Laboratory Diagnosis of AnemiaBy

Dr. Varughese GeorgeDepartment of Pathology

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.

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

Clinical presentation of anaemias

Initial Laboratory Work-Up

• 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.

6

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

13

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

• 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

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

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

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 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

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

Megaloblastic AnemiaMild 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

Macrocytic OvalocytesBlood NRBC Blood

Howell-Jolly body

Teardrop

Schistocyte

Stippled RBC & Cabot Ring

Giant PlateletPap bodies Hypersegmented Neutrophil >5

lobes

Megaloblastic anemia

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 cells-1. Hereditary spherocytosis2. Thalassamia3. Sickle cell anaemia4. Glucose -6-phosphate dehydrogenase deficiency

• Abnormality extrinsic to red cells-1. Immune2. Mechanical etc

Evaluation of haemolytic anaemia

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

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,vaso-

occlusive crisis• Aplastic crisis• Hemolytic crisis• Infections

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

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)

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

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