What is anemia, how do you

diagnose anemia, and how are the

different anemias classified?
► Anemia is the inability of the blood to supply the tissue
with adequate oxygen for proper metabolic function.
► Clinically, anemia is defined as a decrease in normal

concentration of hemoglobin or erythrocytes.

► Anemia is not a disease, but an expression of an
underlying disorder or disease.
► Two common laboratory tests used to measure anemia
◦ Hemoglobin
►Normal values
►Male: 14-18 g/dl
►Female: 12-16 g/dl
►Moderate anemia: 7-10 g/dl
►Severe anemia: <7 g/dl

◦ Hematocrit - the proportion of red cells in whole blood expressed

as a percentage (packed red cell volume)
 Normal values
►Male: 42-52%
►Female: 37-47%
 Newborns less than one week old have hemoglobin of 14-22 g/dl.
 By six months of age, hemoglobin runs: between 11 and 14 g/dl.
 Between 1 year and 15 years of age hemoglobin runs between 11-15 g/dl.
 Normal adult hemoglobin depends on gender:
◦ ♀ 12-16 g/dl
◦ ♂ 14-18 g/dl
 In geriatric age group, men and women have same hemoglobin range:  12-
16 g/dl.

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 Acute blood loss (hemorrhage)
 Accelerated destruction of RBC’s
 Nutritional deficiency (iron, folate or B12)
 Chronic Infections
 Toxicity
 Hematopoietic stem cell arrest or damage
 Hereditary or acquired defect
 Malignancies
► Clinicalhistory
► Physical signs such as pallor, fatigue, weakness and
shortness of breath
► Laboratory tests
◦ CBC
◦ Examination of the blood smear
◦ Reticulocyte - measures effective erythropoiesis
◦ Bone marrow examination
◦ Iron studies - iron, total iron-binding capacity (TIBC), ferritin
◦ Vitamin B12 and folate
◦ Erythropoietin level
 Haemoglobin level at which symptoms and signs of
anemia develop
◦ Speed of onset
◦ Severity of anemia
◦ Age of the patient

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 ↓oxygen carrying capacity
 Shift to right
 ↑ 2,3-DPG
 ↑ Cardiac output
 Circulation shifts to critical areas
 ↑ RBC production
 ↑ Erythropoietin
 ↑ Reticulocyte count

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► Easy fatigability
► Dyspnea on exertion
► Faintness/ Vertigo
► Pallor
► Rapidly bounding pulse
► Dependent edema
► Systolic murmurs
► Hemoglobin
► Hematocrit
◦ Note: the approximate relationship of the hemoglobin to the hematocrit is 1:3.
This may vary with the cause of the anemia and the effect on the RBC indices,
especially the MCV.
► RBC indices
◦ MCV - mean cell volume
► Normal: male - 80-94 fl, female - 81-99 fl)
► Indicates the average size (volume) of the red cells
Calculation: Hct x 10
RBC
► MCH - mean cell hemoglobin weight
◦ Normal: 27-31 pg
◦ A measurement of the hemoglobin content in RBC’s
Calculation: Hgb x 10
RBC

► MCHC - mean cell hemoglobin concentration

◦ Normal: 32-36 %
◦ A measure of the concentration of hemoglobin in the average RBC
Calculation: Hgb x 100
Hct
► Examination of the RBC’s in the peripheral blood smear
◦ Alteration in size of the RBC is called anisocytosis.
◦ Alteration in shape of the RBC is called poikilocytosis.
◦ An RBC with normal hemoglobin content will appear normochromic
(MCHC 32-36).
◦ An RBC with decreased hemoglobin content will appear hypochromic
(MCHC <32).
◦ RBC size is designated as microcytic (small MCV <80), normocytic
(normal size MCV 80-100), or macrocytic (large MCV >100).
 In healthy individuals, about 1% of RBCs lost daily.
 Bone marrow continuously produces RBCs to equal

daily loss.
 Reticulocyte count is a lab measurement of this loss.

Normal retic count is 0.5-2.0% of circulating RBCs.

 Replacement requires functioning bone marrow, normal

RBC maturation and ability to release mature RBCs to

peripheral blood.
 Proper nutrition required (B12, Folate).  Also requires

normal hemoglobin synthesis.

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 Severe anemia (<7 Hb) may see other organ system failures: 
Cardiac and respiratory.

 Do have compensatory mechanism:  See an increase in 2,3-DPG

levels which results in an increase in RBCs’ oxygen carrying
capacity.

 Erythropoietin levels (Epo) useful diagnostic tool. Anemic

people usually respond by increasing erythropoietin levels.

 Erythropoietin is a hormone produced in the kidney. Levels of
erythropoietin varies with oxygen tension in kidney tissues (↓
Oxygen - ↑ Epo, and vice versa)
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 Have a variety of ways - depending on criteria used:
◦ Functional
◦ Morphological
◦ Clinical
◦ Quantitative

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 Decreased RBC production (hypoproliferative)
◦ Defective hemoglobin synthesis
 Fe deficiency
 B12 deficiency
 Folate deficiency
◦ Impaired bone marrow or stem cell function, as in leukemia
 Increased RBC destruction, as in sickle cell anemia or
hemolytic anemia
 Combination of the two (sometimes called “ineffective
erythropoiesis”)

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 Morphological based on sizes and color of RBCs
◦ Normochromic Normocytic
◦ Hypochromic Microcytic
◦ Normochromic Microcytic
◦ Normochromic Macrocytic

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 According to their associated causes:
◦ Blood loss
◦ Iron deficiency
◦ Hemolysis
◦ Infection
◦ Nutritional deficiency
◦ Metastatic bone marrow replacement

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 Quantitatively by:
◦ Hematocrit
◦ Hemoglobin
◦ Blood cell indices
◦ Reticulocyte count

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 Total stores (4-5 g)/ 75-90 mmol
 Hb contains: 2.5 g (45 mmol)= 70% of total body iron
 Storage iron (1-1.5 g): (2/3 as ferritin, 1/3 as hemosiderin)
 Myoglobin : 0.2 g (3.5 mmol)
 Intracellular enzymes: 0.1 g

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 Iron absorbed in GI tract, transported by transferrin to
BM
 Iron absorbed by pinocytosis by RBC precursors
 After 120 days, RBC phagocytosed in spleen

◦ iron is reused
◦ hemoglobin converted to bilirubin
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 Increased Need
◦ Early childhood and adolescence (growth spurts)
◦ Pregnancy (extra 3.8 mg/day over baseline)
◦ Lactation
 Poor Intake/Absorption
◦ Milk baby
◦ Achlorhydria
◦ Inflammatory bowel disease
 Menstruating women
 GI bleeding (most common pathologic cause)
 Tissue loss
 Urinary Loss
 Iatrogenic
 Hypochromic microcytic anemia
 low iron, high transferrin, low ferritin
 small ragged RBC precursors
 lack of stainable iron
► Anemia with macrocytic red cells (MCV > 100 fL)
► Low-normal absolute reticulocyte count
► BM shows intense erythroid hyperplasia w/ abnormal
morphology.
► Macroovalocytes and occasional megaloblasts can be
seen.
► Hypersegmented PMN ( > 5% w/ 5 or more lobes or > 1% w/ 6 or
more lobes.)
►A result of impaired DNA synthesis due to def. in
Folate and/or vitamin B12 (cobalamin).
► Normal B-12 absorption:
◦ Dietary B-12 binds to R factor in saliva and gastric juices.

◦ In duodenum, pancreatic enzymes promote dissociation from R factor and

binding to Intrinsic Factor (IF)

◦ IF-B12 complex taken up by ileal receptor cubilin.

◦ Released into plasma bound to transcobalamines TC I, II, or III.

◦ Enters cells through receptor mediated endocytosis and metabolized into

two coenzymes: adenosyl-Cbl and methyl-Cbl.

◦ FORMS OF VITAMIN B12: Deoxyadenosylcobalamin

(coenzyme B12), methylcobalamin, hydroxycobalamin,
cyanocobalamin
 Sources: dietary
 Daily requirement : 2 microgram
 Normal mixed diet has 5-30 microgram (daily)
 Daily loss is 0.1 % of the total pool of B12 (3-4 mg)
 Bound to transcobalamins in blood.

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► Initial work-up
◦ Check serum B12, folate (serum or red cell), TSH,
Reticulocyte panel, CBC, Fe panel, Social Hx, Neuro exam.
◦
◦ MCV predictive of B12/folate def.

◦ If serum B12 and folate levels are equivocal, check serum

HC and MMA levels.
 In folate def, only HC is elevated (Sn 86%, Sp 99%).
 In B12 def, both HC and MMA is elevated (Sn 94% Sp 99%).

◦ Presence of neurological deficits may also indicate B12 def.

► Clinical Features:
◦ Megaloblastic anemia
◦ Serum B-12 def
◦ Chronic atrophic gastritis
◦ Neurologic manifestations (paresthesias, numbness, weakness, memory loss,
personality changes, ataxia, loss of vibration and position sense, psychosis “megaloblastic
madness”)
◦ Atrophic glossitis
◦ Achlorhydria (lack of HCl in gastric juice)
◦ Elevated serum bilirubin reflective of increased RBC breakdown
due to ineffective erythropiesis.
► Pathophysiology
◦ Autoantibody to IF
►Two types:
 Type I blocks attachment of B-12 to IF
 Type II blocks B-12-IF complex to ileal receptor
►Present in up to 70% of patients with P.A. and Sn approaches 100%.
◦ Autoantibody to gastric parietal cells
►Directed against the H/K-ATPase on cell membrane
►Leads to decline in # of parietal cells and IF production
►Leads to chronic atrophic gastritis and gastric atrophy.
►Found in 90% of patients with pernicious anemia.
► Chronic Atrophic Gastritis
◦ Type A (autoimmune):
►The type involved in P.A. (due to autoantibodies)
►Involves the fundus and body which contain acid-secreting parietal
cells and spares the antrum which contains gastrin-producing cells.
This leads to achlorhydria and high serum gastrin levels.
◦ Type B:
►Involves fundus, body, and antrum
►Usually associated with H.pylori infection.
◦ The progression of Type A chronic atrophic gastritis to
gastric atrophy and clinical anemia is likely to span 20 – 30
years.
► Schilling Test 1

►Give 1mcg of radiolabeled B-12 orally, followed by 1000 mcg of

B-12 IM one hour later to “flush” any absorbed radiolabeled B-12
from tissues. A 24-hr urine is collected to determined how much
radiolabeled B-12 is excreted. Normal is 8-35%.
 On basis of H&H, anemia can be classified as mild, moderate, or
severe.
 On basis of duration of onset, anemia can be classified as either
chronic or acute.
 Rules of Three:
◦ RBC X 3 = Hemoglobin
◦ Hemoglobin X 3 = Hematocrit
 Ratio of Hb and Hct will vary with cause of anemia and affect
the RBC indices, particularly the MCV (Mean Corpuscular
Volume).
 Microscopic examination of peripheral blood smear is required
for evaluation of anemia.  Bone marrow aspirates and smear
evaluation may also be needed.
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 RBC indices include:
◦ Mean Corpuscular Volume (MCV)
◦ Mean Corpuscular Hemoglobin (MCH)
◦ Mean Corpuscular Hemoglobin Concentration (MCHC)

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 Mean cell volume
 MCV is average size of RBC
 MCV = Hct x 10
RBC (millions)
 If 80-100 fL, normal range, RBCs considered
normocytic
 If < 80 fL are microcytic
 If > 100 fL are macrocytic

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 MCH is average weight of hemoglobin per RBC.
 MCH = Hgb x 10
RBC (millions)

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 MCHC is average hemoglobin concentration per RBC
 MCHC = Hgb x 100

Hct (%)
 If MCHC is normal, cell described as normochromic
 If MCHC is less than normal, cell described as
hypochromic
 There are no hyperchromic RBCs

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COMPONENT NORMAL RANGES
WBC 4.8-10.8 x 103/μL
RBC Male 4.7-6.1 x 106/μL; Female 4.2-5.4 x 106/μL
Hgb Male 14-18 g/dL; Female 12-16 g/dL
Hct Male 42-52%; Female 37-47%
MCV 80-100 fL
MCH 27-31 pg
MCHC 32-36%
RDW 11.5-14.5%
Plt 150,000-350,000/μL
Retic 0.5-2.0%

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 Hb is the main component of RBCs and carries oxygen
to tissues.
 Three methods to measure hemoglobin:
◦ Cyanmethemoglobin (recommended method)
◦ Oxyhemoglobin
◦ Iron Content 

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 1. Blood is diluted in a solution of potassium
ferricyanide and potassium cyanide, which oxidizes the
hemoglobin to form methemoglobin.
 2. Then methemoglobin forms cyanmethemoglobin in
the presence of the potassium cyanide.
 3. Absorbance of solution is read in spectrophotometer
at 540 nm.

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 Advantages:
◦ Most forms of hemoglobin are measured
◦ Sample can be directly compared with a standard
◦ Solutions are stable
◦ Method is precise
 Errors in the measurement of Hgb:
◦ Must draw and handle specimen correctly
◦ Reagents must be properly prepared and stored
◦ Equipment failure
◦ Operator error
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 Is packed RBC volume
 Is ratio of RBC volume to volume of whole blood
 Usually expressed in percentage (42%) or as
decimal fraction (.42)
 Venous and arterial hematocrits closely agree
 Specimen of choice is EDTA (ethylenediaminetetra
acetic acid), oxalate or heparin

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 Measurement done by centrifugation or through
calculations performed on many automated
measurements.
 Calculated hematocrit is product of MCV and RBC
count.
 Normal ranges are 42-52% in men and 37-47% in
women.
 Normal ranges also vary among age groups, institutions,
and geographic locations.

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 Problems in measurement of hematocrits include:
◦ Incorrect centrifuge calibration
◦ Choice of sample site
◦ Incorrect ratio of anticoagulant to blood; Improper amount of
blood drawn
◦ Reading errors

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 RBC indices are readily available from the automated
hematology counting devices
 MCV is measured directly or calculated from
hematocrit and RBC count; MCH and MCHC are both
calculated

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 In various anemic states, indices may be altered:
◦ Microcytic Anemia:
 MCV usually 50-80 fL
 MCH usually 15-25 pg
 MCHC usually 22-30%
◦ Macrocytic Anemia:
 MCV usually 100-120 fL

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 Very useful in diagnosing and classifying anemias
 Look for:
◦ Neutropenia
◦ Thrombocytopenia
◦ Hypochromia
◦ Size and shape of RBCs
◦ Unusual leukocytes (hypersegmentation)
◦ Red cell inclusions: basophilic stippling, Howell-Jolly
bodies…

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 Useful in determining response and potential of bone
marrow.
 Reticulocytes are non-nucleated RBCs that still contain
RNA.
 Visualized by staining with supravital dyes, including
new methylene blue or brilliant cresyl blue; RNA is
precipitated as dye-protein complex.
 Normal range is 0.5-2.0% of all erythrocytes.
 If bone marrow responding to anemia, should see
increases in retic count.
 Newborns have higher retic count than adults until
second or third week of life.
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 Bone marrow aspiration and biopsy are important
diagnostic tools in the determination of anemia.

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 Hemoglobin Electrophoresis
 Antiglobulin Testing
 Osmotic Fragility
 Sugar Water Test
 Ham’s Test
 RBC Enzymes
 B12, Fe, TIBC, Folate Levels

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Thank You For Your Attention

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