By : MANINDER KAUR B.

Sc MLT(third)

Moderator: Dr Anshu Palta

Introduction
Hemolytic anemia are the anemia in which there is increased red

cell destruction. Anemia in such cases develops due to inability of the bone marrow to compensate for the degree of destruction. Clinical and laboratory findings in these cases indicates : (a) accelerated destruction of red cells (b) compensatory morrow regeneration. Normal life span of red cells is 120 days and in hemolytic anemia, it may become as low as 15 days.

Red cell destruction

As the red cells reach 120 days, there is loss of membrane sialic

acid and lipids. ATP levels decrease. Spleen is the principle site of red cell destruction. As the red cells pass through spleen repeatedly, there is depletion of glucose from the cell and the surface area decreases. (A) extra vascular destruction: Site of destruction is mainly spleen and this is the major (80-90% of destruction) mechanism of red cell destruction. In hemolytic anemia, this mechanism exaggerated.

 (B) intra vascular destruction : Red cells get destroyed, while in

circulation, with release of hemoglobin in plasma. This is the main pathway in pathologic states like paroxysmal nocturnal hemoglobinuria. Mechanism of extra vascular red cell destruction: In the spleen and bone marrow, reticuloendothelial cells phagocytose the senescent red cells which are broken down and hemoglobin is released. Hemoglobin is broken down into hem and globin and finally amino acids of globin are reutilized, iron is deposited in the marrow and urobilinogen and sterobilinogen are excreted out.

Senescent red cells Phagocytosed by RE cells of spleen Hemoglobin released in RE cells and broken down Hemo + globin

Broken down to amino acids

Iron deposited in marrow

Carried to iron stores like bone marrow

In plasma

Iron + protoporphyrin biliverdin Bilirubin(unconjugated) Conjugated in liver

Re utilized for synthesis of , chains

Bilirubin glucuronide excreted in bile Bilirubin acted upon by bacterial enzymes in intestine Enterohepatic circulation kidney Urobilinogen in urine( 2-4mg/day) absorbed Urobilinogen/stercobilinogen Fecal stercobilinogen

Intra vascular red cell destruction

This is a minor pathway of normal red cell destruction. In intra vascular hemolysis red cells are destroyed in circulation

releasing hemoglobin. Hemoglobin colours the plasma red and also combines with haptoglobin. Hemoglobin is excreted in urine. The characteristic findings of intra vascular hemolysis are: haemoglobinemia (detected by benzidine test ) hemoglobinuria(plasma hemoglobin exceeds the hepatoglobin binding . Hemoglobin excreted in urine. Detected by benzidine test.) Hemosiderinuria (detected in urinary deposit by prussian blue reaction). Decrease in serum hepatoglobin.

Diagnosis of hemolytic process

Confirmation of the hemolytic process is carried out by keeping

in mind general features of hemolytic process. (a)clinical manifestations pallor Jaundice Splenomegaly Gall stone Skeletal abnormalities Leg ulcers (b) Compensatory mechanism to hemolysis : Some of the important compensatory mechanism are : Erythroid hyperplasia in the bone marrow. Reticulocytosis

1)

2)

nucleated RBCs in the peripheral blood. (c) laboratory findings: Increased serum bilirubin(unconjugated) Increased urine urobilinogen Increased serum LDH Hemoglobinuria /hemosiderinuria Peripheral blood findings : blood investigations include : Reticulocyte count Increase in hemolysis. Reticulocyte preparation also demonstrates : HbH inclusions in -Thalassaemia-HbH disease. Heinz bodies in G-6 -PD deficiency-exposure to oxidising drugs/chemicals and Heinz body anemia. Red cell abnormalities: A careful evaluation of peripheral smear is the most important step in diagnosing hemolytic anemia. Presence of abnormal red cells suggests the possible cause of hemolysis.

 E.g. Polychromasia: suggestive of high reticulocyte count. Spherocytes- hereditary spherocytosis , immune hemolytic anemia ,

thermal injury. Target cells : sickle cell anemia ,thalassaemia, HbC, HbD and HbE disease. Sickle cells- sickle cell anemia Schistocytes (fragmented red cells)- microangiopathic hemolytic anemia, hemolytic uremic syndrome. Acanthocytes- pyruvate kinase deficiency. Elliptocytes H.Elliptocytosis.

Tests for various groups of hemolytic anemias

Disease group:

Hemoglobinopathies: Test empoyed : i. foetal hemoglobin estimation. ii. Hb electrophoresis, sickling test. iii. inclusions in reticulocyte pereparation . iv. cytochemical stains for HbF to differentiate thalassemia from HPFH(hereditary persistence of foetal hemoglobin). v. Heinz body formation in unstable hemoglobinopathies.
1)

2)
i. ii.

Spherocytosis : Test empoyed: Osmotic fragility. Acidified glycerol lysis test.

3) i. 4)

Immune hemolytic anemia : Test employed: Coombs test - direct and indirect.

G-6-PD deficiency : Test employed: i. Methemoglobin reduction test. ii. Fluorescent spot test. iii. Quantitative assay.
5) i.

P.K. Deficiency: Test employed: auto hemolysis test, quantitative assay.

Paroxysmal nocturnal hemoglobinuria: Test employed: i. Hams acidified serum test. ii. CD59, CD55 using gel cards or by flow cytometry. iii. Urine for hemoglobin and hemosiderin.
6) 7) i. 8)

Methemoglobinaemia: Test employed: Spectroscopy. Paroxysmal cold hemoglobinuria: Test employed: Landsteiners test.

i.

Thank you