Laboratory diagnosis of anemia

1. Laboratory Diagnosis of AnemiaBy
Dr. Varughese George
Department of Pathology

2. Learning Objectives
At the end of this briefing, you should know
• Clinical presentation of anemia.
• Definition of anemia.
• Approach to diagnosis of anemia
• Classification of anaemia
• Distinct features of each type of anemia.

3. Clinical presentation of anemias
• Fatigue and weakness
• Headache
• Tinnitus
• Numbness and coldness
• Pallor
• Dyspnea and palpitations
• Angina pectoris
• Intermittent claudication
• Hemorrhages in the
fundus of eyes
• Menorrhagia
• Anorexia
• Flatulence
• Nausea
• Constipation

4. Clinical presentation of anaemias

5. Initial Laboratory Work-Up
• Hemoglobin concentration.
• Packed Cell Volume.
• Red cell indices.
• Peripheral Blood Smear.

6. 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.
6

7. Blood parameters (Laboratory Normal Range)

8. 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

9. Grading of Anaemia

10. Classification of Anaemias
• Morphological Classification
• Etiological Classification
• Classification based on reticulocyte response.

11. Morphological Classification of Anemias

12. 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)

13. 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
13

14. 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

15. 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.

16. Red cell distribution width
• 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

17. 17
1 Microcytic/hypochromic
3
1 2
2 Macrocytic/Normochromic
3 Normocytic/Normochromic
Morphologic Categories of Anemia
N.B. The nucleus of a small
lymphocyte (shown by the arrow)
is used as a reference to a normal
red cell size

20. Macrocytic anemia
• Low/normal reticulocyte
count, macrocytosis(oval
and round)
• Elevated MCV,MCHC
• Basophilic stippling
• Howell-jolly bodies
• Cabot rings
• Pancytopenia
• Hypersegmented
neutrophils
• Bone marrow-
megaloblastic
maturation,sieve like
chromatin,nuclear-
cytoplasmic
asynchrony,maturation
arrest

21. Normocytic Normochromic anaemia
with effective erythropoiesis

22. Etiological Classification of Anaemias

23. Basic Approach to a diagnosis of anemia

24. Evaluation of microcytic hypochromic anaemia

25. Evaluation of macrocytic anaemia

26. Evaluation of normocytic anaemia

27. Evaluation of haemolytic anaemia

28. A simplified approach to diagnosis of haemolytic
anaemias

29. Reticulocyte Count
(In the Diagnosis of Anemia)
• Reticulocytes are non-
nucleated 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.
29

30. Fe++ deficiency anemia
• 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

31. 31
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.

32. 32
Macrocytic Ovalocytes
Blood NRBC Blood
Howell-Jolly body
Teardrop
Schistocyte
Stippled RBC &
Cabot Ring
Giant Platelet
Pap bodies Hypersegmented Neutrophil >5
lobes
Megaloblastic anemia

33. 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.)

34. 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)

35. Anemia of chronic disease
• Decreased serum iron,decreased total iron
binding capacity and normal or raised ferritin
• Increased marrow storage iron
• ESR is high

36. 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

37. 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.

38. Hemolytic anemia
• Abnormality intrinsic to red cells-
1. Hereditary spherocytosis
2. Thalassamia
3. Sickle cell anaemia
4. Glucose -6-phosphate dehydrogenase
deficiency
• Abnormality extrinsic to red cells-
1. Immune
2. Mechanical etc

39. Evaluation of haemolytic anaemia

40. Hereditary spherocytosis
• Inherited defect in the red cell membrane
cytoskeleton (spectrin, ankyrin or band 3)
leading to the formation of spherocytic red
cells.
• Autosomal dominant
• Mild to moderate anaemia
• Intermittent jaundice
• Splenomegaly
• Pigment gall stones
• Peripheral smear-microspherocytes
• Screening test-osmotic fragility

41. Thalassemia
• Decreased or absent globin
chains
• Alpha and beta
thalassemias
• Microcytic
hypochromic,target
cells,basophilic stippling
• Reticulocytosis
• Hb F elevated in
electrophoresis

42. 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,vaso-
occlusive crisis
• Aplastic crisis
• Hemolytic crisis
• Infections

43. Sickle cell anaemia
• 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

44. Glucose-6-phosphate dehydrogenase
deficiency
• 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)

45. Glucose-6-phosphate dehydrogenase
deficiency
• Asymptomatic
• Neonatal jaundice
• Acute hemolytic anaemia
• Chronic hemolytic anaemia
• On peripheral smear-
polychromasia,fragmented red
cells,spherocytes,bite cells,half
ghost cells
• Biochemical-increased
bilirubin,hemoglobinemia and
hemoglobinuria

46. Glucose-6-phosphate dehydrogenase
deficiency
• Screening tests-fluorescent spot
test,methemoglobin reduction test and dye
decolorisation test

47. 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)

48. 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

49. SUMMARY
• Microcytic hypochromic anaemia-iron
deficiency
• Macrocytic hyperchromic-megaloblastic
anaemia
• Normochromic normocytic-hemolytic
anaemia
• Pancytopenia-megaloblastic and aplastic
anemias