Here are the answers to the MCQs: 1. a 2. D 3. d 4. b 5. a 6. a 7. b 8. b 9. a 10. b

1. LABORATORY TESTING OF
SPHEROCYTIC ANAEMIA
Dr Brijendra Bahadur Singh

2. Spherocytes are round,
slightly smaller than
normal red blood cells,
and lack central pallor
•Decreased surface area
to volume ratio.
•Most surface tension
efficient and least
flexible.

3. +ve -ve
Family history +ve family history -ve
Immune mediated
hemolytic anemia
OFT
Coombs test
High retic count
Spherocytes in PBS
Retic. count
Heinz bodies+
HS
HS
confirmed
EMA, dye binding
test, SDS PAGE
Bite cell
Unstable
Hb
G6PD assay
G6PD def.
Isopropanol
test, Heat
denaturation
test
Unstable Hb
disease

4. • Hereditary Spherocytosis
• Immune Hemolytic Anemia
• Oxidant damage
- G6PD deficiency
- Unstable Hemoglobin
• Thermal injury
• Toxin
- Clostridium
- Bee, Spider, Snake venom

6. • Caused by defect in proteins that disrupt the vertical
interactions between the transmembrane protein and
underlying protein cytoskeleton.
• 75% of families inherited as autosomal dominant trait
• 25% of cases inherited as non dominant

7. • Gene mutations where the defective proteins disrupt the
vertical linkages include:
• ANK1 – ankyrin (40 - 50% of cases)
• SPTA1- α spectrin( ≤ 5 % of cases)
• SPTB- β spectrin(15 -30 % of cases)
• EPB42- protein 4.2(≤ 5 % of cases)
• SLC4A1- band 3(20 -30 % of cases)

9. • Clinical manifestations : - Splenomegaly
- Anemia
- Jaundice
• Complete blood count result :
-↓ hemoglobin
-↑ MCHC
-↑RDW
-↑ reticulocyte count
• PBS finding: Spherocytes, polychromasia
• Direct antiglobulin test result – negative
• Indicators of hemolysis – ↓ serum haptoglobin, ↑ serum LDH, ↑ serum indirect
bilirubin.
• Bone marrow shows erythroid hyperplasia

11. Osmotic fragility test
Eosin-5’-maleimide binding test
SDS-PAGE
Ektacytometry
Acidified glycerol lysis test
Autohemolysis test
Cryohemolysis test

12. Osmotic Fragility Test
• It gives an indication of the surface area/volume ratio of the erythrocytes.
As this ratio falls cells become more sensitive to osmotic lysis.
• Red blood cells that are spherocytic , for whatever cause, take up less water
in a hypotonic solution before rupturing than do normal blood cells.

13. • When RBCs are placed in hypotonic solution water is drawn into RBC
• This result in swelling of cell, which acquire a spherical shape
• After a critical volume is reached membrane first leaks small molecules like
potassium ions
• Membrane pores increase in diameter, larger molecules like Hb leave the cell
• The amount of Hb in supernatant is measured colorimetrically and compared
with sample of completely lysed cells

14. • Stock solution of 10% Nacl (100gm in 1 ltr)
• Defibrinated or heparinzed blood sample( as other anticoagulants
likely to induce morphological changes which may affect the result)
• The test should be set up within 2 hours of collection, or within 6
hours if the blood is refrigerated

15. • Prepare a series of saline dilutions of 0.90%, 0.70%, 0.60%, 0.55%, 0.50%,
0.45%, 0.40%, 0.35%, 0.20% & 0.10%. (more convenient to make 1%
saline solution by taking 1 part of stock solution + 9 parts of distilled water
and then make further dilutions)
• Add 5 ml of each saline solution to respective test tubes & add 5 ml of
distilled water to last test tube
• Add 50 μliter of defibrinated or heparinized blood to each tube & mix
immediately by inverting tubes several times, avoiding foam

16. TEST TUBE
1% buffered
NaCl in ml. D.W in ml
Final conc(%)
% hemolysis
1 0.00 10 0.00
2 1.0 9.0 0.10
3 2.0 8.0 0.20
4 3.0 7.0 0.30
5 3.5 6.5 0.35
6 4.0 6.0 0.40
7 4.5 5.5 0.45
8 5.0 5.0 0.50
9
10
5.5
6.0
4.5
4.0
0.55
0.60
11 7.0 3.0 0.70
12 8.0 2.0 0.80
13 9.0 1.0 0.90

17. • Wait for 30 mins at room temperature
• Mix again and centrifuge for 5 mins @1200rpm
• Remove the supernatant and estimate the amount of lysis in the supernatant
using spectrophotometer at 540 ηm
• Use tube 13(or with 0.90% saline) as blank and assign value of 100% lysis
to 1st tube(or tube DW ) – Express the readings of other tube as % of
value of 1st tube
• Percent of hemolysis = O.D. of supernatant x 100
O.D. supernatant 1st tube

19. • EMA is fluorescent dye that binds to transmembrane protein
band 3, Rh, RhAg and CD47in the RBC membrane
• HS patients show a lower mean fluroscence intensity( MFI)
than RBCs from normal control and from patients with
spherocytes due to immune-mediated hemolysis
• Sensitivity- 93% - 96%
• Specificity- 94% - 99%

20. • Variation in membrane surface area and cell water content can
be determined by osmotic gradient ektacytometry
• Ektacytometer is a laser-diffraction viscometer that records the
laser diffraction pattern of a suspension of RBCs exposed to
constant shear stress in solutions of varying osmolality from
hypotonic to hypertonic
• An RBC osmotic deformability index is calculated and plotted
against the osmolality of the suspending solution

21. Cryohemolysis Test- HS cells are particularly sensitive to cooling
at 0⁰C in hypertonic solution. % hemolysis is calculated after
patient’s RBCs are incubated in buffered 0.7mol/l sucrose first at 37
⁰C for 10 minutes and then at 0⁰C for 10 mins.
– Normal cells show 3% - 15% hemolysis
– RBCs in HS greater than 20% hemolysis
• SDS-PAGE- Identify membrane protein deficiencies by separation
of various proteins in solubilized RBC membranes with quantitation
of the proteins by densitometry

22. • Acidified Glycerol Lysis test- measures the amount of hemolysis after
patient RBCs are incubated with a buffered glycerol solution at an acid pH
• Autohemolysis Test- Patient RBCs and serum are incubated for 48 hours
with and with out glucose.
– Normal controls : - <5% hemolysis at the end of 48 hours incubation period
- < 1% hemolysis if glucose is added.
– In HS hemolysis : 10% - 50%

23. Autoimmune Drug induced Alloimmune
hemolytic anemia hemolytic anemia hemolytic anemia
-Warm AIHA - Drug dependent -Hemolytic transfusion
-Cold agglutinin reaction
disease - Drug independent -Hemolytic disease of
-Paroxymal cold new born
hemoglobinuria
-Mixed AIHA

24. Warm AIHA Cold agglutinin
disease
Paroxymal cold
hemoglobinuria
Mixed-Type
AIHA
Ig Class IgG IgM IgG IgG, IgM
Optimum
reactivity temp
37⁰C 4⁰C 4⁰C 4-37⁰C
Sensitization
detected by
DAT
IgG or IgG
+C3d
C3d C3d IgG and C3d
Complement
activation
Variable Yes Yes Yes
Hemolysis Extravascular
primarily
Extravascular;
rarely
intravascular
Intravascular Both
Auto Ab
specificity
Pan reactive
or Rh
complex
I(most), i(some)
Pr(rare)
P Panreactive;
unclear
specificity

25. • May be either primary or secondary in etiology
– Primary-idiopathic in nature
– Secondary-due to an underlying disease
• Lymphoproliferative disorders-CLL, B lymphocytic lymphoma
• Non lymphoid neoplasm- Thymoma, Ca-lung, colon, kidney,ovary
• Autoimmune diseases- SLE, RA, PAN
• Infection- Viral
• Immunodeficiency disorder

27. Mechanism of Destruction
• Primarily due to extravascular hemolysis
• Antibodies bind to surface of RBC membrane
• Fc portion of antibody binds to macrophages
– Interaction → spherocytes
• Spherocytes become trapped in spleen and are destroyed

28. Cold Agglutinin Disease
• Due to development of an IgM antibody
• Antibody active at cold temperature (4°C) and not usually physiologically
significant
• Either primary or secondary in etiology
– Primary-idiopathic in nature
– Secondary-due to an underlying disease
• Infection- Mycoplasma pneumoniae, Infectious mononucleosis
• Lymphoproliferative disorder- CLL, B lymphocytic lymphoma

30. Mechanism of destruction
• Intravascular hemolysis
– IgM antibodies activate the compliment system →cytolysis
• Extravascular hemolysis (predominantly)
– C3b and iC3b rather than the Fc portion of IgM are recognized
– Hemolysis occurs in the liver via action of Kupffer cells

31. • ↓hemoglobin
• ↓ serum haptoglobin level
• ↑reticulocyte count
• ↑ indirect serum bilirubin and LDH
• MCV may be ↑
• Hemoglobinuria(intravascular hemolysis or severe extravascular
hemolysis)
• PBS- Polychromasia, spherocytes, occasionally RBC agglutination,
nucleated RBCs, schistocytes and erythrophagocytosis

32. Principle of Antiglobulin Test
• The incomplete antibodies (IgG) attach to red cell membrane
by the Fab portion of the immunoglobulin molecule (IgG)
• The IgG molecules attached to the red cells are unable to
bridge the gap between sensitized red cells which are separated
from each other by the negative charge on their surface and the
sensitized red cells do not agglutinate

34. • Important intracellular enzyme for protecting hemoglobin &
other cellular protein and lipids from oxidative denaturatation
• Gene located on X chromosome
• Deficiency is the most common RBC enzyme defect
• G6PD-deficient RBCs can’t generate sufficient NADPH to
reduce glutathione and thus can’t effectively detoxify
hydrogen peroxide

36. • History: Recent infection, administration of drugs, ingestion of
fava beans
• Clinical manifestation: Chills, fever, headache, nausea, backpain
and abdominal pain, jaundice and dark urine
• Indicator of hemoylsis: ↓serum haptoglobin, ↑serum LDH, ↑
serum indirect bilirubin, hemoglobinemia and hemoglobinuria

37. • CBC results: ↓ hemoglobin, ↑reticulocyte count
• PBS finding: Polychromasia, RBC morphology varies from
normal to marked anisocytosis, poikilocytosis, spherocytosis
or schistocytes, bite cells and heinz bodies
• DAT result: Negative

40. Quantitative spectrophotometric assays
Fluorescent spot test.
Dye reduction assays.

41. • Gold standard for determining G6PD deficiency
• Based on direct measurement of NADPH generated
• G-6-P+ NADP G6PD 6-Phosphogluconate + NADPH
• Rate of NADPH formation ≈ G6PD activity and measured as
increase in absorbance at 340nm
• Activity is measured as a ratio of G6PD activity per gram of
hemoglobin(g Hb)
• Cut off point for G6PD deficiency set as < 20% of normal activity
(< 4.0 IU/g Hb)

42. • Based on the principle that NADPH generated is fluorescent
• Blood and glucose-6-phosphate/NADP reagent are incubated
and spotted on filter paper at timed intervals
• Normal G6PD activity – moderate to strong fluorescent spots
under UV light
• Decreased or no activity – display weak or no fluorescence

43. • NADPH produced by enzymatic reaction reduces a dye, giving
a visually observed colour change
• In normal G6PD activity, NADPH generated reduces the dye
to formazan product, brown black in colour
• Senstivity of 98% in detecting deficient specimen with G6PD
deficiency < 4% IU/g Hb

44. •Peripheral blood film of
microspherocytes seen in
Clostridium perfringens
sepsis
•regular spherocytes are
usually smaller than
normocytic red blood cells,
microspherocytes are even
smaller than that
•usually seen in critically ill,
septic patients with severe
C. perfringens infection

45. MCQ

46. 1. In hemolysis mediated by IgG antibodies, which abnormal
RBC morphology is typically observed on the peripheral
blood film?
a- Spherocytes
b- Nucleated RBCs
c- RBC agglutination
d- Macrocytes

47. 2- In autoimmune hemolytic anemia, a positive DAT is evidence
that an:
A- IgM antibody is in the patient’s serum
B- IgG antibody is in the patient serum
C- IgM antibody is sensitizing the patient’s RBCs
D- IgG antibody is sensitizing the patient’s RBCs

48. 3- The most important finding in the diagnostic investigation of
a suspected autoimmune hemolytic anemia is:
a- Detection of a low hemoglobin and hematocrit
b- Observation of hemoglobinemia in a specimen
c- Recognition of a low reticulocyte count
d- Demonstration of IgG and /C3d on the RBC surface

49. 4- In HS a characteristic abnormality in the CBC results is:
a- increased MCV
b- increased MCHC
c- decreased MCH
d- decreased platelet and WBC count

50. 5- An altered shape of spherocyte in HS is due to :
a- An abnormal RBC membrane protein affecting vertical
protein interactions
b- Defective RNA synthesis
c- An extrinsic factor in Plasma
d- Abnormality in the globin composition of the hemoglobin
molecule

51. 6- Which of the following results are consistent with HS?
a- Increased osmotic fragility, negative DAT result
b- Decreased osmotic fragility, positive DAT result
c- Increased osmotic fragility , positive DAT result
d- Decreased osmotic fragility, negative DAT result

52. 7- Increased osmotic fragility is seen in all of the following except
a- Hereditary spherocytosis
b- Autoimmune immune hemolytic anemia
c- Hereditary elliptocytosis
d- Thalassemia

53. 8- A patient experiences an episode of acute intravascular
hemolysis after taking primaquine for the first time. The
physician suspects that the patient may have G6PD deficiency
and orders an RBC G6PD assay 2 days after the hemolytic
episode begins. How will this affect the test result?
a- No effect
b- False increase due to reticulocytosis
c- False decrease due to hemoglobinemia
d- Absence of enzyme activity

54. 9- All of the following are indicators of hemolysis except
a- Increased serum haptoglobin
b- Decreased hemoglobin
c- Increased serum indirect bilirubin
d- Increased LDH

55. 10- Immune mediated hemolytic anemia is due to a(n):
a- Structural defect in the RBC membrane
b- Allo- or autoantibody against an RBC antigen
c- T cell immune response against an RBC antigen
d- Obstruction of blood flow by intravascular thrombi

56. 1- a
2- d
3- d
4- b
5- a
6- a
7- d
8- b
9- a
10- b