1. ANEMIA & Its Laboratory Diagnosis
Dr: Ahmad Sh. Silmi
IUG-Faculty of Health Science

2. Objectives At The End Of This Lecture You Will Be Able To:
Define anemia
Discuss the causes & clinical significance of different categories of anemia
Describe the classification of anemia
Explaining:
Microcytic anemia
Macrocytic anemia
Normochromic normocytic anemia
Discuss the laboratory findings for each category of anemia
Perform basic laboratory tests for the diagnosis of anemia 2

3. Chapter Outline 1. Definition of anemias 2. Classification of anemias
2.1. Hematologic Response to Anemia
2.2. Signs of Accelerated Bone Marrow Erythropoiesis
2.3. Physiologic Response to Anemia
2.4. Methods of classification
2.5. Anemia Diagnosis/Cause
2.6. Lab Investigation of Anemia
3. Types of anemia
3.1 microcytic hypochromic anemia
3.2. macrocytic normocytic anemias
3.3. normocytic anemias
3.4. normocytic anemias due to hemoglobinopathies 3

4. 1. Definition of Anemia True anemia: Pseudo or dilutional 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
Blood volume = RBC mass + plasma volume
An increase in plasma volume may cause a dilutional or pseudo anemia (with low Hgb & HCT values) even though the RBC mass is normal ....can occur during pregnancy or caused by volume overload (IVs), congestive heart failure.
IV=intravenous fluid 4

5. Definition of Anemia cont’d
Discuss relationship of RBC mass and plasma volume in differentiating true from pseudo anemia. 5

6. 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. 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

8. 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.

9. 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

10. 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

11. Signs of Accelerated Bone Marrow Erythropoiesis
The marrow becomes hyper-cellular 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.

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

13. 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.
Physiologic response to anemia: the patient's ability to adapt to the hypoxia of anemia will depend on their age,cardio/pulmonary function, the rate the anemia has developed (can compensate easier if slow onset), and underlying disease.
1. Symptoms - decreased oxygen delivery to the tissues/organs causes fatigue, faintness, weakness, dizziness, headaches, dyspnea, poor exercise tolerance, leg cramps. Physical findings - pallor, rapid pulse, neurologic problems…see table. 13

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

15. 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

16. Diagnosis of anemia
Before making a diagnosis of anemia, one must consider:
Age
Sex
Geographic location

17. 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
Tiredness

18. Patient history cont… 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

19. 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

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

21. Lab investigation cont’d……
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

22. 4. Hemoglobin Electrophoresis
Diagnosis of anemia cont’d…..
4. Hemoglobin Electrophoresis

23. 6. Osmotic Fragility Test
Lab investigation cont’d……
5. Antiglobulin Testing
6. Osmotic Fragility Test

24. Methods of Anemia Classification
Several Schemes Of Classifying Anemias Exist
Morphologic
Based on RBC morphology
Anemia is divided into three groups mainly on the basis of the MCV (RBC indices)
Pathophysiologic
Anemia is divided using three main causes/mechanisms
Impaired erythrocyte formation (Aplastic anemia, IDA, Sidroblastic 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
Anemia classification - No "perfect" classification method so a combination is used.
A. Morphologic ‑ anemias are divided into three groups on the basis of the MCV....uses the RBC indices (RBC size & haemoglobin content).
B. Etiologic ‑ anemias are divided using two main causes/mechanisms.
Two general mechanisms are involved in anemia states:
1. Decreased delivery of red cells to the circulation:
■Anemias due to defective maturation or decreased production.
This group of anemias generally have inappropriately low retic counts  the bone
marrow fails to respond appropriately due to injury, replacement, or lack of essential
haematopoietic factors (such as iron or folic acid).
2. Increased loss of red cells from the circulation:
■Anemias due to acute bleeding or accelerated destruction.
This group of anemias usually have high retic counts  the bone marrow can respond
and haematopoietic factors are sufficient. Anemia in this group results when red cell loss exceeds the bone marrow's capacity to increase its activity.
Note: If bone marrow compensation is adequate, anemia does not develop. 24

25. Methods of Classification cont’d
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
Increased destruction of RBCs (hemolytic anemias)
Anemia results when red cell destruction exceeds the bone marrow’s capacity to increase its activity 25

26. Morphologic Categories of Anemia
Normocytic Normochromic anemia (normal red cell indices)
Blood loss anemia
Hemolytic anemia
Aplastic anemia
Chronic diseaes
Renal insufficciency

27. Morphologic Categories of Anemia
Microcytic hypochromic ( low red cell indices)
Iron deficiency anemia
Sideroblastic anemia
Lead poisoning
Thallassemia
Chronic diseases

28. Morphologic Categories of Anemia
Macrocytic Normochromic ( high MCV and MCH, normal MCHC)
Megaloblastic anemia
Liver disease
Post splenectomy
Hypothyroidism
Stress erythropoiesis

29. Morphologic Categories of Anemia1 2 3
1 Microcytic/hypochromic
2 Normocytic/Normochromic
3 Macrocytic/Normochromic
N.B. The nucleus of a small lymphocyte (shown by the arrow) is used as a reference to a normal red cell size 29

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
Protoporphyrin
Iron
Iron deficiency
Chronic inflammation or malignant (ACD)
Sideroblastic anemia
Heme
Globin +
Thalassemia ( or )
Hemoglobin

33. Microcytic/Hypochromic Anemias
Normal RBC maturation is shown for comparison
RBC maturation in microcytic anemias
Normoblastic RBC maturation  normocytic red cells
Abbott Manual 33

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.
Iron deficiency is that state in which the iron content of the body is inadequate to sustain iron stores or maintain normal levels of iron‑containing pigments (e.g. hemoglobin). Normally, transferrin supplies iron to the developing RBCs in the bone marrow for hemoglobin production. Most is recycled iron. Transferrin levels are regulated by iron availability. *Increased synthesis of transferrin occurs in iron deficient states. When transferrin is <16% saturated with iron (normal is about 33%), the production rate of RBCs and hemoglobin is reduced resulting in anemia with microcytic/hypochromic red cells and low retic count. (Due to lack of iron delivered to the bone marrow). 34

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. Estimated daily iron requirements
Units are mg/day
Adult men
Post menopausal female
Menstruating female
Pregnant female
Children
Female (age 12-15)

38. The distribution of body iron
Amount of iron in average adult Male (g) Female (g) % of total Hb
ferritin & hemosiderin
Myoglobin
Heme enzyme
Transferrin-bound iron

39. 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:
Storage iron decreases/ low serum ferritin; serum iron & TIBC are normal, no anemia, normal red cells.
Serum iron decreases/TIBC increases (increased transferrin); no anemia, normal red cells.
Anemia with microcytic/hypochromic red cells = IDA. 39

40. 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

41. Clinical signs and symptoms
Spoon‑shaped nails (koilonychia), brittle nails and hair.
Bone marrow findings (not usually performed):
1. No storage iron in macrophages - negative Prussian blue iron stain; it is normal to have iron present in the bone marrow.
2.No stainable iron in immature RBCs.
3. RBCs are small with decreased hemoglobin concentration…blue cytoplasm with ragged margins.
Treatment: Identify/treat underlying cause.
1. Oral iron is given; see increased retic count post-therapy.
2. May see dimorphism following treatment…a dual red cell population with older microcytic red cells along with the newly produced normocytic red cells. 41

42. 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
Iron studies - includes serum iron level, TIBC, and serum ferritin; useful to differentiate the microcytic anemias or detect iron overload (repeat transfusions or haemochromatosis).
a. Iron circulates bound to the transport protein transferrin which carries iron to bone marrow red cells for haemoglobin synthesis or to tissue sites (liver, spleen, bone marrow) for storage as ferritin
b. Iron studies:
(1) Serum iron level - measures the amount of iron bound to transferrin; normally about 33% of transferrin binding sites are occupied with iron (% saturation).
(2) Total iron binding capacity (TIBC) - measures the total amount of iron transferrin can bind when fully saturated ; an indirect measure of the amount of transferrin protein.
(3) Serum ferritin level - indirectly reflects storage iron in tissues without doing a bone marrow or tissue biopsy. 42

43. 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)
Iron studies - includes serum iron level, TIBC, and serum ferritin; useful to differentiate the microcytic anemias or detect iron overload (repeat transfusions or haemochromatosis).
a. Iron circulates bound to the transport protein transferrin which carries iron to bone marrow red cells for haemoglobin synthesis or to tissue sites (liver, spleen, bone marrow) for storage as ferritin
b. Iron studies:
(1) Serum iron level - measures the amount of iron bound to transferrin; normally about 33% of transferrin binding sites are occupied with iron (% saturation).
(2) Total iron binding capacity (TIBC) - measures the total amount of iron transferrin can bind when fully saturated ; an indirect measure of the amount of transferrin protein.
(3) Serum ferritin level - indirectly reflects storage iron in tissues without doing a bone marrow or tissue biopsy. 43

44. 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. 44

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

46. ABSENT IRON STORES IN BONE MARROW IN IRON DEFICIENCY
Normal control
Iron deficiency

47. Iron Deficiency Anemia
Lab findings
Low RBC, Hgb, Hct
Low MCV, MCH, MCHC
Normal WBC and PLT
Blood findings in untreated IDA:
1. Mild to severe anemia (11 to 5 g/dl Hgb); microcytic/hypochromic RBCs.
2. Low RBC indices (MCV fl; MCH < 27 pg; MCHC <32%).
3. Aniso with high RDW; ovalocytes/pencil cells, may see target cells, normal or elevated platelet count.
4. No basophilic stippling - if present, rules out IDA and helps differentiate IDA from thalassemia minor. No pappenheimer bodies (composed of iron) are seen in IDA.
5. Low retic count – red cell production is restricted by lack of iron.
6. Low serum iron, high TIBC, low serum ferritin (stores), low % saturation of transferrin. *Increased transferrin synthesis occurs in iron deficient states.
Clinical symptoms (due to decreased tissue iron):
1. Pica – cravings for ice, dirt, laundry starch, clay
2. Tongue atrophy/glossitis - raw and sore.
3. Spoon‑shaped nails, brittle nails and hair.
4. Numbness and tingling.
Blood smear 47

48. 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
Blood findings in untreated IDA:
1. Mild to severe anemia (11 to 5 g/dl Hgb); microcytic/hypochromic RBCs.
2. Low RBC indices (MCV fl; MCH < 27 pg; MCHC <32%).
3. Aniso with high RDW; ovalocytes/pencil cells, may see target cells, normal or elevated platelet count.
4. No basophilic stippling - if present, rules out IDA and helps differentiate IDA from thalassemia minor. No pappenheimer bodies (composed of iron) are seen in IDA.
5. Low retic count – red cell production is restricted by lack of iron.
6. Low serum iron, high TIBC, low serum ferritin (stores), low % saturation of transferrin. *Increased transferrin synthesis occurs in iron deficient states.
Clinical symptoms (due to decreased tissue iron):
1. Pica – cravings for ice, dirt, laundry starch, clay
2. Tongue atrophy/glossitis - raw and sore.
3. Spoon‑shaped nails, brittle nails and hair.
4. Numbness and tingling. 48

49. Ovalocytes - Pencil forms No RBC inclusions
Iron Deficiency
Wright’s stained blood smear
Ovalocytes - Pencil forms No RBC inclusions
Bone marrow findings (not usually performed):
1. No storage iron in macrophages - negative Prussian blue iron stain; it is normal to have iron present in the bone marrow.
2.No stainable iron in immature RBCs.
3. RBCs are small with decreased hemoglobin concentration…blue cytoplasm with ragged margins.
Treatment: Identify/treat underlying cause.
1. Oral iron is given; see increased retic count post-therapy.
2. May see dimorphism following treatment…a dual red cell population with older microcytic red cells along with the newly produced normocytic red cells. 49

50. B. Sideroblastic Anemia (SA)
This group of anemias are characterized by:
Defective protoporphyrin synthesis (blocks) resulting in iron loading
A hypochromic anemia due to deficient hemoglobin synthesis.
Block(s) in protoporphyrin synthesis leads to iron overload and microcytic/hypochromic anemia 50

51. 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.

52. Sideroblastic Anemia (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.

53. Sideroblastic Anemia (SA)
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.
Decreased transferrin synthesis occurs in iron overload states.
Bone marrow: ringed sideroblasts (Hall mark of Sideroblastic Anemia)

54. Sideroblastic Anemia (SA)
Blood
Bone marrow
Bone marrow
Instructor note: explain by comparing the diagram (upper part) with stained preparations (lower part). Be aware that the diagram at the top is similar with the stained preparation at the bottom
Ringed Sideroblast
Sideroblast
Pappenheimer bodies
RBC with iron Wright’s stain
NRBC with iron Prussian blue stain
NRBC with ring of iron Prussian blue stain 54 54

55. Basophilic stippling/stippled RBCs
Sideroblastic Anemia (SA)
Basophilic stippling/stippled RBCs
Blood
Pappenheimer bodies Wright’s stain
Blood
Pappenheimer bodies Prussian blue iron stain
Blood 55

56. Bone marrow findings (if done):
Ringed sideroblasts demonstrated with Prussian blue stain.
Increased stainable iron in macrophages.
100x
Ringed Sideroblasts Prussian blue iron stain
Bone marrow
10x
Increased stainable iron Prussian blue iron stain
Bone marrow 56

57. C. Anemia of chronic disease
Anemia of chronic disease (ACD) – inability to use iron and decreased response to EPO
Very common anemia
Associated with systemic disease, including chronic inflammatory conditions:
Rheumatoid arthritis
Chronic renal disease
Thyroid disease
Malignancies
Tuberculosis
Chronic fungal infections etc

58. 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 Hunts 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

59. Lab Diagnosis Blood findings Early stage: normocytic normochromic.
Late stage: hypochromic microcytic.
Leukocytosis
Abundant storage of iron in macrophage (Prussian blue)

60. Target cells/Codocytes
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
Target cells/Codocytes
Beta
Alpha
Thalassemias - Quantitative defect
Group of inherited disorders in which globin chains of normal structure are produced but the production rate of one type of chain is diminished....● decreased globin chain synthesis. All thalassemias are characterized by microcytic/hypochromic rbc's and target cells.
NOTE: Hemoglobinopathies - presence of variant hemoglobins (e.g. Hgb S) due to production of globin chains with abnormal structure…..a qualitative defect.
B. Thalassemias are genetic mutations classified according to the particular globin chain whose synthesis is suppressed.
1. Impaired beta chain synthesis = Beta thalassemias and impaired alpha chain synthesis = Alpha thalassemias
2. Occurs in Greek/Italian (beta), Asian (alpha) and African (both) ancestries. 60 60

61. 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 A2 = 2α & 2δ
Hgb F = 2α & 2γ
Hgb A = 2α & 2β
97% 1% 2%

62. 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 62

63. 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.
Major  severe anemia; no α (or β) chains are produced, so cannot make normal hemoglobin (s).
Minor/trait  mild anemia; slight decrease in normal hemoglobin types made.
The impaired alpha or beta globin synthesis results in an unbalanced number of chains produced which leads to:
1. RBC destruction in beta thalassemia major - lack of beta chains results in excess alpha chains that precipitate causing rigid red cells. (NOT beta thalassemia minor condition).
2. Compensatory hemoglobins in beta thalassemias - increased hemoglobins F or A2 are produced to compensate for deficient beta chain synthesis.
3. Unstable or non-functional hemoglobins in alpha thalassemias – when alpha chain production is absent or decreased, excess beta and/or gamma chains accumulate to form:
a. Hemoglobin H - beta4 - unstable; precipitates in red cells.
b. Bart's hemoglobin - gamma4 – useless, won't release oxygen.

64. 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 thalassemias - Abnormal beta gene produces abnormal mRNA = deficient β chain production.
Occurs in Mediterranean area - Greeks, Italians.
A.Beta thalassemia major (homozygous) = (Dr.) Cooley's anemia. Both beta genes inherited are abnormal....parents have β thalassemia minor.
1. Marked decrease or **absence of β chain production results in an α chain excess. Little or no hemoglobin A is produced.
a. Excess α chains combine and precipitate on surface of the RBC membrane  Heinz bodies cause membrane damage and rigidity = massive RBC destruction occurs.
b. Many of these abnormal RBCs (70-80%) are destroyed in the bone marrow…....called ineffective erythropoiesis. Cells that are released into blood show pitting and fragmentation…the spleen removes these rigid RBCs.
c. RBC destruction results in increased erythropoiesis in the bone marrow and extramedullary sites to compensate and causes increased RBC breakdown products and iron accumulation in tissues. 64

65. Beta Thal Major (Homozygous)
CLINICAL FINDINGS
Lab Findings
Severe anemia, target cells, nucleated red cells
RBC inclusions
No hemoglobin A; compensatory Hgb F
Stippled NRBC
NRBC
Target cell
Wright’s stained blood smear
HJB
Clinical findings:
a. *Symptomatic by 6 months of age.
b. Hepatosplenomegaly - due to extramedullary hematopoiesis.
c. Mongoloid appearance, stunted growth; bone marrow in skull expands and causes ‘hair on end’ effect on X-ray.
d. Iron overload causes organ failure, e.g. cardiac - high iron is due to the ongoing RBC destruction and multiple blood transfusions that are required.
e. Jaundice due to high bilirubin levels.
Lab findings:
a. SEVERE anemia - Hgb g/dl.
b. Mk micro/hypo red cells; mk aniso/poik; MCV fl.
c. Targets, bizarre/fragmented rbc's, teardrops. Nuc rbc's or more.
d. Normal red cells in circulation are transfused red cells (dimorphic population)
e. Basophilic stippling, Howell‑Jolly bodies, Pappenheimer bodies, Heinz bodies (composed of unused α chains)
f. Increased serum iron and iron stores, low TIBC; elevated bilirubin level.
g. Retics are increased but not high enough to replace destroyed rbcs....the bone marrow can't keep up.
h. Hemoglobin electrophoresis: ▪NO hemoglobin A; Hemoglobin A2 varies. ▪Compensates with hemoglobin F up to 90%  reactivates gamma chain production (fetal life). 65

66. Beta Thal Major (Homozygous)
Pap bodies
NRBC
Transfused RBC
Target cell
Blood smear
Howell-Jolly body
Target cells
Blood smear
Transfused RBC
Hypercellular Bone Marrow (10x)
Treatment
Transfusion
Splenectomy
Iron chelation
Bone marrow:
Hypercellular due to ↑ RBC precursors; low M:E ratio; increased storage iron.
Treatment:
Used to rarely reach adulthood but therapy has improved.
a. Transfusion dependent (causes dimorphic blood picture).
b. Splenectomy (~4yo) – to keep rigid red cells circulating longer.
c. Iron chelating agents given to help iron overload problem. 66

67. 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
Wright’s stained blood smear
Stippled RBC
Target cell
Beta thalassemia minor (heterozygous)
One β gene inherited is abnormal, other gene is normal.
1. **Deficient rate of β chain synthesis - often discovered by accident; easily confused with IDA but normal iron tests.
2. Lab findings:
a. MILD anemia - Hgb g/dL, often with a high RBC count.
b. Micro/hypo red cells and target cells; may have ovalocytes; NO nucleated red cells; normal RDW.
c. Heinz bodies are not a problem as in beta major; the few excess alpha chains are removed during rbc maturation.
d. Basophilic stippling often seen - *not present in IDA.
e. Iron studies are usually normal (so no pappenheimer bodies).
f. Hgb electrophoresis:
▪Slight decreased hemoglobin A; Hemoglobin F varies.
▪Compensates with Hgb A2 3-7% (mild anemia so usually not F).
Ovalocytes 67

68. 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 thalassemias - Due to deletion of varying # of alpha genes = decreased α chain production. Occurs mainly in Asian ancestries.
A. Barts Hydrops Fetalis = Alpha thalassemia major (homozygous)
All four alpha genes are deleted (--/--); normal (αα/αα).
1. Complete **absence of α chain production.
a. No normal hemoglobins produced. SEVERE anemia.
b. Incompatible with life ‑ die in utero or shortly after birth....hydrops fetalis.
2. Fetus produces Barts hemoglobin composed of γ4 (~ 80%) which is useless as oxygen carrier; fetus dies of hypoxia. 68

69. 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
Hgb H disease = Alpha thalassemia intermedia
Deletion of three alpha genes, one normal gene (--/-α).
1. **Moderate decrease in α chains yields β chain excess.
a. Produces Hemoglobin H - composed of β4, an unstable hemoglobin which precipitates in red cells  Heinz bodies.
b. Rigid red cells with Heinz bodies are destroyed in spleen.
Lab findings:
a. MODERATE anemia - Hgb 8-9 g/dL, micro/hypo rbcs.
b. Targets, basophilic stippling; chronic lifelong anemia.
c. Hemoglobin electrophoresis:
▪Decreased hemoglobin A ~80%
▪Hemoglobin H 4-30%; Bart's hgb in cord blood. 69

70. 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
Alpha thalassemia trait/minor
Deletion of 1 or 2 alpha genes, 2 or 3 normal genes.
1. **Slt decrease in α chain production - hard to diagnose but few, if any, problems.
2. Slight if any anemia; micro/hypo red cells with few targets.
Hemoglobin electrophoresis:
▪Essentially normal - Slight decreased hemoglobin A; normal A2 and F.
▪Hemoglobins H and Bart's detected in cord blood only. 70

71. Beta Thalassemias
Beta thal major – severe anemia, NRBCs; beta thal minor – mild, usually no nucRBCs 71

72. Alpha Thalassemias
Alpha thal major – severe; alpha thal intermedia – moderate anemia; alpha thal minor - mild 72

73. Differential Diagnosis of Microcytic Anemia+
HGB Synthesis Defects
Review defects in hemoglobin production; cannot rely on target cells or ovalocytes/pencils to differentiate; the presence of RBC inclusions is helpful 73

74. Distinguishing features between iron def (IDA) and thalassemia
Mentzer index: MCV/RBC <13 favors thalassemia
The RBC count in thalassemia is more than 5.0 x 106/μL (5.0 x /L) and in IDA is less than 5.0 x 106/μL (5.0 x 1012/L)
MCV usually less than 70 in TT, more than 70 in IDA
The red cell distribution width (RDW) in IDA is more than 17% and in TT is less than 17%.

75. 2. Macrocytic Normocytic Anemias75

76. Macrocytic Normocytic Anemias
Wright’s stained blood smear

77. 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

78. 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
Megaloblastic anemias –are due to vitamin B12 OR folate deficiency which causes impaired nuclear maturation.
DNA synthesis is defective causing abnormal nuclear maturation, while RNA synthesis is normal so maturation of the cytoplasm is not affected.
This leads to a state of unbalanced growth, called maturation asynchrony. Because the nucleus lags behind the cytoplasm.
Cytoplasmic components, especially hemoglobin, are synthesized in excessive amounts during the delay between cell divisions.
A decreased number of cell divisions occur resulting in macrocytic red cells. An enlarged and fragile cell is the final product.
All hematopoietic cells can exhibit the maturation defect. The cells produced are fragile and many die in the bone marrow (particularly the red cells)….ineffective hematopoiesis.
Vitamin B12 (cobalamin) and folic acid (folate) are two essential coenzymes necessary for normal DNA synthesis and are almost always the cause of the Megaloblastic anemias.
Vitamin B12 is also needed for normal myelin synthesis so this deficiency can impair the central nervous system (CNS). 78

79. Megaloblastic Anemia
RBC maturation in microcytic anemias…IDA
Normoblastic RBC maturation  normocytic red cells
Megaloblastic RBC maturation  macrocytic red cells 79

80. Megaloblastic Anemia Lab Findings 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.
Lab findings in vitamin B12 or folate deficiency:
Mild to SEVERE anemia; MCV fl; increased MCH, normal MCHC. Low RBC and HGB values with mildly decreased WBC and PLT counts (fragile cells) due to ineffective hematopoiesis.
Low retic - due to high RBC death in bone marrow and blood.
Macrocytic ovalocytes and teardrops; marked aniso and poik is typical.
Schistocytes/microcytes - due to RBC breakage upon leaving the bone marrow.
Advanced anemia: multiple Howell-Jolly bodies, nucRBCs, basophilic stippling, pappenheimer bodies, Cabot rings.
Megaloblastic anemias are noted for markedly increased LD levels and high bilirubin & iron levels due to destruction of fragile red cells in the bone marrow and blood.
Hypersegmented neutrophils (>5 lobes) - 1st change to appear and last to disappear. (Normal segmented neutrophils have 2-5 lobes; hyperseg neutrophils have 6 lobes).
May also see giant band neutrophils as well as giant platelets. 80

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

82. Vitamin B12 deficiency
Occur as a result of one of the following conditions
Nutritional Coballamin deficiency
Abnormal intragasteric events ( i.e. inadequate proteolysis of food Coballamin)
Loss or atrophy of gastric mucosa ( deficient IF)
Abnormal events in the small bowel lumen
Inadequate pancreatic protease
Competing agents like fish tape worms

83. 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.
Two RBC populations Dimorphism
Macrocytic RBCs
Microcytic RBCs
Folic Acid deficiency:(Low serum and RBC folate levels) Dietary deficiency - the leading cause of folate deficiency; develops quickly (2-4 mos) due to low stores as compared to vitamin B12.
Poverty/customs in tropical areas.
Alcoholic cirrhosis - due to poor diet and alcohol is a folate antagonist; a frequent problem in alcoholics.
Increased folate requirements
Pregnancy - very common; may have concurrent iron deficiency which causes a dimorphic red cell picture with micro and macro RBCs produced…MCV is unreliable (falsely normal) with high RDW.
Infancy - rapid growth; heated milk destroys folate.
Disorders associated with rapid proliferation – high folate use by rapidly dividing cells; leukemias and severe hemolytic anemias like sickle cell disease.
Malabsorption syndromes
Non‑tropical sprue - wasting disorder.
Celiac disease of children - atrophy of intestine.
Dilantin - anti-convulant drug which competes for folate absorption.
Regional enteritis - bowel inflammation
Drug induced - impaired use of folic acid is often seen in cancer/leukemia patients receiving chemotherapeutic anti- folate drugs, e.g. Methotrexate.
Treatment for megaloblastic anemia:
1.Give correct treatment....B12 (intramuscular) or folate (oral).
2.Following therapy - see a marked retic response. May see dimorphism/dual red cell population with old macrocytic red cells and newly released normal red cells. Hypersegmented neutrophils will disappear last.

84. 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

85. 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

86. 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
Polychromatophilic RBCs Wright’s stain
NRBC
Non‑megaloblastic macrocytic anemia
Accelerated erythropoietin - a regenerating marrow or marked response following red cell destruction or hemorrhage causes:
Release of bone marrow retics into the blood; a very high number of retics in circulation causes:
An increased MCV (since retics are slightly larger than mature red cells). 86

87. 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.
Echinocytes
Acanthocytes
Target cells
Liver disease (including alcoholics) - complex and multiple problems:
Variable anemia, often macrocytic with round macrocytes.
Target cells/acanthocytes - due to abnormal lipid metabolism. Echinocytes are also commonly found on the smear in liver disease.
Iron deficiency can develop due to chronic blood loss or poor diet.
Folate deficiency due to poor diet and/or alcohol abuse.
Sideroblastic anemia or stomatocytes due to alcohol.
Coagulation abnormalities due to decreased production of hemostatic proteins...the liver produces almost all factors including thrombopoietin.
Splenomegaly in cirrhosis may cause low number of platelets (hypersplenism).
Note: the high bilirubin levels in patients with end-stage liver disease may interfere with the photometric measurement of hemoglobin.
Stomatocytes, Alcoholic 87

88. Differential Diagnosis of Macrocytic Anemia
Blood smear
Megaloblastic and non- Megaloblastic
Perform B12 and folate levels
Specific morphology
Differential diagnosis of Macrocytic anemias: The macrocytic anemias are differentiated into Megaloblastic and non-megaloblastic by performing vitamin B12 and folic acid levels.
Specific morphologic findings and history provide additional clues.
Macrocytic ovalocytes and hyper segmented neutrophils are key findings indicative of megaloblastic anemia.
It is very important to identify the specific cause of a megaloblastic anemia due to the CNS damage associated with Vitamin B12 deficiency. 88

89. 3. Normocytic Normochromic Anemia

90. 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

91. 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
Aplastic Anemia [no growth]
Defined as a condition of blood pancytopenia due to primary bone marrow failure....causes ●decreased production of ALL cell lines.
2. Bone marrow failure may be due to:
a. damage to the bone marrow stem cells.
b. damage to the bone marrow microenvironment so unsatisfactory for cell growth.
c. bone marrow suppression by immune mechanisms.
3. The ‘injury’ also affects hematopoietic cells in the liver and spleen so extramedullary hematopoiesis does NOT occur to compensate for bone marrow failure.
4. Types of Aplastic anemia:
a. Primary/Idiopathic ‑ 50%, no cause of injury identified.
b. Secondary/Acquired ‑ 50%
(1) Chemical or physical agents - benzene, radiation, insecticides
(2) Drugs - chloramphenicol, sulfonamides
(3) Infections - viral
c. Congenital/hereditary ‑ Fanconi's syndrome
(1) Aplasia plus dwarfism, skeletal abnormalities, mental retardation, abnormal skin pigmentation. High association with leukemia development. 91

92. 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.

93. Lab diagnosis of Aplastic Anemia
Normal RBCs No Platelets
Blood
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
Bone marrow, decreased # precursor cells
10X
Peripheral blood findings:
a. Pancytopenia...low WBC, low RBC/HGB, low PLT.
b. Mod-severe anemia with normocytic or slight macro rbcs.
c. NO evidence of damaged red cells on blood smear.
d. No nucRBCs, no inclusions; low retic/no polychromasia.
e. No RBC breakdown/destruction products.
f. Normal or increased iron – a result of repeated blood transfusions and iron not used for hemoglobin production.
6. Bone marrow:
a. Hypocellular - < 30% cellularity (decreased cells, increased fat).
b. M:E ratio ~3:1 - no change since all cell precursors are decreased in number. Marrow storage iron may be high (since not being used).
7. Complications:
a. Bleeding risk - due to low # of platelets.
b. Infection risk - due to low # of neutrophils.
8. Treatment:
a. Support - red cell and platelet transfusions, antibiotics.
b. Remove toxic agent if identified.
c. Steroids, growth factors to stimulate hematopoiesis. GM-CSF acts to stimulate production of neutrophils, eosinophils, monocytes, erythrocytes and platelets.
d. Bone marrow transplant; some success with immunosuppressive therapy.
9. Prognosis is poor - die of infection/cerebral bleeds. May develop acute leukemia. 93

94. 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

95. Intravascular hemolysis: in the circulation
B. Hemolytic anemia
Two main mechanisms for RBC destruction in HA
Intravascular hemolysis: in the circulation
Extravascular hemolysis: in RE system (reticuloendothelial system)

96. Extravascular hemolysis
Aged RBC 120 day
Abnormal RBC
During destruction RBC releases Hgb
Hgb
Exstravascularly removed
by Macrophage (RES)
in BM, liver and spleen
Protoporphyrin
Unconjugated bilirubin
liver (glucuronic acid)
conjugated bilirubin
gut reabsorbed &
Excreted as urobilin & urobilinogen
Iron
reabsorbed
Globin
Amino acid
Protein synthesis

97. Extravascular hemolysis
Lab Features
Increased RBC break down
Serum bilirubin increase
Stool urobilinogen increase
Blood urobilinogen increase
Urine urobilinogen increase

98. 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

99. Intravascular hemolysis
Lab Features
Hemoglobinemia and hemoglobinuria
Hemosiderin uria
Reduced/absent serum haptoglobin

100. 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

101. 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 on smear, high MCHC
Increased OF test
Spherocytes
Normocytic/Normochromic Hemolytic anemias due to Membrane Defects
Problems with membrane loss/↓ flexibility/abnormal permeability
A. Hereditary Spherocytosis ‑ inherited autosomal dominant; most common membrane defect.
1. Characterized by variable degree of anemia, splenomegaly, and spherocytes on the blood smear....hallmark finding.
2. Defect – decreased spectrin & increased permeability of rbc membrane to sodium ions, results in increased glycolysis to pump sodium out of the RBC and loss of membrane fragments → rigid spherocytes are trapped/removed in the spleen.
3. Lab findings:
a. Hgb varies; 9‑12 to 3‑4 g/dl in a crisis.
b. Few to many spherocytes; MCV normal or slightly reduced; MCHC may be >36%. HS is still classified as a normocytic/normochromic anemia.
c. Increased retics/polychromasia.
d. Increased Osmotic Fragility test.
e. Increased bilirubin...jaundice.
f. Negative direct antiglobulin test (DAT) - no antibody is involved.
Bone marrow shows erythroid hyperplasia (increased RBC precursors).
101

102. H. 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%
H Ovalocytosis
Normocytic ovalocytes
Hereditary Elliptocytosis/Ovalocytosis ‑ autosomal dominant.
1. Characterized by greater than 25% ovalocytes on the smear.
2. Membrane defect - polarization of cholesterol or hemoglobin at ends rather than around pallor and increased sodium permeability. Abnormal shape occurs in the mature RBC.
3. Most persons are asymptomatic with no anemia due to normal RBC lifespan. Normal Osmotic Fragility test.
‑15% develop a mild hemolytic anemia due to decreased RBC lifespan. Increased Osmotic Fragility test, increased retics. May treat with splenectomy if severe. [May be related to Hereditary pyropoikilocytosis.]
102

103. 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
H Stomatocytosis
Hereditary Stomatocytosis ‑ group of inherited types.
1. Characterized by 20‑30% stomatocytes on the smear.
2. Membrane defect - abnormal permeability to sodium and potassium cause swelling.
3. Great variability in inherited types resulting in mild to severe hemolytic anemia. Increased Osmotic fragility test, increased retics. May remove spleen.
103

104. Hereditary Acanthocytosis
Defect is increased membrane cholesterol due to abnormal plasma lipids
Numerous acanthocytes on smear
Mild anemia
Also known as abetalipoproteinemia
H Acanthocytosis = Abetalipoproteinemia
Hereditary Acanthocytosis = Abetalipoproteinemia – autosomal recessive inheritance. [abeta = no beta lipid transport proteins]
1. Characterized by numerous acanthocytes on smear.
2. Defect – often increased cholesterol in membrane due to abnormal plasma lipids.
3. Mild anemia, normal Osmotic Fragility test.
4. Serum contains no Beta lipoprotein to transport lipids.
5. Neurological and retinal abnormalities; impaired fat absorption.
104

105. PK deficiency G-6-PD Deficiency
B. 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
PK Deficiency
Echinocytes
Normal RBCs if no exposure to oxidant
G-6-PD Deficiency
Pyruvate Kinase Deficiency - Northern Europeans, Japan, Mexico; autosomal recessive.
1. Most common enzyme deficiency in the EMB pathway. PK is necessary to generate ATP......ATP is needed for membrane maintenance and for the cation pump (controls volume by controlling ratio of Na+ and K+).
In PK deficiency - lack of ATP and backup of intermediates in the EMB pathway causes impairment of cation pump and decreased red cell deformability  decreased lifespan.
Fairly severe hemolytic anemia, reticulocytosis, echinocytes may be found on smear.
Diagnose with enzyme assay. Splenectomy may help anemia.
D. G-6-PD Deficiency (Glucose‑6‑phosphate dehydrogenase) – follows malarial belt; African, Mediterranean, Asian populations; X-linked inheritance pattern...seen in males; females carriers.
1. Most common enzyme deficiency in the HMP shunt.
2. G-6-PD enzyme causes reduction of NADP → NADPH indirectly.
a. NADPH is required as a cofactor to keep glutathione reduced.
b. Glutathione protects vital enzymes and hemoglobin from oxidation.....it neutralizes oxidants, its becomes oxidized (GSSG), and must be reduced back to its functional form (GSH) by NADPH.
3. In G-6-PD deficiency - Heinz bodies form (methemoglobin (Fe+3)  denatured hemglobin) because reduced glutathione levels are not maintained due to decreased NADPH generation (so unable to protect hemoglobin).
a. Exposure to oxidants induce Heinz body formation, which adhere to the rbc membrane, inducing rigidity, splenic ‘pitting’ and consequent RBC lysis = hemolytic crisis.
b. Majority of persons with G-6-PD deficiency have no clinical problems (NOT anemic) unless exposed to oxidants such as primaquine, sulfonamides, nitrofurans, mothballs, or develop a severe infection.
4. Normal G-6-PD variant is G-6-PDB (100% activity)….red cells have enough G-6-PD enzyme to last 120 days. Young red cells (retics) have highest enzyme concentration;enzyme concentration decreases with red cell aging.
a. Two variants ‑ African and Mediterranean, both have variable degree of G-6-PD enzyme in red cells.
b. **Enzyme concentration determines the extent of red cell destruction that occurs upon oxidant exposure.
African variant (G-6-PDA‑)
a. Mod deficiency of enzyme (5-15% activity)….sufficient enzyme for ~60 days. Young red cells have G-6-PD; old red cells lack enzyme.
b. Asymptomatic unless exposed to an oxidant (above) which overwhelms protective mechanisms  form Heinz  red cell lysis
c. Self-limiting hemolytic episode - old cells which lack G-6-PD are preferentially destroyed; young rbcs with enzyme are not affected.
6. Mediterranean variant (G-6-PDMed)
a. Severe deficiency of enzyme (<1% activity)….enzyme lasts <20 days. All red cells lack G-6-PD (young and old rbc's).
b. Hemolytic crisis is easily precipitated by oxidants (as previously noted above) and by eating fava beans, called favism.
c. Not self-limiting, severe hemolysis of young and old red cells; may need blood transfusion.
105

106. G-6-PD Deficiency Blood findings after oxidant exposure: Enzyme assay
Mod to severe anemia
Schistocytes, spherocytes due to pitting out of Heinz bodies by spleen
Enzyme assay
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
Blood findings during hemolytic crisis after exposed to oxidant:
a. Mod to severe normocytic/normochromic anemia.
b. Heinz bodies are present in red cells - Heinz bodies can be demonstrated using a supravital stain such as brilliant green (red cells look like golf balls).
c. Heinz bodies are not visible on a Wright's stained smear.
Schistocytes and spherocytes are present due to pitting out of Heinz bodies by splenic macrophages.
Intravascular rbc destruction occurs....hemoglobinuria.
f. After hemolytic crisis, see a reticulocyte response by the bone marrow.
Diagnose with enzyme assay – should not be performed immediately following a hemolytic episode due to increased # of circulating retics.....would give a falsely high (normal) G-6-PD enzyme result.
106

107. 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 6th of ß
Hgb C = lysine substituted for glutamic 6th of ß
Target cells/Codocytes
Normocytic/Normochromic Hemolytic Anemias due to Hemoglobin Defects
A. Group of inherited disorders in which the globin chains produced are structurally abnormal resulting in variant hemoglobins  Hemoglobinopathies – a qualitative defect.
●Characterized by the presence of target cells.
NOTE: Thalassemias - quantitative defect with decreased production of normally structured globin chains, i.e. no amino acid substitution. Characterized by micro/hypo red cells and target cells.
B. We will discuss disorders in which the Beta chain is abnormal due to an amino acid substitution resulting in an abnormal hemoglobin being made.....substitution causes **changes in hemoglobin solubility (rigid rbc's) and electrophoretic mobility to occur.
Genetic mutation:
1. Hemoglobin S ‑ valine is substituted for glutamic acid at the 6th position of the β chain.
2. Hemoglobin C ‑ lysine is substituted for glutamic acid at the 6th position of the β chain.
107

108. Hemoglobin S Disorders
A. Hemoglobin S disease/Sickle cell anemia/Hgb SS
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
HGB S Disease (Hgb SS)
Sickle cell
Target cell
A. Hemoglobin S Disease/Sickle Cell Disease/Hgb SS [Normal AA]
1. A sickle cell gene is inherited from each parent, both beta chains are abnormal....only Hgb S is produced; no Hgb A.
2. Characterized by chronic hemolysis after 6 months of age and is often fatal before 30 yo.
3. RBCs containing Hgb S will assume the sickle shape when they are deprived of oxygen (deoxygenated) and a low pH.
a. Under low O2 tension, Hgb S molecules interact  polymers. Polymers form rods  bundles  cells show projected tactoids = sickle cells.
b. The sickle cell is a rigid non‑deformable cell....it becomes impeded in circulation, causes blocks which restrict blood flow in vessels and leading to organs; this increases tissue hypoxia and further lowers tissue pH c. Result is increased sickle formation with damage to tissues and organs (failure)......cycle Sickle cells can be reversed to normal if reoxygenated.
a. BUT the red cell loses reversion capabilities after being repeatedly sickled due to loss of membrane in capillaries...stays in sickle shape.
b. We see irreversible sickle forms on the blood smear.
c. Red cells containing hemoglobin S have a very short lifespan; hemolysis occurs in the blood circulation and bone marrow.
5. Clinical findings:
a. Vascular occlusive disease = Sickle cell crisis.
(1) Plugging of small vessels by masses of sickle cells.....causes joint, chest, stomach pain; occlusion affects brain, can cause stroke.
(2) Crisis is precipitated by infections, high altitudes, respiratory problems, dehydration.
b. Ocular changes, skin ulcers, hand-foot syndrome.
c. High susceptibility to infection (pneumonia/meningitis).
d. Hyposplenism - repeated infarcts by sickle cells damage the spleen…can no longer remove RBC inclusions.
6. Lab findings:
a. SEVERE anemia - Hgb 6-10 g/dl, usually normocytic.
b. Targets - few to several; irreversible sickle cells are present.
c. Increased retics but not high enough to equal destruction.
d. NucRBC's, pappenheimer bodies, basophilic stippling, Howell‑Jolly bodies.
e. Positive Hgb S screen.
f. Increased bilirubin, decreased haptoglobin, elevated iron levels, decreased OF test, decreased erythrocyte sedimentation rate (ESR).
g. Hgb electrophoresis:
▪NO hemoglobin A; Hemoglobin A2 varies
▪ >80% hemoglobin S.
▪Compensates with hemoglobin F 2-20%.
7. Bone marrow shows erythroid hyperplasia, decreased M:E ratio, high iron stores.
8. Treatment:
a. Lifelong transfusion support; hydroxyurea, penicillin.
b. Avoid infections, dehydration, hypoxia.
108

109. Hemoglobin S Disorders
B. Hemoglobin S trait/Sickle cell trait/Hgb SA
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
Target cells only NO Sickle cells
B. Sickle Cell Trait/Hgb SA - most common in U.S.
1. One sickle cell gene is inherited; one β chain is abnormal, one β chain is normal.
2. Asymptomatic, not typically anemic, normal Hgb/RBC levels; no nucRBCs.
3. Lab findings:
a. Normocytic/normochromic red cells with few targets.
b. Usually no sickle cells; decreased O2 tension can result in complications so important to know of trait condition….screen for the presence of Hgb S.
c. Positive Hgb S screen.
d. Hgb electrophoresis:
▪Hemoglobin A ~ 60%; hemoglobin S ~ 40%.
▪A2 and F normal.
(Abn hgb produced at lower rate than normal hgb so not expected 50:50 ratio).
4. Thought that sickle trait condition imparts a selective advantage to its carriers  resist P. falciparum. The following conditions are prevalent in the malarial belt: Hemoglobin S trait, Hereditary Ovalocytosis, G-6-PD deficiency, Duffy negative red cells, and thalassemia minor. Target cells are present when sickle cells are seen.
HGB S Trait (Hgb SA)
109

110. 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
HGB C Disease (Hgb CC)
Target cell
Lab findings
Mild anemia
Many target cells
Intracellular C crystals
No Hgb A, >90% Hgb C
Decreased OF
Hemoglobin C Disorders - 2nd most prevalent hemoglobinopathy; the C gene is found among the (West) African population.
A. Hemoglobin C Disease/Hgb CC
1. Inherit C genes from both parents; both β chains are abnormal.....only hemoglobin C is produced, no hemoglobin A.
2. C crystals polymerize differently and look like blocky Hgb packed rods in the red cells....intracellular.
a. Formation is not due to decreased oxygen tension.
b. RBCs with "coffin-shaped" crystals are rigid; mildly reduced lifespan.
3. Lab findings:
a. Mild anemia, may be microcytic.
b. Many targets (90%), folded cells, few spherocytes.
c. C crystals present but usually only a few.
d. Splenomegaly common, decreased OF test, negative Hgb S screen.
e. Hgb electrophoresis:
▪NO hemoglobin A.
▪ >90% hemoglobin C.
▪May compensate with hemoglobin F but < 7%.
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111. 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
HGB C Trait (Hgb CA)
Target cells only NO C crystals
B. Hemoglobin C Trait/Hgb CA
1. Inherit one C gene; one abnormal β chain, one normal β chain.
2. Asymptomatic, not anemic.
3. Lab findings:
a. Normocytic/normochromic red cells with targets (40%).
b. No C crystals; folded cells may be present.
c. Hgb electrophoresis:
▪Hemoglobin A ~60%; hemoglobin C ~ 40%.
▪Normal hemoglobin A2 and F.
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112. HGB SC Disease (Hgb S & Hgb C)
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
SC Crystals
Target cells
HGB SC Disease (Hgb S & Hgb C)
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
C. Hemoglobin SC Disease/Hgb SC
1. Double heterozygote ‑ inherit sickle gene from one parent, inherit C gene from other parent; both β chains are abnormal....no hemoglobin A produced (one beta gene codes for hgb S and other for hgb C).
2. Intermediate in severity between Hgb SS and Hgb SA; painful.
3. Lab findings:
a. Mod anemia, normocytic/normochromic rbc's.
b. Sev targets (85%), folded cells.
c. SC crystals usually abundant...bizarre, appear more dense than sickle cells with fingerlike projections that protrude from red cell.
d. May see occasional sickle cell or C crystal but the majority are SC crystals.
e. Positive Hgb S screen.
f. Splenomegaly, decreased OF test.
g. Hgb electrophoresis:
▪NO hemoglobin A.
▪About 50% hemoglobin S and 50% hemoglobin C.
▪Compensates with hemoglobin F up to 7%.
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113. 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

114. 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
Blood
Warm autoimmune hemolytic anemia = WAIHA
1. An altered immune response causes the production of an autoantibody - a warm reacting
IgG antibody directed against ‘self’ antigens on the rbc membrane; the Ab attaches to Ag.
2. RBCs are coated with the IgG autoantibody/C3 or IgG alone. Macrophages have
receptors for Ig/C3 and partially phagocytize the red cell causing loss of membrane
fragments  spherocytes. The rigid red cells are ultimately destroyed.
3. Causes:
a. Primary/Idiopathic - cause of antibody production is unknown.
b. Secondary to disease which alters the immune response such as *chronic lymphocytic leukemia, lymphoma, systemic lupus.
[Also drugs]
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115. Warm Autoimmune HA (WAIHA)
Ingestion of coated RBC
RBC
Electron Microscopy
Lab findings
Mod to severe anemia, spherocytes, high MCHC
Erythrophagocytosis
Looks similar to H spherocytosis but positive DAT
Increased OF, bilirubin
Erythroid hyperplasia
Blood
4. Lab findings:
a. Mod to severe anemia with few to many spherocytes.
b. MCHC may be >36%;
c. ●WAIHA blood picture looks similar to Hereditary spherocytosis.
d. Increased retics/polychromasia, nucRBCs, few schistocytes
e Erythrophagocytosis - ingestion of antibody/complement coated red cells by monocytes or neutrophils.
f. Positive DAT
g. Increased OF result due to the spherocytes.
h. Increased bilirubin; increased plasma hgb and decreased haptoglobin if severe.
i. Bone marrow (if done) - erythroid hyperplasia.
5. Treatment:
a. Treat underlying disease if possible.
b. Give steroids to suppress immune response.
c. Difficult to transfuse! No compatible blood.
RBC
Monocyte with ingested RBC
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116. 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
RBC Agglutination
100x
Cold autoimmune hemolytic anemia = CAIHA = Cold Hemagglutinin Disease
1. Altered immune response causes production of an autoantibody - a cold reacting IgM antibody directed against ‘self’ antigens on RBC membrane; the Ab attaches to Ag.
2. RBCs are coated with IgM antibody/C3  agglutination. RBCs may be lysed by complement or macrophages with C3 receptors engulf the red cell = red cell destruction.
3. Causes:
a. Primary/idiopathic - cause unknown.
b. Secondary to *mycoplasma pneumonia, lymphoma or infectious mono.
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117. 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)
4. Cold agglutinin titers (IgM antibody) must be >1:1000 for any clinical problems.
Symptoms of acrocyanosis/Raynaud's phenomenon - the agglutination of red cells in extremities....ears, toes, nose  tissue damage  gangrene.
Lab findings:
a. Anemia severity varies with seasons.
b. The IgM antibodies cause RBC agglutination; IgM is best agglutinator. May see agglutination, e.g., RBC clumping, both macroscopically and microscopically.
c. Reticulocytosis
d. Positive DAT (specific to detects complement-coated red cells)
e. Hemolysis is intravasular during attack.
f. RBC agglutination causes problems obtaining valid RBC count and MCV measurements on automated instruments. Blood sample must be warmed.
6. Avoid exposure to the cold; steroids given.
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118. 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
Hemolytic transfusion reaction
Incompatible blood transfusion - recipient has antibodies to antigens on donor red cells received; donor cells are destroyed.
**ABO is worst - massive intravascular hemolysis which is complement-induced lysis; increased plasma hgb, hemoglobinuria.
3. Can be life-threatening....renal failure, respiratory failure, and DIC may develop.
4. Cells are immediately lysed; none on blood smear.
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119. 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
Hemolytic Disease of the Newborn (HDN)
Caused by maternal IgG antibodies; the IgG abs are directed against antigens on baby’s red cells; IgG abs cross placenta and destroy fetal red cells. Only IgG antibodies cross.
HDN due to Rh incompatibility (Erythroblastosis fetalis) [immature-RBCs-fatal]
a. First, Rh negative mother is exposed to Rh antigen....forms IgG antibody.
b. Next pregnancy, baby is Rh positive (has Rh antigens on red cells).
c. Mother's powerful IgG antibody crosses the placenta and lyses/destroys the baby’s red cells.
d. Lab findings:
(1) Severe - anemia with low hemoglobin <8.0 g/dl.
(2) High number of Nucleated RBC's (>10) - baby tries to compensatefor RBC destruction.
(3) Positive DAT
(4) Kernicterus – extremely high bilirubin levels can cause brain damage.
e. Treatment - exchange transfusion in utero or at birth.
f. HDN due to Rh is no longer a common problem with the use of Rh immune globulin (RhoGam).
3. HDN due to ABO incompatibility
a. Mother is type O with anti-A and anti-B in her serum.
b. Baby is type A or B, has A or B antigens on red cells.
c. Mother’s IgG Antibody (usually very little) crosses placenta and coats the baby’s red cells.
(1) Mild/Asymptomatic - anemia is uncommon, with a HGB (>14 g/dl).
(2) Spherocytes present - a few coated rbcs are phagocytized.
(3) May have slight to moderately elevated bilirubin level.
e. Treatment – phototherapy
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120. 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
Fibrin Strands
RBC
RBC fragmentation on fibrin strands
Schistocytes
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121. 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
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122. END OF ANEMIA