Erythropoiesis 2.

Hexose Monophosphate shunt/Pentose

phosphate pathway
Red Blood Cells  is a metabolic pathway parallel to
- mature red blood cell glycolysis. It generates NADPH and
- non-nucleated, bi concave disc-like cell pentoses (5-carbon sugars) as well as
- 100 RBCS = 3 to 8 platelets ribose 5-phosphate, the last one a
- 1000 RBCS = 1 white blood cells precursor for the synthesis of nucleotides
 10% glycolysis occurs in this pathway
Gender: Conventional SI  Aerobic pathway
Normal
Values: Male
5.5 to 6.5 5.5-6.5 x  Provides reduced glutathione to prevent
mil/mm3 1012/L hemoglobin denaturation
4.5 to 5.5 4.5-5.5 x  G6PD deficiency yields Heinz bodies
Female
mil/mm3 1012/L

Erythrocyte Membrane
 Protein (50%)
 Integral Protein: Glycophorin A
 Contains Sialic acid
 Pheripheral protein
 Contains spectrin
 Lipid (40%)
 External surface: phosphatidylcholine, 3. Rapoport-Leubering Pathway
glycolipid and sphingomyelin  responsible for generation of 2,3-BPG which
 Internal surface: regulates hemoglobin affinity for Oxygen
phosphatidylethanolamine,
phosphatidylinositol and
phosphatidylserine
 Cholesterol
 Carbohydrate (10%)

Pathways of Metabolism of Red Blood Cells

1. Embdenmeyerhoff pathway
 Major source of red cell energy
 Yields 2 ATPs
 ATP controls Na and K and prevent
oxidation of membrane lipid
 Hemoglobin
- found in the red blood cells of the body.
- Each red blood cell (RBC) contains
approximately 280
- million hemoglobin molecules

Function of Hemoglobin4
 The main function of red blood cells is the
transport of oxygen from the lungs to the body's
cells.
 carries oxygen. In capillaries, oxygen is released
to be used by the body's cells.
Composition
 A hemoglobin molecule is composed of a protein
group, known as globin, and four heme groups,
each associated with an iron atom.
 1 heme=1 mole of Oxygen
 1 hemoglobin molecule=4 moles Oxygen
 In the lungs, each iron atom combines reversibly
with a molecule of oxygen.

4. Methemoglobin Reductase Pathway

 Maintains hemoglobin in Ferrous state to be
functional
Breakdown of Red Blood Cells
- As RBC cells age: decrease enzymes, ATP,
size and increase density
- Culling- destruction of senescent red cells by
spleen
- Pathways:
 Extravascular
 Intravascular
Synthesis of Hemoglobin Molecule

How is the normal Oxygen level maintained in  Heme Portion: begins in the mitochondria
the body? with the formation of D-ALA from glycine and
succinyl coenzyme A
 Globin portion: produced on specific
ribosome in the cytoplasm of the red blood
cells
 The globin in each hemoglobin molecule
consists of four polypeptide chains which
determine the type of hemoglobin formed.
 Gene involved for Alpha & Zeta chains:
chromosome 16
 Gene involved for Beta, Delta, Epsilon &
Gamma chains: chromosome 11
 Example:

 Sickle Cell Anemia

 90%-100% HbS
 Some HbA2, & HbF
 Sickle Cell Trait
 55%-60% HbA
 40%-45% HbS

Normal Hemoglobin
Clinically Significant Variant Hemoglobins-
 Hemoglobin A Qualitative Defect in Hgb: Substitution of amino
- This is the designation for the normal acids in the Beta Chain
hemoglobin that exists after birth.
 Hemoglobin C.
- Hemoglobin A is a tetramer with two alpha
- Hemoglobin C results from a mutation in the
chains and two beta chains (a2b2).
beta globin gene and is the predominant
 Hemoglobin A2
hemoglobin found in people with hemoglobin
- This is a minor component of the
C disease (a2bC2).
hemoglobin found in red cells after birth and
- Hemoglobin C disease is relatively benign,
consists of two alpha chains and two delta
producing a mild hemolytic anemia and
chains (a2d2).
splenomegaly. Hemoglobin C trait is benign.
 Hemoglobin F
 Lysine replaces GLUTAMIC ACID at 6th
- Hemoglobin F is the predominant
position
hemoglobin during fetal development.
- The molecule is a tetramer of two alpha
Clinically Significant Variant Hemoglobins-
chains and two gamma chains (a2g2). Qualitative Defect in Hgb: Substitution of amino
acids in the Beta Chain
Clinically Significant Variant Hemoglobins-
Qualitative Defect in Hgb: Substitution of amino  Hemoglobin E
acids in the Beta Chain - This variant results from a mutation in the
hemoglobin beta chain.
 Hemoglobin S
- People with hemoglobin E disease have a
- This the predominant hemoglobin in people
mild hemolytic anemia and mild
with sickle cell disease. The alpha chain is
splenomegaly.
normal.
- Hemoglobin E trait is benign. Hemoglobin E
 The disease-producing mutation exists in the
is extremely common in S.E. Asia and in
beta chain, giving the molecule the structure,
some areas equals hemoglobin A in
a2bS2. People who have one sickle mutant
frequency
gene and one normal beta gene have sickle
 Lysine replaces GLUTAMIC acid at 26th
cell trait which is benign.
position
 Valine replaces GLUTAMIC ACID at 6th
position
 Clinically Significant Variant Hemoglobins:  Having two different variant genes from the
Quantitative defect in Hgb alpha globin gene cluster or two different
variant genes from the beta globin gene
 Hemoglobin Constant Spring. cluster (a gene for hemoglobin S and one for
- is a variant in which a mutation in the hemoglobin C)
alpha globin gene produces an alpha Hemoglobin SC disease
globin chain that is abnormally long. - Patients with hemoglobin SC disease inherit a
 The quantity of hemoglobin in the cells is low gene for hemoglobin S from one parent, and a
for two reasons. First, the messenger RNA for gene for hemoglobin C from the other.
hemoglobin Constant Spring is unstable. - Hemoglobin C interacts with hemoglobin S to
Some is degraded prior to protein synthesis. produce some of the abnormalities seen in
Second, the Constant Spring alpha chain patients with sickle cell disease.
protein is itself unstable. - On average, patients with hemoglobin SC
 The result is a thalassemic phenotype. disease have milder symptoms than do those
From family of ethnic Chinese background with sickle cell disease.
from the Constant Spring district of Jamaica Sickle/beta-thalassemia
- In this condition, the patient has inherited a
Clinically Significant Variant Hemoglobins: gene for hemoglobin S from one parent and a
Quantitative defect in Hgb gene for beta-thalassemia from the other.
 Hemoglobin H - Has large quantity of normal hemoglobin
- Hemoglobin H is a tetramer composed of produced by the beta-thalassemia gene.
four beta globin chains. - Thalassemia genes produce normal
 Forms in people with three-gene alpha hemoglobin, but in variably reduced amounts
thalassemia as well as in people with the Hemoglobin E/beta-thalassemia
combination of two-gene deletion alpha - The combination of hemoglobin E and beta-
thalassemia and hemoglobin Constant Spring. thalassemia produces a condition more severe
than is seen with either hemoglobin E trait or
Clinically Significant Variant Hemoglobins: beta-thalassemia trait.
Quantitative defect in Hgb - moderately severe thalassemia (thalassemia
 Hemoglobin Barts intermedia)
 Hemoglobin Barts develops in fetuses with - most common in people of S.E. Asian
four-gene deletion alpha thalassemia. background.
 During normal embryonic development, Alpha thalassemia/Hemoglobin
the episilon gene of the alpha globin gene Constant Spring.
locus combines with genes from the beta - Has alpha globin gene cluster
globin locus to form functional hemoglobin - The alpha globin gene cluster on one of the
molecules. two chromosomes 16 has both alpha globin
 The episilon gene turns off at about 12 genes deleted.
weeks, and normally the alpha gene takes - On the other chromosome 16, the alpha1 gene
over. With four-gene deletion alpha has the Constant Spring mutation.
thalassemia no alpha chain is produced. - produces a severe shortage of alpha globin
 From St. Bartholomew's Hospital in London. chains.
The hospital has the fond sobriquet, St. Barts, - The excess beta chains associate into
and the hemoglobin was named tetramers to form hemoglobin H.
"hemoglobin Barts."
RBC Disorders
Compound Heterozygous Conditions
A. Cell Size
 Hemoglobin is made of two subunits derived
from genes in the alpha gene cluster on  Anisocytosis
chromosome 16 and two subunits derived - variation in size of red cells
from genes in the beta gene cluster on - sign of regeneration
chromosome 11.  RDW
- numerical expression that correlates with
the degree of anisocytosis
 NV: 11.5 – 14.5% o Normochromic
NORMOCYTIC: - Normal MCV  Normal MCHC= 31-36%
- 80-100 fL o Hypochromic
MICROCYTIC: - MCV <80 fL  central pallor area exceeds 1/3 of the
MACROCYTIC: - MCV>100fL diameter of the cell
 Decreased MCHC= <36%
B. Cell Shape
o Hyperchromic
 Poikilocytosis  no central pallor
- variation in shape of RBCs  Increased MCHC = >36%
o Acanthocyte o Polychromasia/Polychromatophilia
- thorn cell, spur cell  Blue-gray coloration
- irreversibly thorny, rounded  Indicates young red cell/presence of
- RBC with irregularly arranged 5-10 reticulocytes
spicules of various lengths  Increased erythropoietic activity seen in
o Burr cell hemorrhage and hemolysis
- reversibly spiculated elongated cells o Anulocyte (pessary cell, ghost cell)
o Crenated RBC (Echinocyte)  RBC with just a thin rim of Hgb and a
-has 10-30 short and blunt spicules that are large clear central area
evenly distributed over the surface of the
red cell
o Elliptocyte (Ovalocyte)
-seen in pernicious anemia
o Blister cell
-with single or multiple vacuoles or
markedly thinned areas at the periphery
o Keratocyte/Helmet Cells
- triangular cells
o Pyknocyte
- distorted, contracted, spiculated RBC
o Sickle cell/Drepanocyte/Menisocyte
- crescent shaped RBC
o Spherocyte
- spherical RBC with diminished diameter
o Stomatocyte/Mouth Cell
- mouth-like clear central area
o Schistocyte
-fragmenting or disintegrating RBC
o Stellar cell/Astrocyte
-star like RBC
o Tear Drop/Dacryocyte
-pear-shaped cell
o Target Cell/ Leptocyte/ Platycyte/
Codocyte/ Bull’s Eye Cell/Mexican Hat
Cell/Greek Helmet Cell
- a central and peripheral condensation of
Hgb with a clear zone in between
o Racket Cell
-pear shaped like tear drop but with longer
tail or projection
C. Hemoglobin Content
 Anisochromasia
- variation in hemoglobin content
  Erythrocytosis
- Increase in RBC above 6.5mil/mm3 due to
chronic heart disease, lung disease and high
altitude
 Oligocythemia
-general decrease of RBC in the circulation
and RBC forming organs in the body

G. Hemoglobinopathies
 Hemoglobin S
 Hemoglobin C
 Hemoglobin O-Arab
 Hemoglobin D and G
 Hemoglobin SC
D. Abnormal RBC Inclusion Bodies
H. Classification of Anemias cited by
 Howell-Jolly bodies Turgeon, 2012
 Cabot’s ring  Decreased production of red blood cells
due to:
 Marrow damage
 Leukemias
 Leukoerythroblastosis
 Aplastic anemia
 Decreased Erythropoietin
 Basophilic Stipplings  Inflammatory process
 Heinz-Ehrlich Bodies  Renal disease
 Siderocytic Granules  Hypothyroidism
 Maragliano Bodies  Nuclear Maturation
 Malarial Stipplings  Vitamin B12 deficiency
 Crystals  Pernicious anemia
 Hemoglobin H inclusions  Folic Acid Deficiency
 Refractory Macrocytic anemia
 Hemoglobin Zurich Inclusions
 Di Guglielmo’s anemia
E. Miscellaneous RBC Appearance  Cytoplasmic Maturation Abnormality
 Rouleaux Formation  Cytoplasmic Maturation Abnormality
- red cells rolls/ stacks of coins  Severe Fe Deficiency
 Defect in Heme Synthesis
 Partially hemolyzed RBC
 Sideroblastic Anemia
- lightly colored and malformed
 Defect in Globin Production
 Thalassemia
F. Terms Describing RBC Involvement  Hemolytic Anemia
 Acute
 Polycythemia
 Hemorrhage
- general term for the increase of RBC
 Chronic
concentration in the peripheral blood
 Congenital
 Polycythemia vera
 Hemoglobinopathies
- is essentially a myeloproliferative disorder
 Enzyme Defects
- Characterized by abnormal proliferation of
 Glucose-6-Phosphate
erythroid, myeloid and megakaryocytes in the
Dehydrogenase Deficiency
bone marrow and increased in RBCs
 Acquired
 Erythremia
 Heinz body Anemia
- increased in RBC count above 6.5mil/mm3
 Auto-immune Hemolytic Anemia
  Aplastic Anemia Normal Values of Blood Cell Indices
- Inability of the bone marrow to replace lost
 MCV: 80 to 100 femtoliter
red blood cells
(Mean cell volume)
 Thalassemia Increased MCV is seen in Megaloblastic
- Abnormal production rate of one of the anemia and nonmegaloblastic anemia such
polypeptide chains of hemoglobin molecule as liver disease, hypothyroidism
 Pernicious Anemia Decreased MCV is seen in IDA, defective
- Due to lack of an intrinsic factor needed for Fe utilization (chronic disease) &
the normal absorption of Vit. B12 thalassemia

 MCH: 27 to 31 picograms/cell
Spur Cell
(Mean cell hemoglobin)
- Small, dense RBC with few irregularly spaced Is rarely used
projections of varying lengths Increased MCH is seen in macrocytic
anemias
Decreased MCH is seen in hypochromic
and microcytic anemias unless RBCs are
spherocyte

 MCHC: 31 to 36 grams/deciliter (g/dL) or 320

to 360 grams per liter (g/L)
(Mean cell hemoglobin concentration)
Increased MCHC is seen in hyperchromic
cells (spherocytes)
Decreased MCH is seen in hypochromic
cells (IDA, defective Fe utilization and
Red Blood Cell Morphology
thalassemia

 RDW: 11.5-14.5% (numerical expression that

correlates with the degree of anisocytosis)
(Red cell distribution width)
Hypochromia Grading
 1+ = area of central pallor is one-half of
cell diameter
 2+ = area of pallor is two-thirds of cell
diameter
 3+ = area of pallor is three-quarters
 4+ = thin rim of hemoglobin
What Abnormal Results Mean
These test results indicate the type of anemia:
Blood Cell Indices
Microcytic - Fe deficiency
- Red blood cell (RBC) indices are part of the hypochromic - Thalassemia
anemia - Lead poisoning
complete blood count (CBC) test.
- They are used to help diagnose the cause of Normocytic - Fe deficiency, Aplastic
Normochromic anemia
anemia.
Anemia - Sepsis, acquired hemolytic
Why the Test is Performed? anemia
- Acute blood loss
- Hemoglobin transports oxygen. RBCs carry Microcytic - Renal disease (loss of
hemoglobin and oxygen to our body's cells. normochromic erythropoietin)
- The RBC indices test measures how well the Macrocytic - Vit B12 /folic acid def.
RBCs do this. The results are used to normochromic - Hydantopin ingestion
diagnose different types of anemia. Anemia - chemotherapy
 Interpretation
MCV BELOW normal Microcytic anemia
May be due to:  Normal Values
- thalassemia MCV: 80 to 100 femtoliter
- may be due to low MCH: 27 to 31 picograms/cell
iron levels MCHC: 31 to 36 grams/deciliter (g/dL) or
- lead poisoning 320 to 360 grams per liter (g/L)
MCV NORMAL Normocytic anemia
May be due to:  Results:
MCV-100fL or 1015Liter
- sudden blood loss
- long-term diseases MCH- 29.8 pg or 10-12 grams
- kidney failure MCHC- 34 g/dL
- aplastic anemia
- man-made heart  Interpretation:
valve - Normocytic normochromic anemia; associated
MCV ABOVE normal Macrocytic anemia with Fe deficiency, Aplastic anemia
May be due to: - Sepsis, acquired hemolytic anemia and Acute
- low folate blood loss
- B12 levels ESR
- chemotherapy
Erythrocyte Sedimentation Rate

- refers to the speed of settling of red blood cells

Formula in anticoagulated blood.
- It measures the distance and speed of fall of
RBCs in the plasma in a tube of a standard
bore and length after standing perpendicularly.
- relatively simple
- Inexpensive
- non-specific test
- detects inflammation may be caused by
infection, some cancers, and certain
autoimmune diseases such as juvenile
idiopathic arthritis, lupus, and Kawasaki
disease.
- ESR alone can't be used to diagnose any one
specific disease.

Stages of ESR
A. Initial rouleaux formation
- LAG PHASE
-10minutes
B. Rapid settling of RBC’s
- Decantation Phase
-40 minutes
C. Final Sedimentation of RBC’s
-10 minutes
Factors that Influence ESR
RBC Increased ESR
-Macrocytes
-Anemia
Decreased ESR
-Microcytes
-Poikilocytes
-Polycythemia
 Plasma Increased ESR Methods
Composition -Fibrinogen
-Alpha 1 globin  Standard/original Westergren
-Alpha 2 globin - uses citrate
Decreased ESR  Modified Westergren
-Albumin - Uses EDTA
-cholesterol - Length of tube – 30cm; bore 2.5mm
 Wintrobe and Lansberg
Technical factors Affecting ESR - Uses double oxalate
- Length 11.5cm;bore 3mm
 Tilting
increased for every 3degree angle=30% ESR- WESTERGREN METHOD
increased
 Increased Temperature-increased ESR
 Length and diameter of tube is directly
proportional to ESR result
 Decreased
decreased temperature
20-25 degrees- optimal temperature
 Increased EDTA

Normal Values

Westergren Male  <50y/o =0-15m

Method  > 50y/o =0-20mm
Female  <50y/o =0-30mm
 > 50y/o =0-30mm
Children 0-10mm
Wintrobe Male 0-9mm
Method Female 0-20mm
Children 0-13mm

Variation in ESR Result

 ESR increased
Inflammatory conditions
Acute and chronic infections
Rheumatic fever
Rheumatoid arthritis
Myocardial infarction
Nephrosis
TB, pregnancy after 3rd month ESR- WINTROBE METHOD
Multiple myeloma
Waldrenstrom’s macroglobulinemia
Subacute bacterial endocarditis
Hepatitis
Menstruation

 ESR Decreased
Polycythemia
Presence of RBC abnormalities
(poikilocytosis, spherocytes and sickle cell)
Hemoglobin CC disease
hypofibrinogenemia
OTHER ESR METHOD
 Ves –Matic 20-bench top analyzer
- measures ESR in 20 blood samples
 Zeta Sed Rate (ZSR)
- spins capillary tubes in vertical position in 4-
45s cycle w/in 3 minutes
- ZSR=hct/zetacritx100 (Normal value:4-54%)

OFT

Osmotic Fragility Test

 Measures the ability of RBC to take up fluid

w/o lysing
 Primary factor affecting OFT is the Shape of
Results
RBCs
The larger the amount of RBC membrane  1st tube
(surface area) in relation to the size of the - trace hemolysis in the supernatant
cell the more fluid the cell is capable of - beginning of hemolysis
absorbing before rupturing  1st tube
 Target cells -highest conc. of NaCl w/c hemolysis is
- Has the largest surface area and shows complete
decreased fragility -complete hemolysis
 Spherocyte  Normally, hemolysis should be complete in
- Has the smallest surface area and show 0.3% NaCl and beginning hemolysis should
increased fragility not occur in a concentration over 0.45% NaCl
 Fragility of RBC is increased when the rate
Principle of hemolysis is increased
 Fragility of RBC is decreased when the
 Whole blood is added to varying rate of hemolysis is decreased
concentrations of buffered NaCl solution and
allowed to incubate at room temperature
 The amount of hemolysis in each saline
concentration is then determined
 If RBCs are placed in isotonic solution, fluid
will neither enter nor leave the cell
Indications of OFT Anemia
 Increased - is a condition in which you don't have enough
Hereditary sherocytosis healthy red blood cells to carry adequate
Hemolytic anemias oxygen to your tissues.
 Decreased - is a condition in which your blood has a lower-
Splenectomy than-normal number of red blood cells.
Liver disease - is not a single disease but a condition, like
Fe deficiency anemia fever, with many possible causes and many
Thalassemia forms.
Target cells present Symptoms of Anemia
Reticulocytes present
- decrease of oxygen in the cells or hypoxia.
Hemoglobin- carry oxygen, a decreased
Hemoglobin Tests
production or number of these cells result in
1. Hemoglobin electrophoresis hypoxia.
 Cellulose acetate- alkaline pH 8.6 - Many of the symptoms will not be present with
 Citrate agar-acid pH 6.0-6.3 mild anemia, as the body can often
2. Quantitation of Hgb F compensate for gradual changes in
 Betke mtd of alkali denaturation (NaO hemoglobin.
 Singer mtd of alkali denaturation (KOH) - Vary depending on the cause of your anemia
3. Unstable Hgb but may include:
 heat pptn tes Fatigue
 Isopropyl pptn Pale skin
 G6PD TESTS Fast or irregular heartbeat
- Asorbate cyanide screening test Shortness of breath
- Non specific procedure for detecting Dizziness
deficiencies in pentose phosphate pathway Cognitive problem
 G6PD Fluorescent Screening Cold hands and feet
- Normal: Max fluorescence at 10 min Headache
- Abnormal: little or no fluorescence at all Causes of Anemia

Test for Paroxysmal Nocturnal Hemoglobinuria  Infection

(PNH)  Certain disease
 Certain medications
- Acquired disorder in which patients red cells  Poor nutrition
are abnormally sensitive to destruction by Anemia occurs when your blood doesn't
normal constituents in plasma have enough red blood cells. This can
 Increased sensitivity of rbc to lysis happen if:
 Your body destroys red blood cells
 Sucrose Hemolysis test/sugar water test-  Your body doesn't make enough rbcs
screening  Bleeding causes you to lose rbcs more
- Sucrose provides a medium of low ionic quickly than they can be replaced
strength and promotes the binding of
complement to RBC Maturation and Differential Chart
- = hemolysis

 Acidified Serum Test (Ham’s Test)

- Acidified serum activities complement by
alternate pathway
- + hemolysis
Red Blood Cells How is RBC measured?
 Erythrocyte: 40 – 50 % of human RBCs  Mean corpuscular volume (MCV) test
 Size: 6 – 8 um - measures the average size of the cell.
 Shape: disc-shaped , doughnuts without holes  Mean corpuscular hemoglobin (MCH)
in the center test
- measures the cell’s content of hemoglobin
 Mean corpuscular hemoglobin
concentration (MCHC) test
- measures the ratio of hemoglobin to cell
size.
Diagnostic Approach to Anemia

Normocyte: Normal Erythrocyte

Laboratory Diagnosis
- There are various blood tests and other
diagnostic tests or procedures to find out what
type of anemia and how severe it is.
What RBCs do?
A. Complete Blood Count
 enables red blood cells to carry oxygen from - Hgb and Hct level
your lungs to all parts of your body - RBC & WBC count
 to carry carbon dioxide from other parts of - Differential Count
the body to your lungs so that it can be - Platelet
exhaled. - Cell Indices
B. Blood Smear and Differential
- If results of the CBC indicate anemia, it may be
followed up with an examination of a Blood
Smear or a Differential.
- Results from these tests may give clues as to
the cause.
- Several other tests may be run to help
determine the cause of the anemia and to guide
treatment.
C. Hemoglobin electrophoresis
- is a blood test that can detect different types of
hemoglobin.
- It uses the principles of gel electrophoresis to
 Most blood cells, including red blood cells, separate out the various types of hemoglobin and
are produced regularly in your bone is a type of native gel electrophoresis.
marrow — a red, spongy material found - Electrophoresis uses an electrical current to
within the cavities of many of your large separate normal and abnormal types of
bones. hemoglobin in the blood.
- Hemoglobin types have different electrical
 To produce hemoglobin and red blood cells,
charges and move at different speeds. The
your body needs iron, vitamin B-12, folate
amount of each hemoglobin type in the current is
and other nutrients from the foods you eat.
measured.
Types of Normal Hemoglobin Reticulocyte Production Index (RPI)
1. Hemoglobin F (fetal hemoglobin). - The problem arises because the reticulocyte
- found in fetuses and newborn count is not really a count but rather a
- Hemoglobin F is replaced by hemoglobin A percentage: it reports the number of
(adult hemoglobin) shortly after birth reticulocytes as a percentage of the number of
- Some diseases, such as sickle cell red blood cells.
disease, aplastic anemia, and leukemia (have - In anemia, the patient's red blood cells are
abnormal types of hemoglobin and higher depleted, creating an erroneously elevated
amounts of hemoglobin F.) reticulocyte count.
2. Hemoglobin A - The idea of the RPI is to assess whether the
- most common type of hemoglobin in adults bone marrow is producing an appropriate
- Some diseases, such as severe forms response to an anemic state.
of thalassemia, may cause hemoglobin A - Reticulocyte production should increase in
levels to be low and hemoglobin F levels to be response to any loss of red blood cells. It
high. should increase within 2-3 days of a major
3. Hemoglobin A2 acute hemorrhage.
- This is a normal type of hemoglobin found in - RPI corrects the retic count for the degree of
small amounts in adults. anemia.

Types of Abnormal Hemoglobin

1. Hemoglobin S - RI = Retic Count *Hematocrit/Normal
- present in sickle cell disease. Hematocrit
2. Hemoglobin C
- does not carry oxygen well.
3. Hemoglobin E
- found in people of Southeast Asian descent.
4. Hemoglobin D
- present in a sickle cell disorder.
5. Hemoglobin H (heavy hemoglobin).
- may be present in certain types of
thalassemia.
Interpretation
D. Reticulocyte Count
- measures the number of young red blood - The reticulocyte index (RI) should be between
cells in your blood. 1.0% and 2.0% for a healthy individual.
- shows whether your bone marrow is making - RI < 2% with anemia indicates loss of red
red blood cells at the correct rate. blood cells, but decreased production of
 Differential Diagnosis of Retic Count reticulocytes (ie, and inadequate response to
should be determined to distinguish correct the anemia) and therefore red blood
between underproduction from cells.
hemolysis. - RI > 3% with anemia indicates loss of red
a. Low or normal Retic Ct - in blood cells (from causes such as destruction,
underproduction bleeding, etc.), with an increased
b. High Retic Ct - when bone marrow is compensatory production of reticulocytes to
responding to blood loss, hemolysis or iron replace the lost red blood cells.
deficiency.
- Normal Value: Reticulocyte Count NV 0.5- E. Serum iron / serum ferritin /TIBC tests
1.5% - Transferrin level and total iron-binding
- Absolute RC – the number reticulocytes capacity tests also measure iron levels
actually circulating NV 25,000 -75,000/mcl Ferritin
- Absolute RC = Retic Ct x total numbers of - is a protein that binds to iron
RBC - most of the iron stored in the body is
bound to Ferritin
 - Ferritin is found in the skeletal muscles,  Moderate anemia is considered when
and bone marrow. Only a small amount of hemoglobin is between 8.0 - 9.5 g/dL
ferritin is found in the blood.  Severe anemia is considered for
- The amount of ferritin in the blood shows hemoglobin concentrations below 8.0
how much iron is stored in your body. g/dL
Iron Deficiency Anemia
F. Occult Blood Test
- A test to check the stool for blood . - caused by a shortage of the element iron in
G. Endoscopy your body
- For this test, a tube with a tiny camera is - caused by blood loss, such as from heavy
used to view the lining of the digestive tract. menstrual bleeding, an ulcer, cancer, a polyp
H. FNAB- Cytology somewhere in your digestive system
- Bone marrow aspiration and/or biopsy - caused by prolonged use of aspirin or drugs
- A procedure that involves taking a small known as nonsteroidal anti- inflammatory
amount of bone marrow fluid (aspiration) drugs (NSAIDs).
and/or solid bone marrow tissue (called a core
biopsy), usually from the hip bones, to be
examined for the number, size, and maturity of
blood cells and/or abnormal cells.
I. Schilling Test
- is the classic test for Pernicious Anemia
- it is no longer widely used, as there are safer
and more efficient methods.
- this test can distinguish PA from other forms
of B12 deficiency
Part one of the Schilling test consists of Vitamin Deficiency Anemia
taking an oral dose of radio labelled B12 - In addition to iron, your body needs folate and
and having the radioactivity of the urine vitamin B-12 to produce sufficient numbers of
measured over a 24-hour period. healthy red blood cells.
Second part of the test is a repeat of the - A diet lacking in these and other key nutrients
first, with the addition of oral intrinsic factor. can cause decreased red blood cell
With lower-than-normal amounts of intrinsic production.
factor produced in PA, the addition of - Additionally, some people may eat enough B-
intrinsic factor in the second test allows the 12, but their bodies aren't able to process the
body to absorb more B12, producing a vitamin. This can lead to vitamin deficiency
higher urine radioactivity anemia.
J. DNA analysis
- used to investigate alterations and mutations Megaloblastic (Pernicious) Anemia
in the genes that produce hemoglobin
components. - Caused by digestive diseases, folate
- confirm the diagnosis and establish the exact malabsorption, medication - induced folic acid
genetic type of thalassemia. deficiency, folic acid deficiency
K. Isoelectric focusing and high-performance - The inner contents of each cell are not
liquid chromatography (HPLC) completely developed.
- use similar principles to separate hemoglobins - This malformation causes the bone marrow to
- can be used instead of or in various produce fewer cells, and sometimes the cells
combinations with hemoglobin electrophoresis to die earlier than the 120-day life expectancy.
determine the types and quantities of hemoglobin a. Macrocytes or very large RBCs that have
present. an MCV >100 fL.
b. the inner contents of each cell are not
Types of Anemia (Categorized according to:) completely developed
c. Seen in megaloblastic anemias, such
o Cause of the infection/condition B12/folate as deficiency.
o Morphology of RBCs d. Occurs in anemia of liver disease.
 Poikilocytes - red cells of unequal shape e. RBCs are oval in shape
 Anisocytes - red cells of unequal size.
o Hemoglobin Concentration
 Mild anemia is considered when
hemoglobin is between 9.5 - 13.0 g/dL
  Methe malbuminemia
 hemoglobinuriaHemoglobinanemia
 methemalbuminemia,
 hemoglobinuria
 hemosiderinuria (where there is significant
intravascular hemolysis).
- HA can be inherited or acquired. Common
causes of inherited hemolytic anemia are
sickle cell anemia and thalassemia.

Causes of Hemolytic Anemia

Aplastic Anemia A. Intrinsic causes:

- Hereditary (inherited) hemolytic anemia can
- a rare but serious blood disorder in which the be due to:
body's bone marrow doesn't make enough  Defects of red blood cell membrane
new blood cells because the bone marrow's production (as in hereditary spherocytosis
stem cells are damaged resulting to and hereditary elliptocytosis.
pancytopenia.  Defects in hemoglobin production (as in
- cause is unknown; also is called bone marrow thalassemia, sickle-cell disease and
failure congenital dyserythropoietic anemia)
- AA is not a type of cancer but may be  Defective red cell metabolism (as in
associated with certain cancers (especially glucose-6-phosphate dehydrogenase
those affecting the bone marrow, such as deficiency and pyruvate kinase deficiency)
leukemia) or cancer treatments. A small
number of patients with AA may develop B. Extrinsic causes:
leukemia. - Acquired hemolytic anemia may be causes by
- AA can be inherited or acquired. Acquired immune-mediated causes, drugs, and other
aplastic anemia is much more common than miscellaneous causes
the inherited type.  Immune-mediated causes could include
- causes of inherited aplastic anemia are transient factors as in Mycoplasma
fanconi anemia (FA), dyskeratosis pneumoniae infection (cold agglutinin
congenita (DC), Diamond-Blackfan disease) or permanent factors as
syndrome in autoimmune diseases like autoimmune
- It can develop suddenly or slowly. The disorder hemolytic anemia (itself more common in
tends to get worse over time, unless its cause diseases such assystemic , rheumatoid
is found and treated. Treatments for aplastic arthritis, hodgkin disease and chronic
anemia lymphocytic leukemia)
- include blood transfusions, blood and marrow  Paroxysmal nocturnal hemoglobinuria
stem cell transplants, and medicines. (PNH), sometimes referred to as
Marchiafava-Micheli syndrome, is a rare,
Hemolytic Anemia acquired, potentially life-threatening
disease of the blood characterized by
- Due to problems that cause the red blood cells complement-induced intravascular
(RBCs) to die or be destroyed prematurely. hemolytic anemia.
- Normally, red cells live in the blood for about  Any of the causes of hypersplenism
four months. In hemolytic anemia, this time is (increased activity of the spleen) such as
shortened, sometimes to only a few days . portal hypertension
- Due to hemolysis, the abnormal breakdown of  Acquired hemolytic anemia is also
RBCs, either in the blood vessels encountered in burns and as a result of
(intravascular hemolysis) or elsewhere in the certain infections.
human body (extravascular)  Lead poisoning resulting from the
- Increased breakdown of hemoglobin, which environment causes non-immune
may result in: hemolytic anemia.
 increased bilirubin level (mainly indirect-  Runners can suffer hemolytic anemia due
reacting) with jaundice to "footstrike hemolysis", owing to the
 increased fecal and urinary urobilinogen destruction of red blood cells in feet at foot
 Hemoglobinanemia Hemoglobinanemia, impact.
 Methemalbuminemia Hemoglobinanemia,
  Low-grade hemolytic anemia occurs in Sideroblastic Anemia
70% of prosthetic heart valve recipients,
and severe hemolytic anemia occurs in 3% -disease in which the bone marrow produces ringed
 March hemoglobinuria - also known as sideroblasts rather than healthy RBCs
march hematuria -word "march" is in - caused either by a genetic disorder or indirectly as
reference to the condition arising in part of myelodysplastic syndrome which can evolve
soldiers who have been marching for long into hematological malignancies.
periods; the condition was first documented - Sideroblasts are atypical abnormal nucleated
in 1881. erythroblasts (precursors to mature red blood cells)
Sickle Cell Anemia with granules of iron accumulated in perinuclear
mitochondria Sideroblasts are seen in aspirates
- also called sickle cell disease (SCD), of bone marrow .
drepanocytosis - Ring sideroblasts are named so because of the
- is a hereditary blood disorder characterized arrangement of the iron granules in a ring form
- by red blood cells that assume an abnormal, around the nucleus.
rigid, sickle shape.
- an inherited disorder that leads to the
production of hemoglobin S (Hb S or Hgb S),
an abnormal hemoglobin variant.
- Sickled RBCs are generally short-lived, only
lasting about 10-20 days
a. Shapes vary but show thin, elongated
pointed ends, and appear crescent shaped.
b. Contains hemoglobin S Cell shape is
Erythrocytic Morphology and Associated
caused by cell membrane alterations due
Diseases (Size and Shape)
to hemoglobin S.
c. Decreased osmotic fragility. Normocytes (normal erythrocytes)

Thalassemia Macrocytes

- An inherited blood disorder characterized by - Erythrocytes that have an MCV >100 fL.
less hemoglobin and fewer red blood cells - Seen in megaloblastic anemias, such
- PBS –rbcs are microcytic, target cells look like B12/folate as deficiency.
bull’s eye - Occurs in anemia of liver disease.
- Several types of thalassemia exist, including
alpha-thalassemia, beta-thalassemia, Cooley's
anemia and Mediterranean anemia.
A. Alpha Thalassemia
- is caused by a change in the genes for the
alpha globin component of hemoglobin.
- most common among people of Southeast
Asian and Chinese descent.
- two types: hemoglobin H disease and alpha
thalassemia major Microcytes
B. Beta Thalassemia (Cooley's Anemia)
- Are small RBC’s less than 6 micrometers in
- is caused by a change in the gene for the
diameter.
beta globin component of hemoglobin.
- Shows an MCV >80 fL
- Seen in iron deficiency anemias, thalassemias,
hemolytic anemias, sideroblastic anemias, and
chronic disorder anemias.
Echinocytes Target Cells
- Codocytes or Mexican hat.
- Crenated RBC
- Show a central area of hemoglobin surrounded
- Show uniform round bumps or spikes on the
by colorless ring and a peripheral ring of
RBC surface
hemoglobin.
- Usually indicates artifacts of staining or
- Considered artifacts if appears in only one
increased platelets
section of the smear.
- No pathology is indicated.
- Seen in liver disease, Hb SS,SC, S
- Caused by changes in cellular osmotic
thalassemia, thalassemia, iron deficiency, and
pressure.
postplenectomy

Burr Cell Spherocytes

- Keratocytes. - Slightly smaller (6.2-7.0 microliter) than normal
- Irregular in size with spiny projections. RBC.
- Seen in renal insufficiency, liver disease, - Show no central pallor.
ulcers, and heparin therapy. - Normal MCV, increased MCHC, and increased
- Caused by cell membrane breakup due to osmotic fragility.
cytoplasmic vacuoles. - Seen in burns, hereditary spherocytosis, and
- Can be confused with crenated erythrocytes. extravascular hemolytic processes.
- Microspherocyctes are frequently seen in
severe burn cases.
- Spherocyctes and microspherocytes result
when the RBC surface area decrease due to a
decrease in cell volume.

Acanthocytes
- Appear as small, densely stained RBCs with Teardrops
multiple irregularly spaced spikes or clublike - Dacryocytes
projections. - Tennis racquet cell
- Associated MAHA (microangiopathic hemolytic - Show a tapered and round end. Slightly
anemia), alcoholic liver diseases, hereditary smaller than normocytes
acanthocytosis, and abetalipoproteinemia. - Usually microcytic and/or hypochromic
- Caused by excessive cholesterol in the - Abnormality is associated with
membrane. myeloproliferative syndrome, pernicious
 anemias, ineffective erythropoiesis, - May be found whenever blood vessel
thalassemia, and myelophthisic anemia. pathology is present.
- Seen in DIC, burns, renal transplant rejection
and hemolytic processes.

Stomatocytes
Sickle Cells - Characterized by an elongated or slit like area
of central pallor that resembles a mouth.
- Shapes vary but show thin, elongated pointed - Stomatocytes results from increased sodium
ends, and appear crescent shaped. and decreased potassium concentration within
- Contains hemoglobin S. the cytoplasm of RBC.
- Seen in sickle cell anemia. - Seen in hereditary stomatocytosis, obstructive
- Cell shape is caused by cell membrane liver disease, alcoholism.
alterations due to hemoglobin S.
- Decreased osmotic fragility.

Elliptocytes
Helmet Cells
- Ovalocytes
- Keratocytes - Cells have rod, cigar, or sausage shape.
- Interior portion of the cell is hollow, resembling - Caused by membrane integrity defect.
a helmet. - Seen in hereditary elliptocytosis, iron
- These cell fragments are formed in the spleen deficiency anemias, megaloblastic anemia,
and intravascular fibrin clots. thalassemia, and sickle cell anemia.
- Associated with microangiopathic hemolytic
anemia.

Schistocytes Nucleated RBC

- RBC fragment. - nRBCs
- RBC fragmentation results from passage - Most are orthochromic normoblast but can
through damaged or altered blood vessels. appear in any erythrocytic stage of maturation.
- Variety of shapes: triangular, comma-shaped, - Indicate some type of bone marrow stimulation
helmet shaped. or increased erythropoiesis.
- Microcytic - Normally found in newborns.
- Seen in acute blood loss, leukemias,
hypoxia,megaloblastic anemias, heart disease,
and myelofibrosis.
- As few as one nRBC should be reported when
seen on adult peripheral slide.

Heinz Bodies
- Seen in glucose6phosphate dehydrogenase
deficiency.
- Results from denatured hemoglobin.
- Multiple inclusions ranging in size from 0.3 to
Erythrocyte Inclusions and Associated Disease 2.0 micrometer.
Howell-Jolly Bodies
- Appear as small, round fragment (1 to 2
micrometer in diameter) of nuclear material
(DNA) that may be single or multiple.
- Caused by nuclear disintegration.
- Fragment stain reddish/blue to purple.
- Not seen in normal erythrocytes.
- Seen in sickle anemia, megaloblastic anemia,
alcoholism, splenectomy, hemolysis, and Pappenheimer bodies
hemoglobinopathies.
- Small irregular, dark-staining granules (iron
granules) clumped together at one end or
region.
- Caused by an accumulation of ribosome,
mitochondria, and iron fragments.
- Seen in sideroblastic anemia, thalassemia,
hemosiderosis, and megaloblastic anemia.

Basophilic Stippling
- Multiple, tiny, fine, or coarse inclusions
(ribosomal RNA remnants) throughout the cell.
- Stain dark blue to purple.
- Seen in thalassemias, megaloblastic anemias,
lead poisoning, alcoholism, and disorders that
increase erythropoiesis. Role of Medical Technologist in the
- Larger more coarse granules hold greater Diagnosis of Anemia
pathological importance. Allied health professional
- recognition of signs and symptoms and
correlation of laboratory results with clinical
manifestations
Frontline morphologist
- evaluation and identification of abnormal cells
in the peripheral blood smear

Cabot Ring Laboratory scientist

- performance and interpretation of basic and
- Thin, blue to reddish-purple, single to multiple
state of the art laboratory assays
ringlike structure that may appear in loop or “8”
Clinical researcher
shapes.
- investigation of future and potential laboratory
- Seen in megaloblastic anemia, lead poisoning,
tests foraccurate, quick and reliable diagnosis
and dyserythropoiesis.
- Caused by fagments of nuclear material.