3) Peripheral smear : (15,19) In a well spread, fixed and stained peripheral smear; see whole film under low magnification, later selected areas be examined under oil emersion. In India anemic patient's peripheral smear on careful examination can be of importance to detect malarial parasite. Microcytic hypochromic red cells suggest iron deficiency. Macrocytic red cells with large hypersegmented neutrophils, suggest megaloblastic anemia (vitamin B12 or folic acid deficiencies). Marked leucopenia and thrombocytopenia are seen in aplastic anemias. Toxic granulations in neutrophils are indicative of infection. Immature leucocytes are observed in leukemias.

4) Response to iron therapy : (21) Rise in hemoglobin (by 1.0 g/dl or hematocrit by 3% in 1-2 months), in an individual on iron therapy indicates iron deficiency anemia.

5) Red blood cells count and indices:

• i) Red cell count - Both erythrocytes and leucocytes are counted in whole blood. As the number of red cells is more than 500 times than the leucocytes, the error introduced is negligible. In automated analyzer, the observed precision is <1 % (cv) as compared to manual method with value of >11% (cv).
  
  
• ii) Mean corpuscular volume (MCV) - is highly reproducible, less subjective to sampling error in finger prick, as dilution by the fluid does not influence the cell volume. It is a useful red cell index providing insights into path physiology of red cell disorders (classification of anemia). MCV is directly measured on the automated analyzer (by dividing the summation of red cell volumes by the erythrocyte count; cv <1%). But can be calculated from the erythrocyte count and the hematocrit (cv around 10%). MCV is higher at birth, decreased rapidly during first 6 months of life. Decreased MCV is observed in iron deficiency, thalassemia, infection and chronic diseases. Disproportionately low MCV with normal hemoglobin suggest thalassemia minor (22). Increased MCV is observed in megaloblastic anemia and liver disorders. Mentzer index- the ratio of MCV to RBC count in million; values < 13% is in thalassemia minor in 85%; while > 13% in 85% chances in iron deficiency (23). In contrast, MCH and MCHC do not add much significant clinical information.
  
  
• iii) Red cell distribution width (RDW) - Quantifies the red cell volume heterogeneity estimated by the more modern analyzers and reflect the range of red cell size measured with in a sample. It is useful in characterizing the microcytic anemias: iron deficiency - (high RDW, normal to low MCV) and uncomplicated heterozygous thalassemia (normal RDW, low MCV). Value > 15% proved highly sensitive (71 to 100%) but relatively non-specific (50%) for iron deficiency.
  
  
• iv) Percentage hypochromic mature RBC (hyPom), - can be measured on new automated analyzer (24).
  
  
• v) Reticulocyte: a) Reticulocyte hemoglobin content (CHr) decrease is an early sensitive indicator of iron deficiency erythropoiesis. It is also reliable in assessment of iron therapy response (25). b) Presently automated hematology analyzer has automated reticulocyte counting and size (Ret Y) measurement as part of the testing. Ret Y is a sensitive indicator of iron deficiency (26). It correlates closely to sTfR, has highest overall sensitivity and specificity of the panel of tests used in differential diagnosis of iron deficiency vs chronic disease anemia.
  
  

6)Spun hematocrit : (14) When erythrocytes are centrifuged (sediment by slow speed), the supernatant in iron deficiency patient is extremely pale as compared to its normal straw color. This is useful in differentiating iron deficiency from thalassemia syndromes as serum appears darker (with RDW extremely high > 20%).

7)Serum iron levels:(range 50-150 ug/dl) (14) : Over 3 years of age has marked diurnal variation, being higher by 30% in the morning as compared to the night. Value < 30ug/dl in the morning sample after 8 hr fast, suggests iron deficiency. The absolute value for total iron binding capacity (TIBC) is mostly increased in iron deficiency and decreased in anemia of chronic disorders.

                                       serum iron x 100
Transferrin saturation = ____________________

                                              TIBC

Transferrin saturation value (normal 20-45%) is more consistently helpful than either value alone. In both children and adults value < 5% is diagnostic of iron deficiency, while <16% is suggestive of iron deficiency or for anemia of chronic disorders. However it does not reflect iron stores, is related to efficiency of moving iron out of iron processing cells (reticuloendothelial macrophages, hepatocytes or absorptive erythrocytes) and erythron. Non iron deficiency anemia with infection/inflammation may have transferrin saturation <16% rarely <10% but has low TIBC of 200ug/dl. The lower serum iron and TIBC are associated with decreased production of transferrin (B- globulin).