Diagnosis of hereditary and acquired hemolytic anemia

1. DIAGNOSTIC APPROACH TO
HEMOLYTIC ANEMIA
PART I – HEREDITARY CAUSES
By-
Dr. Amita Yadav
MBBS, MD (Pathology)

2. HEMOLYTIC ANEMIA
 Definition:
 Those anemias which result from an increase in RBC
destruction coupled with increased erythropoiesis
 Classification:
 Congenital / Hereditary
 Acquired

6. CLASSIFICATION OF HEMOLYTIC ANEMIAS
INTRACORPUSCULAR
DEFECTS
EXTRACORPUSCULAR
FACTORS
HEREDITARY •HEMOGLOBINOPATHIES
•ENZYMOPATHIES
•MEMBRANE-
CYTOSKELETAL DEFECTS
•FAMILIAL HEMOLYTIC
UREMIC SYNDROME
ACQUIRED •PAROXYSMAL
NOCTURNAL
HEMOGLOBINURIA
•MECHANICAL DESTRUCTION
[MICROANGIOPATHIC]
•TOXIC AGENTS
•DRUGS
•INFECTIOUS
•AUTOIMMUNE

7. CLASSIFICATION-
 MAHA
 Transfusion reaction
 PNH
 Infections
 Snake bite
 Hemoglobinopathies
 Enzymopathies
 Membrane defects
 AIHA
Intravascular hemolysis Extravascular hemolysis

8. HOW IS HEMOLYTIC ANEMIA DIAGNOSED?
Two main principles
 To confirm that it is hemolysis
 To determine the etiology

9. GENERAL FEATURES
OF HEMOLYTIC DISORDERS
 GENERAL EXAMINATION - PALLOR,JAUNDICE,DARK COLOURED
 URINE , BOSSING OF SKULL
 PHYSICAL FINDINGS - SOMETIMES SPLENOMEGALY
 HEMOGLOBIN - FROM NORMAL TO SEVERELY REDUCED
 MCV - USUALLY INCREASED
 RETICULOCYTE COUNT - INCREASED
 BILIRUBIN - INCREASED[MOSTLY UNCONJUGATED]
 LDH - INCREASED
 HAPTOGLOBULIN - REDUCED TO ABSENT

10. HEMOLYTIC FACIES- CHIPMUNK FACIES

11. EXTRAVASCULAR HEMOLYSIS-

12. INTRAVASCULAR HEMOLYSIS-

13. Laboratory Evaluation of Hemolysis
Extravascular Intravascular
HEMATOLOGIC
Routine blood film
Reticulocyte count
Bone marrow
examination
Polychromatophilia
Erythroid
hyperplasia
Polychromatophilia
Erythroid
hyperplasia
PLASMA OR SERUM
Bilirubin
Haptoglobin
Plasma hemoglobin
Lactate dehydrogenase
Unconjugated
, Absent
N
(Variable)
Unconjugated
Absent
(Variable)
URINE
Hemosiderin
Hemoglobin
0
0
+
+ severe cases

14. POLYCHROMATOPHILIC CELLS

15. PATIENT HISTORY
 Acute or chronic
 Medication/Drug precipitants
G6PD
AIHA
 Family history
 Concomitant medical illnesses
 Clinical presentation

17. CASE 1
 6 yr old child presenting with severe pallor,jaundice
growth retardation
 Abnormal facies,hepatosplenomegaly+
 h/o recurrent blood transfusions
 CBC-Hb -3gm%,
 MCV-58FL,
-MCH- 19pg
RDW- 14%
P.S- Microcytic hypochromic
RBCs with
target cells +

18. DIFFERENTIAL DIAGNOSIS-
 Iron Deficiency Anemia
 Thalassemia

19. THALASSEMIA-
 Thalassemia syndromes result from defects in rate
of synthesis of alpha or beta chains.

20. BETA THALASSEMIAS-
 Mutation in Beta globin gene expression
 M.C- deranged splicing of m-RNA
 Thalassemia Major (Cooley’s Anemia)-
 Homozygous or double heterozygous form
 Accumulation of free alpha chains

22. .
Type Genotype Anemia RBC mor-
phology
Hb
electro.
Clinical features
Beta
thalassemia
Minor
B/B+ ,B/B0 Absent or
mild(Hb-10-
12gm/dl)MC
V & MCH
reduced
Mild
anisopoikilo
cytosis with
mild
microcytosi
s and
hypochromi
a,target
cells
,basophillic
stippling
HbA- 90-
93%
Hb A2-
3.5-8%
(Diagno
stic)
HbF- N
to minor
inc.
Asymptomatic
Beta
thalassemia
intermedia
B+/B+,Interac
tion of
alpha,B,HbE,
HbD,HbQ,Hb
S
Moderate(7-
10gm/dl),
MCV & MCH
reduced)
Moderate
changes
HbF-10-
30%
Late onset,Not
transfusion
dependent,mild
splenomegaly,fac
ial and skeletal
change
B
Thalassemi
a Major
B0/B0,B0/B+,
B+/B+
Mod. To
severe(Hb-
3-8gm%)
Dec.MCV,M
Severe
changes,ma
ny nRBCs
HbF -30-
90%
Onset in
infancy,transfusio
n
dependent,sever

23. Type Genotyp
e
anaemia RBC
Changes
Hb Electro. Clinical
Features
Silent
Carrier
aa/a- absent None Hb Bart not
demon.
Asymptomatic
a
Thalassemi
a trait
a-/a-,
aa/--
Absent or mild Microtic
hypochromic
Hb Bart not
demon.
Clinically
normal
HbH
Disease (
beta
tetramer)
a-/-- Progressive
anemia Hb-6-10
gm/dl
Mod.anisopoi
kilocytosis
with mild
microcytosis
and
hypochromia,
target cells
Retic. Prep.
HbH inclusion
HbH Band-
5-35%
Splenomegaly
,moderate
skeletal
deformity
Hydrops
foetalis
(gamma
tetramer)
--/-- Severe Severe
changes,num
erou
erythroblasts
80-100%
Hb Barts
Fatal, pale
and bloated
baby,oedemat
ous placenta

24.  Reticulocyte preparation showing Golf ball HbH
inclusions attached to red cell membrane.

25. INVESTIGATIONS-
 Peripheral blood examination
 Test for inclusion bodies
 Hb Electrophoresis
 Acid elution Test
 Bone marrow examination
 Radiograph

26. PERIPHERAL BLOOD FILMS IN THALASSEMIA
MAJOR

27. NESTROF TEST-

29. ACID ELUTION TEST FOR HBF (KLEIHAUER’S TEST) –
A.RED CELLS CONTAINING HBF ARE STAINED PINK.
B.GHOST CELLS INDICATING PRESENCE OF HBA

31. A.GIEMSA STAINED BM –MACROPHAGES LADEN WITH
GOLDEN BROWN GRANULES OF HEMOSIDERIN
PIGMENT
B.PERL’S STAIN DEMONSTRATES IRON STORES

32. BONE CHANGES IN THALASSEMIA MAJOR-
HAIR ON END APPEARANCE DUE TO NEW BONE
FORMATION AND WIDENING AND THICKENING OF
DIPLOE

33. CASE 2
 3 yr old male child presenting with
pallor,jaundice,Severe pain of long bones, fever,
Growth retardation
 CBC- Anemia ,leucocytosis,
 reticulocytosis,

 LAB - S.bilirubin- 5mg%
S.LDH -600 IU/L (normal upto 200)

34. PERIPHERAL SMEAR-
ANISOPOIKILOCYTOSIS,HYPOCHROMIA.PRESENCE OF
DRAPANOCYTES,TARGET CELLS
FEW RED CELLS SHOWING HOWELL JOLLY BODIES AND NRBC

35. WHAT IS THE DIAGNOSIS ?
 SICKLE CELL ANEMIA-
 Characterised by presensce of HbS which imparts
sickle shape to red cells in a state of reduced
oxygen tension.
 Substitution of valine in place of glutamic acid at 6th
position of beta globin chain.
 Altered solubility
 Polymerisation of HbS forming tactoids.

36. SICKLE CELL DISEASE
 Mutation beta globin-6 Glu Val.
 Deoxy HbS (polymerised)
 Ca influx, K leakage
 stiff,viscous sickle cell
 venocclusion decreased RBC survival
microinfarctions,ischemic pain anemia,jaundice,
autoinfarct,leg ulcers gallstones

37. SICKLING DISORDERS-
Type Genotype Clinical
manifesta
tion
RBC morphology Hb Electrophoresis
Sickle Cell
Anemia
βs/βs Moderate
to severe
anemia.Hb
5-10gm/dl.
Mod.to severe
anisopoikilocytosis.sickl
e cells,target
cells,polychromatic cells
,Howell jolly
bodies,reticulocytosis
Hbs Predominant(70-
90%0,HbA(0-
10%),HbF(10-
30%),HbA2 normal
Sickle cell
disease
βs/β0,
βs/β+
Moderate
to severe
anemia
Moderate to severe
changes
Depending upon
hemoglobinopathy
present
Sickle cell
trait
βs/β Mild or no
anemia.
Hb 11-
13%,
Mild changes HbS 25-40%,HbA
60-75%,HbF<1%,

38. CLINICAL MANIFESTATIONS
 Vasoocclusive crisis
 Hemolytic crisis
 Aplastic crisis
 Splenic sequestration crises
 Hand foot syndrome(Dactylitis)
 Acute chest syndrome

39. DIAGNOSIS –
 Peripheral blood examination
 ESR-Low
 Sickling Test
 Solubility test
 Hb electrophoresis-HbS is slow moving as
compared to HbA and HbF
 HPLC
 Estimation of HbF by alkali denaturation test

40. CASE 3
 45 yr old male came to opd in a remote PHC with
burning micturition
 Urine R/M shows numerous pus cells++++
 UTI diagnosed & medical officer gave
cotrimoxazole 2 bd X 5days
 1 wk later,pt developed severe
pallor,palpitation,jaundice
 Lab- increased LDH, S.BILIRUBIN,RETIC COUNT
 P.S- shows polychromasia and irregular cells like
bite cells and blister cells

41.  Bite cells and blister cells in peripheral smear

42. DIFFERENTIAL DIAGNOSIS-
 G-6PD Deficiency
 Unstable hemoglobinopathy
(Positive for Heat instability Test and
Isopropanol precipitation Test)

43. G-6PD DEFICIENCY-
 First enzyme in HMP shunt pathway.
 Protects red cells from oxidant injury.
 Deficiency results in episodes of hemolysis.

45. Definitive risk Possible risk Doubtful risk
antimalarials Primaquine
Dapsone
cholrproguanil
chloroquine quinine
Sulphonamides/
sulphones
Sulphametoxazole
Dapsone
Sulfasalazine
Sulfadimidine
Sulfisoxazole
Sulfadiazine
Antibacterials/
Antibiotics
Cotrimoxazole
Nalidixic acid
Nitrofurantoin
Ciprofloxacin
Norfloxacin
Cholramphenicol
p-Aminosalicylic
acid
Antipyretic/
Analgesics
Acetanilide
Phenazopyridine
[pyridium]
Acetylsalicylic acid
High dose[>3g/d]
Acetylsalicylic acid
[<3g/d]
Acetaminophen

46. HEMATOLOGIC FINDINGS-
 Abrupt fall in Hb to 6-10 gm/dl
 Reticulocyte count increases to 20-50%
 Heinz bodies are seen in reticulocyte preparation
 Peripheral Smear-
 Moderate degree of anisopoikilocytosis,
 polychromatophilia,
 microspherocytes,
 bite cells,
 blister cells
 Hemoglobinuria
 Uribilinogen increased.

47.  Bite cells and blister cells in peripheral smear

49. DIAGNOSIS-
 History
 Peripheral blood smear
 Screening tests-
1.Methemoglobin Reduction Test
2.Fluorescent spot Test
 Quantitative assay of G-6PD ( assess a few weeks
after the acute hemolysis)
 DNA analysis by PCR

50. 2. Pyruvate Kinase Deficiency
 AR
 Deficient ATP production,
 Accumulation of G-3-p,2,3-DPG and
glucose
 Chronic hemolytic anemia
 Clinical features-Pallor,jaundice
 Uncommonly gall stones and/or
splenomegaly
o

52.  Hematologic Finding-
 Moderate anemia
 Peripheral smear-Moderate degree
of anisocytosis,Prickle cells( red
cells with sharp thorn like
projections,few echinocytes.
 Autohemolysis Test
 Quantitative assay

53. CASE 4
 14 YR old female present with anemia, jaundice
 Rt hypochondrial pain
 O/E- vitals stable.pallor+,icterus+,splenomegaly +
 Usg- cholilithiasis
 Lab investigation- elevated LDH, S.Bilirubin
 Peripheral smear shows- Spherocytes

54. DIFFERENTIAL DIAGNOSIS FOR
SPHEROCYTOSIS-
 Hereditary spherocytosis
 Autoimmune hemolytic anemia
 Burn,
 G-6-PD deficiency,
 transfusion reaction,
 Clostridial sepsis,
 Snake/spider bite,
 copper sulphate poisoning,
 microangiopathic hemolytic anemia

55. RED CELL MEMBRANE DEFECTS
1.Hereditary Spherocytosis
 Usually inherited as AD disorder
 Defect: Deficiency of Beta Spectrin or Ankyrin  Loss of
membrane surface area becomes more spherical
Destruction in Spleen

60.  C/F:
Mild to moderate anemia
Intermittent jaundice
Splenomegaly
Pigmented gall stones- in 50-75% of
patients

61. COMPLICATIONS
 Clinical course may be complicated with Crisis:
 Hemolytic Crisis: associated with infection
 Aplastic crisis: associated with Parvovirus infection

62. INVESTIGATIONS-
Mild to moderate anemia
Peripheral smear-
microspherocytes,polychromatophilia
S.Bilirubin increased mainly unconjugated
type
Fecal stercobilinogen increased
S. haptoglobin reduced.
Osmotic fragility Test

63. PERIPHERAL BLOOD PICTURE-

64. OSMOTIC FRAGILITY TEST CURVE-

65. Glycerol Lysis Test
Flow Cytometry based on EMA (
Eosin-5-Maleimide)-mean
fluorescence intesity of EMA tagged
red cells is lower in HS.

67. DIAGNOSTIC APPROACH TO
HEMOLYTIC ANEMIA
PART II – ACQUIRED CAUSES
By-
Dr. Amita Yadav

70. IMMUNE HEMOLYTIC ANEMIA
 Result from RBC destruction due to RBC
autoantibodies: Ig G, M, E, A
 Most commonly-idiopathic
 Classification
1.Autoimmune Hemolytic Anemia
 Warm AI hemolysis:Ab binds at 37degree Celsius
 Cold AI Hemolysis: Ab binds at 0-10 degree Celsius
2.Alloimmmune Hemolytic Anemia
• Hemolytic transfusion reaction
• Hemolytic disease of new born

71. CLASSIFICATION-

72. WARM AND COLD ANTIBODIES-

73. 1.Warm AI Hemolysis:
 Can occurs at all age groups
 F > M
 Causes:
50% Idiopathic
Rest - secondary causes:
1.Lymphoid neoplasm: CLL, Lymphoma, Myeloma
2.Solid Tumors: Lung, Colon, Kidney, Ovary, Thymoma
3.CTD: SLE,RA
4.Drugs: Alpha methyl DOPA, Penicillin , Quinine,
Chloroquine
5. UC, HIV

75. • 2. Cold AI Hemolysis Usually
Ig M directed at the RBC I antigen
• CAUSES-
 Infection: Mycoplasma pneumonia, Infec Mononucleosis
 Neoplasms : waldenstrom macroglobulinemia ,
lymphoma,CLL,kaposi sarcoma, myeloma.
C/F:
Elderly patients
Exacerbations in the winter
Cold , painful & often blue fingers, toes, ears, or
nose ( Acrocyanosis)

76. MECHANISM OF HEMOLYSIS-

77. MECHANISM OF HEMOLYSIS DUE TO DRUGS-

79. CLINICAL FEATURES-
 Progressively increasing pallor
 Mild to moderate jaundice
 Cola coloured urine

80. INVESTIGATIONS-
 History of drug intake
 Hb- low
 Reticulocytes- 4-30%
 Peripheral smear-
 Anisocytosis with polychromatophilia
 Microspherocytes
 S.Haptoglobin-decreased
 S.LDH-elevated
 S.Bilirubin- >4mg/dl
 Urine urobilinogen-increased
 Coomb’s Test

82. CASE 5
 32 yr old presented 4 days history of distention of
abdomen and rt hypochondrial pain and has h/o
passage of dark colored urine early morning
 On USG- hepatomegaly,gross ascites,hepatic vein
thrombosis
Lab : Hb – 7gm%. WBC- 2200, PLC- 80,000
LDH- 600, S.Bilirubin- 4 mg%
urine bile pigment +,heme dip stick++
What is the diagnosis?

84. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
 Acquired chronic H.A
 Persistent intra vascular hemolysis
 H/0 passing dark coloured urine early morning.
 Pancytopenia
 Lab :hemoglobinburia,hemosiderinuria,increased
LDH,bilirubin
 Risk of venous thrombosis
 P.S – polychromatophilia, normocytic normochromic
anemia
 B.M – normoblastic hyperplasia

85. DIAGNOSIS-
-flow cytometry CD59-,CD55-
-Hams’ acidified serum test
-Sephacryl microtyping gel card test
- FLAER(Flow cytometry based assay utilises Fluorescent
labelled inactive variant of protein aerolysin.

86. URINE SAMPLES FROM A TYPICAL PNH
PATIENT-

88. CASE 6
 25 yr old male with RHD – severe MR done MVR,after 10
days presented with pallor, palpitation,jaundice
CBC shows Hb – 7.5 gm %, Hct -22 %
Lab : S.bilirubin -4.5mg%
LDH -600
Retic count 10%
Peripheral smear – Fragmented red cells,schistocytes
(helmet cells,triangular cells),few
spherocytes,polychromasia

89. MICROANGIOPATHIC HEMOLYTIC ANEMIA

90. NON-IMMUNE ACQUIRED HEMOLYTIC
ANEMIA
1. Mechanical Trauma
A). Mechanical heart valves, Arterial grafts: cause shear stress
damage
B).March hemoglobinuria: Red cell damage in capillaries of feet
C). Thermal injury: burns
D). Microangiopathic hemolytic anemia (MAHA): by passage of
RBC through fibrin strands deposited in small vessels 
disruption of RBC eg: DIC,PIH, Malignant HTN,TTP,HUS

93. MICROANGIOPATHIC HEMOLYTIC
ANEMIA(MAHA)
 Other findings - leukocytosis
- thrombocytopenia(DIC,TTP)
- hemoglobinuria
- deranged RFT
- PT,APTT prolonged
(DIC,TTP)

94. HEMOLYSIS DUE TO INFECTIONS-
 P.falciparum malaria
 P.Vivax
 Trypanosomiasis
 Clostridial Sepsis
 Bartonellosis
 E.coli (HUS)
 Leptospirosis