Hema II Chapter 3 Anemia in advance hematology

Hematology

1. 1
CHAPTER 3
ANEMIA AND ITS LABORATORY DIAGNOSIS

2. 2
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
-Normocytic anemia
-Macrocytic anemia
•Discuss the laboratory findings for each category of
anemia
•Perform basic laboratory tests for the diagnosis of anemia

3. 3
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 normochromic anemia
3.3.3. normocytic normochromic anemia
3.3.4. normocytic anemias due to hemoglobinopathies

4. 4
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

5. 5
Definition of Anemia cont’d

6. 6
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.

7. 7
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, etc
–Infection

8. 8
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
O
2
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.

9. 9
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
When does anemia develop???

10. 10
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

11. 11
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 reticulocytes.

12. 12
•If demand exceeds maximal bone marrow activity, RBC
production may occur in extramedullary sites, liver and
spleen (hepatosplenomegaly).

13. 13
3.1.3. Physiologic Response to Anemia
•Ability to adapt to anemia depends on:
–Age
–Cardio/pulmonary function
–Rate at which anemia has developed (can
compensate easier if slow onset),
–Underlying disease

14. 14
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.
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, bone deformity in thalassemia major, spoon nails
in IDA, etc

15. 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. 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. 17
3.1.5. Diagnosis of anemia cont’d
–Tiredness
–Muscle fatigue and weakness
–Headache and vertigo (dizziness)
–Dyspnea (difficult or labored breathing) from
exertion
–GI problems
–Overt signs of blood loss such as hematuria
(blood in urine) or black stools
Patient history cont’d

18. 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 B
12
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. 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:
–Red cells, Leukocytes and/or Platelets
abnormalities

20. 20
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. 21
4. Hemoglobin electrophoresis
3.1.5. Diagnosis of anemia cont’d
5. Antiglobulin testing, AHG (Coomb’s test)
6. Osmotic fragility test

22. 22
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
due to disease or lack of essential supplies

23. 23
Methods of Classification cont’d
II. 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
III.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

24. 24
Pathophysiological classification
2
i
1
ii

25. 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 disease

26. 26
2. Microcytic hypochromic ( low red cell indices)
•Iron deficiency anemia
•Sideroblastic anemia
•Lead poisoning
•Thalassemia
•Chronic diseases
Methods of Classification cont’d

27. 27
3, Macrocytic Normochromic ( high MCV and MCH,
normal MCHC)
•Megaloblastic anemia
•Liver disease
•Post splenectomy
•Hypothyroidism
•Stress erythropoiesis
Methods of Classification cont’d

28. 28
1 Microcytic/hypochromic
3
1
2
2 Macrocytic/Normochromic
3 Normocytic/Normochromic
Morphologic Categories of Anemia
N.B. The nucleus of a small
lymphocyte (shown by the
arrow) is used as a reference
to a normal red cell size

29. 29
1. Microcytic- Hypochromic
Anemia

30. 30
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

31. 31
Iron Protoporphyrin
Heme Globin+
Hemoglobin
• Iron deficiency
• Chronic inflammation o
r malignant (ACD)
• Thalassemia ( or )
• Sideroblastic anemia
The Cause of Microcytic-Hypochromic Anemia

32. 32
RBC maturation in microcytic anemias
Normoblastic RBC maturation  normocytic red cells
Abbott Manual
Microcytic/ Hypochromic Anemias
Normal RBC maturation is shown for comparison

33. 33
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.

34. 34
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)

35. 35
DIETARY SOURCES OF IRONDIETARY SOURCES OF IRON
Inorganic Iron eg
lentils
Organic iron eg beef
DAILY IRON REQUIREMENT 10-15mg/day (5-10% absorbed)

36. 36
Daily Iron cycle (Fig)

37. 37
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
Estimated daily iron requirements
Units are mg/day

38. 38
Iron absorption, Transport and storage
•Iron absorbed from duodenum and jejunum in the GIT
•Moves via circulation to the bone marrow
•Incorporated with protoporphyirin in mitochondria of the
erythroid precursor to make Heme

39. 39
•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

40. 40
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 (degraded form of ferritin
insoluble)
•iron is also found in muscles as myoglobin and in other cells as
iron containing enzymes like cytochromes, catalase

41. 41
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 iron 0.004 0.003 0.1
The distribution of body iron

42. 42
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.

43. 43
CLINICAL FEATURES IRON DEFICIENCYCLINICAL FEATURES IRON DEFICIENCY
•Symptoms eg. fatigue, dizziness, headache
•Signs eg. pallor, Tongue atrophy/ glossitis - raw and sore,
angular cheilosis (Stomatitis)
Koilonychia Glossitis
Angular Cheilosis or
Stomatitis

44. 44
•Clinical signs and symptoms
–Spoon shaped nails (koilonychia), brittle and ridged nails
‑

45. 45
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

46. 46
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)

47. 47
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 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.

48. 48
Bone marrow iron (Tissue iron)
•Tissue biopsy of bone marrow
•Prussian blue stain
•Type of iron is hemosiderin

49. 49
ABSENT IRON STORES IN BONE ABSENT IRON STORES IN BONE
MARROW IN IRON DEFICIENCYMARROW IN IRON DEFICIENCY
Iron deficiencyNormal control

50. 50
Iron Deficiency Anemia
•Lab findings
–Low RBC, Hgb, Hct
–Low MCV, MCH, MCHC
–Normal WBC and PLT
Blood smear

51. 51
Iron Deficiency Anemia
•RBC morphology
–Hypochromia
–Microcytosis
–Anisocytosis
–Poikilocytosis
•Pencil cells (cigar cells)
•Target cells
–no RBC inclusions
•Iron parameters
–Low serum iron,
–High TIBC,
–Low serum ferritin
Blood smear

52. 52
Wright’s stained blood smear

Ovalocytes - Pencil forms
No RBC inclusions
Iron Deficiency

53. 53
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.

54. 54
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

55. 55
Sideroblastic Anemia (SA)

56. 56
Terms:
•Siderocytes are mature RBCs in the blood containing iron
granules called Pappenheimer bodies.
•Sideroblasts are immature nucleated RBCs in the bone
marrow containing small amounts of iron in the cytoplasm.

57. 57
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.

58. 58
SA cont’d
Lab findings:
•Microcytic/hypochromic red cells, low MCV and MCHC,
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 sideroblasts (Hall mark of
Sideroblastic Anemia)

59. 5959
RBC with iron
Wright’s stain
NRBC with iron
Prussian blue stain
NRBC with ring of iron
Prussian blue stain
Pappenheimer bodies
Blood Bone marrowBone marrow
Sideroblast
Ringed Sideroblast
Sideroblastic Anemia (SA)

60. 60
Pappenheimer bodies
Wright’s stain
Blood
Basophilic stippling/stippled RBCs
Blood
Pappenheimer bodies
Prussian blue iron stain
Blood
Sideroblastic Anemia (SA)

61. 61
100x
Ringed Sideroblasts
Prussian blue iron stain
Bone marrow 10x
Increased stainable iron
Prussian blue iron stain
Bone marrow
Sideroblastic Anemia (SA)
Bone marrow findings:
1.*Ringed sideroblasts demonstrated with Prussian
blue stain.
2.Increased stainable iron in macrophages.

62. 62
Types of Sideroblastic anemia cont’d
•Primary cause unknown (can't identify blocks) and
‑
are
irreversible....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 normocytic and/or macrocytic red
cells....MCV is variable and RDW is high.
3.Primary type of Sideroblastic anemia is one of
myelodysplastic syndromes called Refractory Anemia with
Ringed Sideroblast (RARS); may terminate in leukemia

63. 63Wright’s stained blood smear
Stippled RBCs – Lead poisoning
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
–Lead line (gums)
–Chelation therapy (EDTA).

64. 64

65. 65
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

66. 66
ACD pathogenesis
•Lactoferrin is an iron binding protein in the granules of
neutrophils
•Its avidity for iron is greater 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

67. 67
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 (Prussian blue)

68. 6868
Target cells/Codocytes
Beta
Alpha
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

69. 69
Haemoglobin Molecule
Hgb A = 2α & 2β Hgb A
2 =
2α & 2δ Hgb F = 2α & 2γ
•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.
97% 2% 1%

70. 70
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

71. 71
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.

72. 72
Heinz bodies Excess alpha
chains Supravital stain
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)

73. 73
Stippled NRBC
NRBC
Target cell
Wright’s stained blood smear
HJB
Beta Thal Major (Homozygous) ….
•Clinical findings
–*Symptomatic by 6 months of age
–Hepatosplenomegaly - due to extramedullary hematopoiesis.
–Jaundice due to high bilirubin levels.
•Lab findings
–Severe anemia, target cells,
nucleated red cells
–RBC inclusions
–No hemoglobin A; compensatory
Hgb F

74. 74
Pap bodiesNRBC
Transfused RBC
Target
cell
Hypercellular Bone Marrow (10x)
Blood smear
Howell-Jolly
body
Target cells
Blood smear
Transfused
RBC
Beta Thal Major (Homozygous)
•Treatment
–Transfusion
–Splenectomy
–Iron chelation

75. 75
Wright’s stained blood smear
Stippled RBC
Target cell
Beta Thal Minor (Heterozygous)
•One abnormal beta gene
–Slight decreased rate of beta
chain production
–Blood picture can look
similar to iron deficiency
•Lab findings
–Mild anemia, target cells, no
nRBCs, stippled RBCs
–No Heinz bodies
–Normal iron tests
–Compensates with
Hgb
A2
Ovalocytes

76. 76
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

77. 77
Target cells
Wright’s stain blood smear
Heinz bodies Excess
beta chains Supravital
stain
Alpha Thal Intermediat = Hgb H
Disease
•Three alpha genes deleted
–Moderate decrease in alpha chains leads to beta
chain excess…unstable Hgb H
–Moderate anemia

78. 78
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

79. 79
Beta Thalassemias

80. 80
Alpha Thalassemias

81. 81
+
HGB Synthesis Defects
Differential Diagnosis of Microcytic Anemia

82. 82
2. Macrocytic Normocytic
Anemias

83. 83
Macrocytic Normocytic Anemias

Wright’s stained blood smear

84. 84
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

85. 85
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 problem
(CNS)because it is also required for myelin synthesis

86. 86
RBC maturation in microcytic anemias…IDA
Normoblastic RBC maturation  normocytic red cells
Megaloblastic RBC maturation  macrocytic red cells
Megaloblastic Anemia

87. 87
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.

88. 88
Macrocytic Ovalocytes
Blood
NRBC Blood
Howell-Jolly body
Teardrop
Schistocyte
Stippled RBC &
Cabot Ring
Giant Platelet
Pap bodies Hypersegmented Neutrophil >5
lobes

89. 89
Vitamin B12 (Cobalamin) Deficiency

90. 90
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

91. 91
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)

92. 92
Folate (Folic acid) Deficiency:
•Deficient intake.
•Increased needs: pregnancy, infant,
rapid cellular proliferation, and
cirrhosis
•Malabsorption (congenital and drug-
induced)
•Inherited DNA Synthesis Disorders:
Deficient thiamine and factors (e.g.
enzymes) responsible for folate
metabolism.
•Toxins and Drugs:
Two RBC populations
Dimorphism
Macrocytic
RBCs
Microcytic
RBCs

93. 93
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

94. 94
Schilling test
•Used to diagnose pernicious anemia and determine if IF
is available.
•If absorbed a portion of oral dose of vit B
12 (not
used by
the body) ---- excreted in urine
•If not absorbed (malabsorption)…….. Not detected in
urine but pass out in feces

95. 95
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.

96. 96
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

97. 97
•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.
Stage III

98. 98
Polychromatophilic RBCs Wright’s stain
NRBC
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

99. 99
Stomatocytes, Alcoholic Stomatocytes, Alcoholic
Echinocytes
Acanthocytes
Target cells
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.

100. 100
Blood smear
Differential Diagnosis of Macrocytic
Anemia
•Megaloblastic and non-
Megaloblastic
–Perform B12 and folate
levels
–Specific morphology

101. 101
3. Normocytic Normochromic
Anemia

102. 102
Normocytic Normochromic Anemia...
•It includes
•Aplastic anemia due to
BM failure
•Blood loss anemia
•Hemolytic anemia
Is a condition in which the size & Hgb content of RBCs is
normal but the number of RBCs is decreased.

103. 103
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 (b/c the ‘injury’ also
affects hematopoietic cells in the liver and spleen so
extramedullary hematopoiesis does NOT occur to
compensate for bone marrow failure.)

104. 104
Types of aplastic anemia
–Primary/idiopathic = 50%
–Secondary/acquired….chemicals, drugs, infections,
radiation = 50%
–Congenital….Fanconi’s syndrome
•Aplasia plus dwarfism, skeletal abnormalities, mental
retardation, abnormal skin pigmentation.

105. 105
Bone marrow, decreased # Bone marrow, decreased #
precursor cellsprecursor cells
10X10X
Normal RBCs
No Platelets
Blood
Lab diagnosis of Aplastic Anemia
• Normochromic –Normocytic
RBC (normal MCV & MCH)
• Low reticulocyte count & Hgb
• Pancytopenia
• No abnormal cells
• Hypoplasia Bone marrow
• Normal Serum iron, vitamin B12
and folate levels

106. 106
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

107. 107
Hemolytic anemia….
•Two main mechanisms for RBC destruction in HA
–Intravascular hemolysis: in the circulation
–Extravascular hemolysis: in RE system
(reticuloendothelial system)

108. 108
Extravascular hemolysis
Aged RBC 120 day
Abnormal RBC
During destruction RBC releases Hgb

Hgb
Exstravascularly removed
by Macrophage (RES)
in BM, liver and spleen
Iron
reabsorbed
Globin
Amino acid

Protein synthesis
Protoporphyrin
Unconjugated bilirubin
liver (glucuronic acid)
conjugated bilirubin
gut reabsorbed &
Excreted as urobilin &
urobilinogen

109. 109
Extravascular hemolysis…
•Lab features
–Increased RBC break down
•Serum bilirubin increase
•Stool stercobilinogen increase
•Blood urobilinogen increase
•Urine urobilinogen increase

110. 110
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

111. 111
Intravascular hemolysis…
•Lab features
–Hemoglobinemia and Hemoglobinuria
–Hemosiderinuria
–Reduced/absent serum haptoglobin

112. 112
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 Spherocytosis,
Elliptocytosis, acanthocytosis & stomatocytosis)
–Enzyme defect : G6PDH and PK deficiency
–Hgb chain defect (hemoglobinopatheis, qualitative
defect ), abnormal structure

113. 113
Spherocytes
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 & H2O
•Lab findings
–Anemia varies
–Few to many
spherocytes
on smear,
high MCHC
–Increased OF test

114. 114
H Ovalocytosis
Normocytic ovalocytes
Ovalocytosis/Elliptocytosis
•Membrane defect is
polarization of cholesterol or
hemoglobin at ends and
increased sodium permeability
•Over 25% ovalocytes
–Most asymptomatic
–Mild anemia in 10-15%

115. 115
H Stomatocytosis
Hereditary Stomatocytosis
•Membrane defect is
abnormal permeability to
sodium and potassium
•Caused by edema
•20-30% stomatocytes on
blood smear
–Mild to severe

hemolytic anemia

116. 116
H Acanthocytosis =
Abetalipoproteinemia
H. Acanthocytosis
•Defect is increased membrane cholesterol due to
abnormal plasma lipids
•Numerous acanthocytes on smear
–Mild anemia
–Also known as abeta lipoproteinemia. [abeta = no beta
lipid transport proteins]

117. 117
Hypotonic
Osmotic Fragility Test (OF)
•Most commonly used to diagnose Hereditary
Spherocytosis
–Red cells are placed in hypotonic solutions

118. 118
Osmotic Fragility Test (OF)
Decreased Surface:
Volume Ratio “Easy
to Lyse”
Increased Surface:
Volume Ratio “Hard
to Lyse”

119. 119
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.
–Inherited enzyme deficiencies that lead to premature
RBC death

120. 120
PK Deficiency
Echinocytes
Hemolytic Anemias due to Enzyme Defects…
•PK deficiency
–↓ATP impairs cation pump
–Severe hemolytic anemia
–Echinocytes
•G-6-PD Deficiency
–Unable to protect Hgb due to
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
G-6-PD Deficiency

121. 121
G-6-PD deficiency after
exposure to oxidant
Heinz bodies - denatured Hgb
Supravital stain
G-6-PD deficiency
Hemolytic episode
Damaged RBCs
Wright’s stain
G-6-PD Deficiency
•Blood findings after oxidant exposure:
–Moderate to severe anemia
–Schistocytes, spherocytes due to pitting out of Heinz
bodies by spleen
•Enzyme assay

122. 122
Target cells/Codocytes
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

123. 123
HGB S Disease (Hgb SS)
Sickle cell
Target cell
Hemoglobin S Disorders
–Two sickle cell genes
inherited (both beta chains
are abnormal)
–Symptomatic after 6 months
of age
•Lab findings
–Severe anemia
–Targets, sickle cells
–NRBCs, inclusions
–No Hgb A, >80% Hgb S, ↑ F
A. Hemoglobin S disease/Sickle cell anemia/Hgb SS

124. 124
Target cells only NO
Sickle cells
HGB S Trait (Hgb SA)
Hemoglobin S Disorders
–One sickle cell gene inherited
•Lab findings
–Asymptomatic, targets only
–No anemia or sickle cells
–~60% Hgb A, ~40% Hgb S
•Potential problems if hypoxic
B. Hemoglobin S trait/Sickle cell trait/Hgb SA

125. 125
C crystals
HGB C Disease (Hgb CC)
Target cell
Hemoglobin C Disorders
•Lab findings
–Mild anemia
–Many target cells
–Intracellular C crystals
–No Hgb A, >90% Hgb C
–Decreased OF
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.

126. 126
HGB C Trait (Hgb CA)
Target cells only NO
C crystals
Hemoglobin C Disorders……….
B. Hemoglobin C trait/Hgb CA
–One C gene inherited
•Lab findings
–Asymptomatic, no anemia
–Targets, no C crystals
–~60% Hgb A, ~40% Hgb C
–Normal Hgb A2 and F

127. 127
SC Crystals
Target cells
HGB SC Disease (Hgb S & Hgb C)
Hemoglobin SC Disease
•Lab findings
–Intermediate in severity
between Hgb SS & SA
–Several target cells
–Many SC crystals
–No Hgb A, ~50% Hgb S,
~50% Hgb C, ↑ F
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

128. 128
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 AIHA, cold AIHA
–Drug associated
–Infection associated

129. 129
Spherocytes & polychromasia
Blood
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)  spherocytosis
•Cause: Primary (idiopathic) or secondary to disease

130. 130
Ingestion of coated RBC
RBC
Electron
Microscopy
Blood
Monocyte with ingested RBC
RBC
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

131. 131
50x
RBC Agglutination
100x
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

132. 132
Cold Autoimmune HA (CAIHA)….
•Lab findings
–Agglutination of red cells in extremities....ears, toes,
nose  tissue damage  gangrene
–Severity varies with seasons….avoid the cold
–IgM antibodies cause RBC agglutination
–Reticulocytosis
–Positive Direct Antiglobulin Test (detects complement)

133. 133
Hemolytic Transfusion Reaction
•Incompatible blood transfusion
–Recipient has antibodies to antigens on the donor red
cells received
–Donor cells are destroyed
•ABO is worst
–Massive intravascular hemolysis that is complement-
induced lysis
–Cells are immediately lysed
–Can be life-threatening

134. 134
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(Erythroblastosis fetalis)
–Rh negative mother forms Rh antibody after exposure
–HDN due to Rh
•Severe 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

135. 135
Schistocytes
Fibrin Strands
RBC
RBC fragmentation on fibrin strands
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

136. 136
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.

137. 137
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. Anemia with schistocytes
on smear, decreased platelets and depletion coagulation
factors  leads to severe bleeding; can be fatal.
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
.

138. 138
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.

139. 139
Schistocytes & Spherocytes
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

140. 140
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.

141. 141
NCNC 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.

142. 142

143. 143

144. 144