3. Red Blood Cells (RBCs)
• Nutrient Factors
– Iron
– Folic acid
– B12
• Life Cycle and Breakdown
– Finite life span 120 ± 20 days
– Removal of RBCs performed by the
reticuloendothelial (RE) system
4. Generally, Two Different Systems
Have Been Used To Classify
Anemia.
• The Morphologic System;
– (1) Microcytic (MCV < 80), Hypochromic (MCH < 30)
– (2) Macrocytic (MCV >100), Normochromic (MCH 30–34)
– (3) Normocytic (MCV 80–100), Normochromic.
• Red Cell Kinetics
– Hypoproliferative (Decreased Production) vs.
– Increased Destruction or Blood Loss
5. Normal Blood Smear
Central Pallor normally
approximately 1/3 of cell
diameter.
6. Reticulocyte Stain
• Precipitated ribosomal RNA in young red cells.
• Normally Approximately 1-2% of total red cells.
• ~50-100,000/ul absolute count
• If reticulocytes are not elevated, (in setting of anemia), then it
is low, i.e. hypoproductive.
• Elevated reticulocyte index indicates response to hemolysis
or blood loss.
7. Reticulocyte Index
• Reticulocyte “Count” as a percent of red cells. Only
obtained when ordered. (Similar, but distinguished from
polychromatophilia.)
• Reticulocyte Index = (Retic %)*(Pt. Hgb/Nl Hgb)
• (In severe anemia, reticulocytes are released prematurely,
so further divide retic. index by 2.)
• Absolute reticulocyte number=
– (red cell number) X (reticulocyte percent)
– (Normal; approximately 50,000-100,000/ul)
• If reticulocytes are not elevated, (in setting of anemia),
then it is low, i.e. hypoproductive.
• Elevated reticulocyte index indicates response to
hemolysis or blood loss.
8. Microcytic/Hypochromic Red Cells
• Processes which affect
Hemoglobin Synthesis;
– Reduced Globin chain
synthesis (Thalassemias)
– Iron deficiency
– Some Myeloproliferative/
Myelodysplastic Syndromes
(Deficiencies in porphyrin
and heme synthesis)
– Some anemia of chronic
disease
9. Hemoglobin Synthesis
• Key Components
• Globin chain (protein)
• Heme/porphyrin ring
• Iron
10. Iron Deficiency
• Nutritional deficiency rare after childhood.
– May be seen in adolescent females,
especially with onset on menses.
• Essential to identify putative source of blood
loss, i.e. Gyn, GI.
• Rare for iron deficiency from hematuria or
hemoptysis.
• Decreased iron absorption seen with chronic
acid neutralization in stomach from PPIs, due to
reduced oxidation of ferrous to ferric form.
13. Thalassemia;
(Decreased Production of Globin Chains)
• -Thalassemia; Decreased Production of -
globin chains
• -Thalassemia; Decreased Production of -
globin chains.
• Results in decreased hemoglobin synthesis
and a microcytic/hypochromic anemia
• Severity of disease is depending upon the
number of mutant genes.
14. Production of Globin Chains
• Hemoglobin is a
Globin Tetramer
• Hemoglobin can
consist of two alpha
chains and 2 beta
class chains of globin.
• 2 2; Hgb A
• 2 2; Hgb A2
• 2 2; Hgb F
18. -Thalassemia Syndromes
Normal Trait
+ Mild Mild Hgb H, Hydrops
Asymp- Inter- Fetalis
tomatic
Thalassemia minor mediate
19. -Thalassemia Syndromes
Normally four genes for -Globin, two on each of
chromosome 16.
– +; One normal/one mutant on chromosome.
– 0; Both mutant genes on one chromosome.
(Mostly in Asians)
If two genes mutant, Thalassemia minor
– Hemoglobin electrophoresis normal.
If three genes mutant; Hemoglobin H Disease
– Hgb H; 4 Tetramer
If all four genes mutant; Hydrops Fetalis;
– Hgb Barts; 4 Tetramer
20. -Thalassemia
• Two types of mutant alleles;
• 0; Absence of expression. Typically a
mutation in coding region of gene (On
chromosome 11)
• +; reduced expression of globin gene.
Typically promoter mutation.
23. Differentiation of Microcytic
Anemia
In iron deficiency, red cell number, Hemoglobin, and MCV
are all proportionally reduced.
In Thalassemia, the MCV is disproportionately low, and red
cell number is “spared.”
Iron Deficiency Thalassemia Normal
Red Cell Number 3.8 5.8 4.5-6.5 (Male)
(109/ml) 3.9-5.6 (Fem.)
MCV fl 67 60 80-99
Hgb (g/dl) 7.5 10 13.5-17.5 (M)
11.5-15.5 (F)
24. - vs -Thalassemia
• Since only the -chain is synthesized of the -class
of globin chains, the ratio of Hgb A, A2, and F is not
altered in -Thalassemia.
• In -Thalassemia, Hgb A2 ( 2 2 ) and Hgb F ( 2 2)
levels as % of total Hgb are increased due to
selective loss of -globin.
• Hemoglobin electrophoresis can distinguish between
- and -Thalassemia. (best for quantitation by
column chromatography).
25. Thalassemia Syndromes
• Depending on genetic severity;
• Chronic anemia, high out-put cardiac states
• Hepatosplenomegaly due to extramedullary
hematopoiesis.
• Transfusion-Dependent (Thalassemia major)
– Need for iron chelation.
• Due to increased marrow turnover, need for folic acid
replacement.
26. Anemia Of Chronic Disease-
Chronic Inflammatory Block
• Anemia due to decreased red cell production,
related to relatively insufficient levels of
erythropoietin, for the degree of anemia.
• Seen in chronic inflammatory diseases
(Connective Tissue Disease), chronic infection,
etc.
• Classic in chronic renal insufficiency (often
before dialysis requirement).
27. Anemia of Chronic Disease
Prussian Blue (Iron) Stain
In bone
marrow, absent
red cell
precursor iron,
with adequate
storage iron.
Can’t make
diagnosis in
absence of
iron.
28. Distinguishing Between Iron Deficiency
And The Anemia Of Chronic Disease
Iron deficiency Anemia of Chronic
Disease
Serum Iron Decreased Decreased
Serum Transferrin Increased Decreased
Serum Iron Binding Increased Decreased
Capacity
Serum Ferritin Decreased Normal or Increased
Bone Marrow Iron Absent Present
Stores Absent
Cannot definitively diagnose Anemia of Chronic Disease in
absence of adequate iron stores.
31. Macrocytic Anemias
Macrocytic anemias may arise from abnormal
DNA synthesis, producing megaloblastic
changes in the bone marrow.
The major causes include deficiencies in vitamin
B12 and folate.
Alcoholism, (even in the absence of folate or B12 deficiency),
apparently from a direct effect of ethanol on the bone marrow.
Hypothyroidism, (unknown mechanisms), mild macrocytic
anemia.
Myelodysplasia, abnormal maturation of red cell precursors often
produces macrocytosis.
32. Incidence of Various
Macrocytic Anemias
Alcohol abuse 36 %
Vitamin B12 or folate deficiency 21 %
Drug intake 11 %
Accelerated erythropoiesis 7%
Liver disease 6%
Myelodysplasia 5%
Hypothyroidism 2%
Unexplained 12%
Colon-Otero G, et al. Med Clin North Am. 1992; 76:581-597.
33. Megaloblastic Anemia
All cell lines affected.
Macrocytic Anemia (MCV > 100).
Coexisting iron deficiency, thalassemia,
inflammation, may prevent macrocytosis.
Hypersegmented PMN (any 6 lobe, >5% 5
lobed).
Role of folate and vitamin B12 in DNA synthesis.
– Additional role for B12 responsible for neurological and
other manifestations.
36. Vitamin B12 Absorption
Gastric acid separates B12 from food.
Parietal cells produce Intrinsic Factor: Binds
B12
B12:IF complex absorbed in terminal ileum
37. The Schilling Urinary Excretion
Test of Vitamin B12 Absorption
Subjects B12 Given Alone B12 Given with
Intrinsic Factor
Normal 18 (9-36) -
Pernicious anemia 0.5 (0-1.2) 13 (6-31)
Malabsorption 3.6 (0-19) 3.3 (0-10)*
38. Pernicious Anemia Due to Autoimmune
Reaction to Intrinsic Factor/Parietal Cells
Immunofluorescence for
Pernicious Anemia
anti-parietal cell antibodies
Atrophy, loss of
Normal parietal cells,
Stomach gastric glands
40. Normocytic/Normochromic Anemias
• Intrinsic bone marrow disease
• (1) Hypoplasia, (aplastic anemia or pure red cell aplasia)
• (2) Infiltration by abnormal tissue, as in multiple myeloma, leukemia,
fibrosis, or metastatic malignancy
• (3) Myelodysplastic disorders, in which abnormal maturation occurs.
• Diminished Erythropoietin (Anemia of
Chronic Inflammatory Block or Anemia of
Chronic Disease)
41. Hemolytic Anemia
• Premature destruction of red blood
cells
– Red cells normally circulate approximately
100 to 120 days.
• Intravascular vs. extravascular
hemolysis.
• May be immune or non-immune
mediated.
42. Hemolytic Anemia, Laboratory Findings
• Reticulocyte index is >3% and the
absolute reticulocyte count is
>100,000/mm3.
• Immature erythrocytes prematurely leave
bone marrow. The indirect bilirubin is
elevated and accounts for >80% of the
total bilirubin.
• The serum LDH level is increased.
• Serum haptoglobin is diminished.
43. Causes of Hemolysis
(by Site of Abnormality)
• Intrinsic to the red cell Extrinsic to the red cell
• Abnormal hemoglobins Immunologic
– Warm antibody
– Sickle cell anemia
– Cold antibody
– Hemoglobin C, E, etc. – Drugs
• Enzyme defects Mechanical
– Glucose-6-phosphate – March hemoglobinuria
dehydrogenase deficiency – Traumatic cardiac hemolytic anemia
– Pyruvate kinase deficiency, etc. – Microangiopathic hemolytic anemia
• Membrane abnormalities Infectious
– Hereditary spherocytosis, – Malaria
elliptocytosis – Clostridium perfringens infection
– Acanthocytosis Chemicals
– Paroxysmal nocturnal Hypersplenism
hemoglobinuria
45. Hereditary Spherocytosis
• Due to Abnormality of red cell cytoskeleton
– Most mutations in Ankryn, Band 3 (Also Spectrin,
Band 4.2)
• Common autosomal Dominant
– Approx 1/5000 (Not just Northern Europeans)
• Clinically; Anemia, Jaundice, splenomegaly
• Osmotic Fragility Test
• Management; Splenectomy
46. Glucose 6-Phosphate
Dehydrogenase Deficiency
• Enzyme defect in hexose-
monophosphate shunt
pathway.
• Normally responsible for
generation of reducing agents
in cells (Reduced glutathione).
• RBC sensitive to oxidant
stress, drugs, infections,
malaria, etc.
• Heinz bodies; denatured
(oxidized) hemoglobin, usually
seen on reticulocyte stain.
• X-Linked inheritance;
– Mediterranean (Severe)
– African (Mild)
– Asian (Varied)
52. Leukoerythroblastosis/
Myelophthisic
• Marrow invasion with abnormal cells (carcinoma, advanced
myeloproliferative disease, fibrosis, etc.)
• Immature red and white cells (promyelocytes, myelocytes,
metayelocytes, teardrop cells.
Nucleated rbc
Myelocyte
53. Teardrop Cells
Most prominently in thalassemias, marrow
infiltration by fibrosis or malignancy.
– From distortion of the erythrocytes as they
travel through the vasculature of an abnormal
bone marrow or spleen.
54. Approach To Anemia
• Have a system, based on rates of production/destruction and
morphology
– Morphology
• (1) Microcytic/Hypochromic
• (2) Macrocytic
• (3) Normocytic/Normochromic.
– Red Cell Kinetics
• Hypoproliferative (Decreased Production) vs.
• Increased Destruction or Blood Loss
• LOOK AT YOUR PATIENTS’ SMEARS.
• Do not treat anemia with empiric iron therapy.
55.
56. Burr Cells (Echinocytes)
• Blunt, fairly symmetrical projections.
• Prominent in renal failure from any cause
– (may also occur with liver diseases, especially when uremia
coexists.)
• (Can develop as a storage artifact)
57. Spur Cells (Acanthocytes)
• Several irregularly sharp projections of unequal length.
• Most of the cells are also small and lack central pallor.
• Prominent in liver disease, usually alcoholic cirrhosis, with an
increase in the cholesterol:phospholipid ratio in the red cell
membrane, leading to hemolysis.
58. Bite Cells (Degmacyte)
• A semicircular defect in their edge that resembles a bite mark.
• Oxidative destruction/precipitation of hemoglobin, often in
patients with a deficiency of the enzyme glucose-6-phosphate
dehydrogenase (G6PD) or unstable hemoglobins.
60. Red Cell Agglutination
• Red Cells coated with antibodies
– Typically IgM, but can be IgG
• Extensive clumping typically develops with high levels of IgM, which are
present in Waldenstrom’s macroglobulinemia or in cold-agglutinin
hemolytic anemia from such causes as infectious mononucleosis and
Mycoplasma pneumoniae infection.
