed &
Excreted as urobilin &
110
Protein synthesis urobilinogen
 Extravascular hemolysis

• Lab features
–Increased RBC break down
• Serum bilirubin increase
• Stool urobilinogen increase
• Blood urobilinogen increase
• Urine urobilinogen increase
111
 Intravascular hemolysis
• Red cells are destroyed in blood vessels and
Hgb is released into the circulation:
Free Hgb

Saturates plasma haptoglobin

Excess free Hgb is filtered by the glomerules (kidney)

(if rate of hemolysis saturates renal reabsorption capacity)

Free Hgb enters urine

Fe is released in bladder tubule

Renal tubule loaded with hemosiderin 112

 Intravascular hemolysis
• Lab features
– Hemoglobinemia and hemoglobinuria
– Hemosiderin uria
– Reduced/absent serum haptoglobin

113
 1. Hereditary hemolytic anemia
• This is a congenital hemolytic anemia. some of
which present at birth and other later in life,
while still others may remain silent unless a
physiological stress is super imposed
• Result of intrinsic red cell defects
– Membrane defect (Hereditary Shperocytosis,
Elliptocytosis and sickle cell anemia)
– Metabolic defect : G6PDH and PK defic
– Hgb chain defect (hemoglobinopatheis) :
sickle cell anemia
114
 A. 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 Spherocytes
on smear, high MCHC
– Increased OF test 115
 H Ovalocytosis/Elliptocytosis
• Membrane defect is
H Ovalocytosis
polarization of
cholesterol or
hemoglobin at ends
and increased sodium
permeability
• Over 25% ovalocytes Normocytic ovalocytes
– Most asymptomatic
– Mild anemia in 10-
15%
116
 Hereditary Stomatocytosis
• Membrane defect is
abnormal
permeability to
sodium and
potassium
• Caused by edema
• 20-30%
stomatocytes on H Stomatocytosis
blood smear
– Mild to severe
hemolytic anemia 117
 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
118
 Osmotic Fragility Test (OF)
• Most commonly used to diagnose
Hereditary Spherocytosis
– Red cells are placed in hypotonic solutions

Hypotonic

119
Osmotic Fragility Test (OF)

Decreased Surface:
Volume Ratio “Easy
to Lyse”

Increased Surface:
Volume Ratio
“Hard to Lyse”
120
 B. Defect red cell metabolism
(Enzyme defect)
• G6PD deficiency
– G6PD is the only source of NADPH in red cell
– NADPH is reduced for the production of reduced
Glutathione.
– Hgb and RBC membrane are usually protected
from oxidant stress by reduced glutathione (GSH)
– In G6PD deficiency NADPH and GSH synthesis is
impaired, rendering the red cells vulnerable to
oxidant stress.
– Most individuals with G6PD are asymptomatic
except during oxidant stress resulting from drugs
or other causes.
121
 B. Hemolytic Anemias due to Enzyme
Defects
• Inherited enzyme deficiencies that lead to
premature RBC death

122
 Hemolytic Anemias due to Enzyme
Defects
PK Deficiency
• PK deficiency
– ↓ATP impairs cation pump
– Severe hemolytic anemia
– Echinocytes Echinocytes

• G-6-PD Deficiency
– Unable to protect Hgb due to G-6-PD Deficiency
decreased NADPH
– No clinical problems unless
exposed to oxidants
– Exposure to oxidants induce
Heinz body formation and
RBC destruction
Normal RBCs if no
exposure to oxidant 123
 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

124
Supravital stain Wright’s stain
 C. Normocytic anemias due to
hemoglobinopathies
• Inherited hemoglobin defect with production of structurally
abnormal globin chains;
– 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

125
 Hemoglobin S Disorders
A. Hemoglobin S disease/Sickle cell anemia/Hgb SS

– Two sickle cell genes

inherited (both beta Target cell
chains are abnormal)
– Symptomatic after 6
months of age Sickle cell

• Lab findings
– Severe anemia HGB S Disease (Hgb SS)
– Targets, sickle cells
– NRBCs, inclusions
– No Hgb A, >80% Hgb S,
↑F 126
 Hemoglobin S Disorders
B. 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 HGB S Trait (Hgb SA)

127
 Hemoglobin C Disorders
A. Hemoglobin C disease/Hgb CC
Two C genes inherited (both β chains are
abnormal)
C crystals polymerize differently and look like
blocky Hgb packed rods in the red
cells....intracellular.
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)
– Decreased OF 128
 Hemoglobin C Disorders
B. Hemoglobin C trait/Hgb CA
– One C gene inherited
HGB C Trait (Hgb CA)
• Lab findings
– Asymptomatic, no anemia
– Targets, no C crystals
– ~60% Hgb A, ~40% Hgb C
– Normal Hgb A2 and F Target cells only NO
C crystals

129
 Hemoglobin SC Disease
Hemoglobin SC disease/Hgb SC
• One sickle gene and one C gene inherited
• Double heterozygote- inherit sickle gene (S) from one
parent and C gene from other parent;
• Both β chains are abnormal
• Lab findings
– Intermediate in HGB SC Disease (Hgb S & Hgb C)
severity between Hgb
SS & SA
– Several target cells
– Many SC crystals SC Crystals
– No Hgb A, ~50% Hgb
S, ~50% Hgb C, ↑ F Target cells
130
 2. Acquired hemolytic anemia
• A variety of acquired conditions result in
shortened survival of previously normal
red cells. These include immune mediated
destruction, red cell fragmentation
disorders, acquired membrane defects,
spleen effects

• Result of extrinsic causes

– Immune HA; warm HIHA, cold AIHA
– Drug associated
– Infection associated 131
 Warm Autoimmune HA (WAIHA)
• Altered immune response causes production of an
IgG warm autoantibody against ‘self’ RBC antigens
– Antibody/complement attaches to RBC
antigen…partially phagocytosed (loss of
membrane)  spherocytes
• Cause: Primary (idiopathic) or secondary to
disease
Spherocytes & polychromasia

132
Blood
 Warm Autoimmune HA
(WAIHA)
• Lab findings
– Mod to severe anemia, spherocytes, high MCHC
– Erythrophagocytosis
– Looks similar to H spherocytosis but positive DAT
– Increased OF, bilirubin
– Erythroid hyperplasia
Ingestion of coated RBC
Blood Electron
Microscopy
RBC

Monocyte with ingested RBC RBC 133

 Cold Autoimmune HA (CAIHA)
• Altered immune response causes production of an IgM
cold autoantibody against ‘self’ RBC antigens
– Antibody/C3 attaches to RBC antigen  agglutination
(lysis by complement or macrophage)
• Primary (idiopathic) or secondary to disease

50x 100x

134
RBC Agglutination
 Cold Autoimmune HA
(CAIHA)
• Lab findings
– Agglutination of red cells in
extremities....ears, toes, nose  tissue
damage  gangre
– Severity varies with seasons….avoid the cold
– IgM antibodies cause RBC agglutination
– Reticulocytosis
– Positive Direct Antiglobulin Test (detects
complement) 135
 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
136
 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 Rh
• Sever anemia
• Many nucleated red cells
• HDN due to ABO incompatibility
– Mother’s ABO blood type is O; baby is type A or B
– HDN due to ABO
• Mild, no anemia
• Spherocytosis 137
 Hemolytic Anemias due to Trauma
• Fragmentation syndromes…most common finding on smear
are schistocytes; anemia varies
• Types of trauma
– Mechanical…prosthetic heart valves/cardiac abnormalities
– Microangiopathic (MAHA)…small vessels
(DIC.........bleeding)
– March hemoglobinuria…forceful contact…. Schistocytes

Schistocytes

Fibrin Strands

RBC
138
RBC fragmentation on fibrin strands
 Normocytic/normochromic Hemolytic
Anemias due to Trauma –
Fragmentation Syndromes
A.When RBCs are exposed to excessive trauma
within the cardiovascular system, they may
undergo fragmentation and lysis.
Schistocytes are the most common finding on the
smear; RBC destruction tests are abnormal.
Severe trauma causes intravascular hemolysis.
B.Three types of trauma:
1. Mechanical trauma
A. Prosthetic heart valves or cardiac
abnormalities fragment red cells.
B. Mod to severe anemia with
schistocytes and polychromasia. 139
 Normocytic/normochromic Hemolytic
Anemias due to Trauma –
2. Microangiopathic hemolytic anemia (MAHA) –
trauma occurs in small vessels

A. Disseminated Intravascular Coagulation (DIC) is a

widespread clotting disorder initiated by conditions
such as OB obstetrical) complications or sepsis.
In DIC, clotting factors and platelets form fibrin 
fibrin deposited in the microvessels fragment red
cells. See anemia with schistocytes on smear,
decreased platelets and depletion coagulation
factors  leads to severe bleeding; can be fatal.
. 140
2. Microangiopathic hemolytic anemia
(MAHA) cont’d……..

B. Hemolytic uremic syndrome (HUS) -

most often occurs in children following GI
infection (E. coli); noted for renal failure
 fibrin damages kidney; hemolytic
anemia with echinocytes and
schistocytes, decreased platelets;
often requires dialysis; can be fatal

141
 Normocytic/normochromic Hemolytic
Anemias due to Trauma –
3. March Hemoglobinuria
A. Transient, occurs after forceful contact of
body with hard surfaces....joggers,
soldiers after long march, bongo drum
players.

B. Hemoglobinuria; schistocytes may be

present on smear.

142
 Hemolytic Anemias due to Infectious
Agents, and Thermal Burns
• Anemia varies, with severe hemolysis
– Schistocytes and spherocytes on blood smear
– Parasitize RBC, elaborate lytic toxins or cause
direct damage to red cell membrane
• Malaria fever
• Closteridal infections..release toxins

Schistocytes & Spherocytes 143

 Normocytic/normochromic Hemolytic
Anemia due to Infectious Agents
A. Malarial infection

1. Damage to membrane occurs when parasite

is pitted out of red cell by splenic
macrophages or the entire RBC is removed;
chills and fever as red cells rupture.

2. P. falciparum causes severe hemolysis

called Blackwater fever.

144
 Normocytic/normochromic Hemolytic
Anemia due to Infectious Agents
B. Clostridial infections - Clostridia elaborate
toxins which damage RBC membrane
causing severe intravascular hemolysis.

Both malaria and clostridial infections are

characterized by schistocytes and
spherocytes on the blood smear; other
infectious organisms can cause
hemolysis, e.g. Toxoplasma, Bartonella,
Babesia, Ehrlichia.
145
END OF
ANEMIA
146