3. DEFINITION
Hemolytic disorders are characterised by signs of
accelerated erythrocyte destruction together with those of
vigorous blood regeneration.
Shortened life span of RBC’s – 15-20 days.
Elevated Erythropoietin
Accumulation of haemoglobin degradation products.
5. SITE OF RBC
DESTRUCTION Intravascular
Red cells rupture within the vasculature releasing free
hemoglobin into circulation
Extravascular
Liver
Spleen
Bone marrow
Splenomegaly is a feature of
extravascular helmolysis
8. Pathogenesis of Extra vascular hemolysis
Extreme changes in shape required for RBC to navigate the splenic
sinusoids successfully
This alterations result in to less deformable RBC
RBC sequestration & phagocytosis by Macrophages located within splenic cords
Globin
Protein pool
Haem
Biliverdin Bilirubin
Unconjugated bilirubin
Conjugated bilrubin
Urobilinogen
Excretion in Faeces & urine and
formation of gall stones
12. General consideration in the
diagnosis of hemolytic anemia
Is the anemia hemolytic ?
If so, is it Intravascular / Extravascular haemolysis?
What is the etiology?
What is the severity of anemia?
13. IS THE ANEMIA HAEMOLYTIC?
Evidence for increased red cell
production-
IN BLOOD - peripheral smear
1.Increased reticulocyte count
2.Circulating nucleated RBC
3.Marked polychromasia.
14. Polychromatophilic red cells(A--green arrows)
Spherocytes (B--blue arrows)
Nucleated red blood cells (D-- orange arrow)
26. INCUBATED OSMOTIC
FRAGILITY TEST
Blood incubated for 24hrs
At 37°C HS cells lose membranes more readily then
normal RBC’s when incubated
Increased sensitivity
Most reliable diagnostic test for HS
27. AUTOHEMOLYSIS TEST
Measures spontaneous hemolysis of blood incubated
at 37ºc for 48 hrs
Measure readings colorimetrically at 540nm
Normal – 0.2-2 %
With added glucose – 0-0.9 %
28. Decreased rate with added Glucose
Hereditary Spherocytosis
PNH
G6PD Deficiency
No response to added glucose
Pyruvate Kinase Deficiency
29. ACIDIFIED GLYCEROL LYSIS
TIME Time taken for 50% hemolysis of a blood sample in a
buffered hypotonic saline glycerol mixture.
Glycerol retards the osmotic swelling of red cells
Rate of hemolysis is measured by rate of fall of
turbidity
Half time for AGLT > 30min for normal RBC’s
HS cells 25-150 sec
30. CRYOHEMOLYSIS
Specific for Hereditary Spherocytosis
Dependent on molecular defects of RBC membrane.
HS cells are particularly sensitive to cooling at 0 °C in
hypertonic saline.
Normal: 3 – 15%
HS - >20%
41. SOLUBILITY TEST
Sickle cell Hb is insoluble in
deoxygenated state in a high
molality phosphate buffer
Crystals formed refract light,
cause solution to be turbid.
Positive test – sickling Hb
Doesn’t differentiate b/w
homozygous & heterozygous
POSITIVE TEST
42. SICKLING TEST
Blood deoxygenated with
reducing substances (sodium
metabisulphite)
Place on slide
Seal coverslip
Immediate sickling – Disease
Sickling in 1 hr – Trait
1. Hb electrophoresis
2. High performance liquid
Chromatography (HPCL)
3. Isoelectric focusing – agar
gel
4. Prenatal diagnosis - PCR
OTHER DIAGNOSTIC
TESTS
44. G6PD DEFICIENCY
INTRODUCTION
1. Its an enzyme of HMP pathway
2. Protects against oxidative stress
3. X linked disorder
4. Deficiency result in to,
Impaired NADPH production
Accumulation of oxidants in cell
Oxidative stress leads to Heinz body formation & extra-
vascular hemolysis
46. FLORESCENT SPOT TEST
Sensitive screening test
Whole blood + G6P + NADP + SAPONIN
Kept on a filter paper
Examine under UV light
Examine the Fluorescence
G6P + NADP 6-Phosphogluconate + NADPH
(Fluoresces)
Lack of fluorescence ------- G6PD Deficiency
47. DYE REDUCTION TEST
Pts blood hemolysate + G6P + NADP + brilliant
cresyl blue ------ incubation
If G6PD present ----- NADP → NADPH
BLUE → COLOURLESS
Time taken is inversely proportional to the amt. of
G6PD present
Controls- normal blood
Specific test
48. OTHER TESTS
ASCORBATE CYANIDE TEST
METHEMOGLOBIN REDUCTION TEST
G6PD ASSAY
Glutathione stability test-Decrease sensitivity in G6PD deficiency.
PCR-To reveal genetic abnormality(Florescent labelled probes are
used to detect mutant G6PD alleles)
50. PAROXYSMAL NOCTURNAL
HEMOGLOBINURIA
Acquired Clonal cell disorder
Somatic mutation in hematopoietic stem cell.
Defect in glycosyl – phosphatidyl inositol (GPI) molecule embedded in cell
membrane
GPI linked proteins – decay accelerating factor (CD 55), Inhibitor of
reactive lysis(CD 59)-Prevents activation of complement.
51. LAB FINDINGS
Anemia
Thrombocytopenia
Hemosiderinuria
Positive sucrose hemolysis test
Positive Ham’s test
Normal Osmotic fragility
52. SUCROSE HEMOLYSIS
TEST
Screening test
Patient’s blood incubated in sucrose solution
Sucrose promotes binding of complement to
RBC
Hemolysis
53. HAM’S TEST(Acidified
serum lysis test)
Patient’s RBCs are exposed at 37°C to action of
normal / patient own serum suitably acidified to
optimal pH for lysis( activate alternate pathway)
10 – 50 % of total RBC ----Lysis
56. PATHOPHYSIOLOGY
RBC’s + IgG Ab
Pass through the spleen
Fc receptor of spleen macrophages attracts
Fc portion of Ab – RBC complex
Phagocytosis of RBC by macrophage
Fragmentation of
RBC membrane
Membrane reseals and
forms spherocytes
Extravascular hemolysis
61. WARM AIHA
Individuals produce Ab against their own erythrocyte Ag
(autoantibodies)
Ab react with red cell Ag best at 37oc.
Lab findings
PBS – Normocytic normochromic anemia
- Reticulocytosis
- Spherocytes,
- Schistocytes, Polychromasia, NRBC’s
- Neutrophilia
- Platelet - normal or decreased
62. Bone marrow
- Normoblastic erythroid hyperplasia
- Erythrophagocytosis
Other tests
- Direct Coombs’ test (DAT)- positive
63. COLD AIHA
Associated with IgM Ab which fixes complement & is reactive
below 32o c
First indication of the presence of unsuspected cold agglutinins
is blood counts.
1. RBC count is inappropriately decreased for Hb%
2. MCV is falsely elevated (due to agglutination)
3. PCV is falsely low
4. MCH & MCHC are falsely elevated
• Visible autoagglutination can be observed in tubes of
anticoagulated blood as the blood cools to room
temperature.
64. When RBC indices go haywire think of the
possibility of cold agglutinin disease
65. PAROXYSMAL COLD
HEMOGLOBINURIA
Lab findings
a) Between the attacks - peripheral blood is normal except for
anemia
b) During the attack – sharp drop in Hb
c)DAT – weakly +ve with anticomplement antisera
- Ab are not detected
d) Indirect coomb’s test may be +ve ,if performed in cold
e) Donath – Landsteiner test
66. DONATH LANDSTEINER(D-L)
Test
PATIENT’S WHOLE
BLOOD
CONTROL TEST
INCUBATE FOR 30
MIN AT
370 C 40 C
INCUBATE FOR 30
MIN AT
370 C 370 C
Centrifuge: Observe plasma for presence of hemolysis
Interpretation
D-L antibodies present No hemolysis Hemolysis
NO D-L antibodies
present
No hemolysis No hemolysis
67. AUTOIMMUNE HEMOLYTIC
ANEMIA
Hemolytic anemia induced by immunization of an
individual with RBC Ag’s from another individual
.
Eg: 1. Hemolytic transfusion reactions
2. Hemolytic disease of new born
68. DRUG INDUCED AIHA
It is the result of an immune mediated hemolysis precipitated by
ingestion of certain drugs.
MECHANISM:
1. Drug adsorption (hapten type)
2. Immune complex formation
3. Autoantibody induction
4. Membrane modification
Fragmented cells, spherocytes, blister cells and punctate basophilia
blood film of a patient with Pyruvate Kinase Deficiency? Thorny Apple sputnik cells, polychromasia
Reticulocytosis
Blister cells (arrows)
Heinz bodies 9new methylene blue stain) (Inset)
