Laboratory Diagnosis of Anemia
By
Dr. Varughese George
Department of Pathology
Learning Objectives
At the end of this briefing, you should know
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Clinical presentation of anemia.
Definition of anemia.
Approach to diagnosis of anemia
Classification of anaemia
Distinct features of each type of anemia.
Clinical presentation of anemias
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Fatigue and weakness
Headache
Tinnitus
Numbness and coldness
Pallor
Dyspnea and palpitations
Angina pectoris
Intermittent claudication
Hemorrhages in the
fundus of eyes
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Menorrhagia
Anorexia
Flatulence
Nausea
Constipation
Clinical presentation of anaemias
Initial Laboratory Work-Up
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Hemoglobin concentration.
Packed Cell Volume.
Red cell indices.
Peripheral Blood Smear.
Definition of Anaemia
• Anaemia is defined as a reduction in the concentration
of circulating haemoglobin below the level that is
expected for healthy personsof same age and sex in the
same environment.
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Blood parameters (Laboratory Normal Range)
Blood parameters (Laboratory Normal Range)
•In full term infants, hemoglobin is 18.0 ± 4.0 g/dl
•Children - 12.5 ± 1.5 g/dl
•Children(6-12 years) - 13.5 ± 2.0 g/dl
Grading of Anaemia
Classification of Anaemias
• Morphological Classification
• Etiological Classification
• Classification based on reticulocyte response.
Morphological Classification of Anemias
Hematocrit
• Proportion of the volume of red cells relative
to the volume of blood
• Rules of Three:
– RBC X 3 = Hemoglobin
– Hemoglobin X 3 = Hematocrit
Packed cell volume (PCV) or Haematocrit (Hct)
Men - 0.45 ± 0.05 l/l (40-50%)
Women - 0.41 ± 0.05 l/l (38-45 % in non- pregnant women
36-42 % in pregnant women)
Mean Corpuscular Volume
• Dividing the total volume of red cells by the
number of red cells
• Index for average size of red cells
• Normal range - 92 ± 9 fl
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Mean Corpuscular Hemoglobin
• Average amount of haemoglobin in each red
cell.
• It is expressed in picograms or pg.
• Normal range - 29.5 ± 2.5 pg
Mean Corpuscular Hemoglobin Concentration
• This represents the average concentration of
haemoglobin in a given volume of packed red
cells.
• Normal range – 330 ± 15 g/l
• MCHC raised in hereditary spherocytosis.
• Decreased in hypochromic anaemia.
Red cell distribution width
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Variation in red cell size
Normal range - 12.8% ± 1.2%
Low in B-thalassemia trait
High in iron deficiency anaemia
Normal in anaemia of chronic disease
Morphologic Categories of Anemia
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1 Microcytic/hypochromic
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2 Macrocytic/Normochromic
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3 Normocytic/Normochromic
N.B. The nucleus of a small
lymphocyte (shown by the arrow)
is used as a reference to a normal
red cell size
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Macrocytic anemia
• Low/normal reticulocyte
count, macrocytosis(oval
and round)
• Elevated MCV,MCHC
• Basophilic stippling
• Howell-jolly bodies
• Cabot rings
• Pancytopenia
• Hypersegmented
neutrophils
• Bone marrowmegaloblastic
maturation,sieve like
chromatin,nuclearcytoplasmic
asynchrony,maturation
arrest
Normocytic Normochromic anaemia
with effective erythropoiesis
Etiological Classification of Anaemias
Basic Approach to a diagnosis of anemia
Evaluation of microcytic hypochromic anaemia
Evaluation of macrocytic anaemia
Evaluation of normocytic anaemia
Evaluation of haemolytic anaemia
A simplified approach to diagnosis of haemolytic
anaemias
Reticulocyte Count
(In the Diagnosis of Anemia)
• Reticulocytes are nonnucleated RBCs that still
contain RNA.
• Visualized by staining with
supravital dyes, including
new methylene blue or
brilliant cresyl blue.
• Useful in determining
response and potential of
bone marrow.
• Normal range is 0.5-2.5%
of all erythrocytes.
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Fe++ deficiency anemia
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Low hemoglobin and low packed cell volume
Low MCV,MCH and MCHC
Microcytosis & hypochromia are hallmarks
RDW is increased
Serum ferritin is less than 15 micro gram/dl
Serum iron is low,TIBC is increased and transferrin saturation is less
than 10 percent
• Free erythrocyte protoporphyrin is increased.
• Increased soluble transferrin receptor in serum
• Bone marrow-micronormoblastic,absence of stainable iron in bone
marrow on Perls Prussian blue reaction
Megaloblastic Anemia
Mild to severe anemia,
– Increased MCV & MCH, normal MCHC
– Low RBC, HGB, WBC and PLT counts (fragile cells) due
to ineffective hematopoiesis.
– Low reticulocyte count
– Macrocytic ovalocytes and teardrops;
– Marked anisocytosis and poikilocytosis
– Schistocytes/microcytes - due to RBC breakage upon
leaving the BM
– Erythroid hyperplasia - low M:E ratio (1:1)
– Iron stores increased.
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Megaloblastic anemia
Howell-Jolly body
Teardrop
Schistocyte
Blood
Macrocytic Ovalocytes
Stippled RBC &
Cabot Ring
NRBC
Blood
Giant Platelet
Pap bodies
Hypersegmented Neutrophil >5
lobes
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Tests
• Folate and B12 levels
• Schilling test may be useful to establish
etiology of B12 deficiency
– Assesses radioactive B12 absorption with and
without exogenous IF
• Other tests if pernicious anemia is suspected
– Anti- parietal cell antibodies, anti-IF antibodies
– Secondary causes of poor absorption should be
sought (gastritis, ileal problems, etc.)
Anemia of chronic disease
• Normocytic anemia with ineffective erythropoiesis
(reduced reticulocyte count)
• Normochromic
• Results from
– Chronic inflammation (e.g. rheumatologic disease):
Cytokines released by inflammatory cells cause
macrophages to accumulate iron and not transfer it to
plasma or developing red cells (iron block anemia)
– Inflammation
– malignancy
• Bone marrow suppression (EPO is elevated)
Anemia of chronic disease
• Decreased serum iron,decreased total iron
binding capacity and normal or raised ferritin
• Increased marrow storage iron
• ESR is high
Normochromic, normocytic anemia with
effective erythropoiesis
INCREASED reticulocyte count
• Acute blood loss
– Very acutely, with hypovolemia,
may have normal blood counts,
will become anemic with
volume replenishment
• Hemolytic anemia
– Increased reticulocyte
production cannot keep pace
with loss of RBCs peripherally.
• Response to specific therapy in
nutritional anemias
Aplastic anemia
• Pancytopenia caused by bone marrow
failure…decreased production of all cell
lines and replacement of marrow with
fat.
• Inherited- Fanconis anaemia, Dyskeratosis
congenita
• Acquired - Idiopathic,drugs like
NSAIDs,chloramphenicol,benzene,parvo
virus,hepatitis and EB virus.
Hemolytic anemia
• Abnormality intrinsic to red cells1. Hereditary spherocytosis
2. Thalassamia
3. Sickle cell anaemia
4. Glucose -6-phosphate dehydrogenase
deficiency
• Abnormality extrinsic to red cells1. Immune
2. Mechanical etc
Evaluation of haemolytic anaemia
Hereditary spherocytosis
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Inherited defect in the red cell membrane
cytoskeleton (spectrin, ankyrin or band 3)
leading to the formation of spherocytic red
cells.
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Autosomal dominant
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Mild to moderate anaemia
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Intermittent jaundice
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Splenomegaly
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Pigment gall stones
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Peripheral smear-microspherocytes
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Screening test-osmotic fragility
Thalassemia
• Decreased or absent globin
chains
• Alpha and beta
thalassemias
• Microcytic
hypochromic,target
cells,basophilic stippling
• Reticulocytosis
• Hb F elevated in
electrophoresis
Sickle cell anaemia
• Presence of Hb S
• Point mutation in 6th place of beta
chain
• Substitution of valine for glutamic acid
• On deoxygenation,sickle cells are
formed
• Chronic hemolytic anaemia,vasoocclusive crisis
• Aplastic crisis
• Hemolytic crisis
• Infections
Sickle cell anaemia
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Sickling test is positive.
Solubility test is positive.
Electrophoresis shows HbS.
In sickle cell trait, electrophoresis shows 60
percent of Hb A and 40 percent Hb S
Glucose-6-phosphate dehydrogenase
deficiency
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X linked disorder
Reduced activity of G6PD
Inability to remove H2O2
Accumulated H2O2 leads to
oxidation of hemoglobin
with precipitation of globin
chains
• Heinz bodies
• Red cells with heinz bodies
destroyed in
spleen(extravascular
hemolysis)
Glucose-6-phosphate dehydrogenase
deficiency
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Asymptomatic
Neonatal jaundice
Acute hemolytic anaemia
Chronic hemolytic anaemia
On peripheral smearpolychromasia,fragmented red
cells,spherocytes,bite cells,half
ghost cells
• Biochemical-increased
bilirubin,hemoglobinemia and
hemoglobinuria
Glucose-6-phosphate dehydrogenase
deficiency
• Screening tests-fluorescent spot
test,methemoglobin reduction test and dye
decolorisation test
Immune hemolytic anaemia
• Warm antibody-persons over 50 years,mild
jaundice and splenomegaly,red cells coated
with IgG,spherocytes.Seen in autoimmune
disorders,lymphoma
• Cold antibody-acrocyanosis,IgM.Seen in cold
agglutinin disease, Paroxysmal cold
hemoglobinuria (PCH)
Coomb’s test
• Detects presence of either antibody on RBC or
of antibody in serum
• Helpful in determining if a hemolytic anemia is
immune-mediated
SUMMARY
• Microcytic hypochromic anaemia-iron
deficiency
• Macrocytic hyperchromic-megaloblastic
anaemia
• Normochromic normocytic-hemolytic
anaemia
• Pancytopenia-megaloblastic and aplastic
anemias