Laboratory approach to Microcytic hypochromic anemia

1. Dr.Nikhil
Laboratory approach to Microcytic Hypochromic
anemias

2. Skeleton
 Definition of Anemia
 Patient History in case of Anemia
 Clinical Findings of Anemia
 Types of Microcytic Hypochromic Anemia
 Laboratory Approach to Microcytic Hypochromic
Anemia

4. Definition of Anemia
 Anemia : is defined as reduction in the concentration
of circulating Hemoglobin below the level that is
expected for healthy persons of same age and sex in
the same environment.
 Anemia is not a disease it is a manifestation of disease.

5. WHO Definition of Anemia
 World Health organization(WHO) has defined anemia
as Hb<13.0 g/dl for men and <12.0g/dl for women
 For Pregnant women <11.0 g/dl .

6. Grading of Anemia
Mild Anemia Hemoglobin from
lower limit of
normal to 10.0 g/dl
Moderate
Anemia
7.0-10.0g/dl
Severe Anemia <7.0 g/dl
Critical Value

7. Clinical Findings of Anemia

8. MCV
 Average Volume of a single red cell.
 Normal range 80-100 fl.
 Calculated as :
 PCV x 10
Red cell count

10. RDW
 It is a measure of degree of variation in red cell size
(Anisocytosis ) in a blood sample.
 Normal
11.6-14 %

11. Normal Range of Iron Profile
 S.Iron (65-180 ug/dl) in Males
S.Iron (50-180 ug/dl) in Females
 Transferrin saturation 30 % to 50 %
 Total iron Binding capacity 250 to 450 ug/dl
 S.ferritin 50-200 ug/l

12. Morphological Categories of Anemia
1)Microcytic Anemia
3)Normocytic Anemia
2)Marocytic Anemia
Size of the RBC is compared with
The nucleus of small lymphocyte
MCV<80 fl is Microcytic
MCV>100 fl is Macrocytic

13. •Normochromic Vs
Hypochromic RBC’s
•Defined by area of Central
Pallor
•Upto 1/3rd the Size of RBC is
Normochromic
•>1/3rd of RBC Size is
Hypochromic

14. Microcytic Hypchromic anemia
 When the average cell size(MCV ) is reduced the
anemia is called as microcytic anemia
 It is usually associated with hypochromia

15. Types Of Microcytic Hypochromic Anemia
 Iron Deficiency Anemia
 Sideroblastic Anemia
 Thalassemia
 Anemia of Chronic disease
 Porphyrias
 Lead Poisoning
 Congenital Atransferrinemia
 DMT1 Mutation
 Glutaredoxin5
 Aceruloplasminemia

16.  LAB APPROACH TO MICROCYTIC
HYPOCHROMIC ANEMIA

17. Laboratory Approach to Anemia
 Complete Blood Count
Low Hemoglobin –Anemia
Abnormalities restricted to red cell series
Reticulocyte count
Raised Not Raised Red cell Indices
Investigate for Blood loss Hemolytic anemia

18. Laboratory approach to anemia
 Complete Blood Count
Low Hemoglobin –Anemia
Associated abnormalities of White Blood cells and
platelets
Bone Marrow Examination to assess for
Megaloblastic anemia ,Aplastic Anemia
,Myelodysplasia,Myelofibrosis ,myelophthsis ,Leukemia

20. Case presentation 1
 A 29 year Old woman presents with a History of fatiguability,
weakness and lethargy was found to Have a hemoglobin of 7.8 g/dl
,with a reticulocyte count of 0.4%
MCV 70 fl
MCH 20 pg
MCHC26 g/dl
RDW20
S.Iron15 ug/dl
TIBC 460 ug/ dl
S.ferritin15 ug/l
The most likely cause of Anemia is ?

21.  IRON DEFICIENCY ANEMIA

22.  Children (13-15 yrs.): 20-
30 mg/d
 Pregnant women : 40 mg/d

23.  Upper part of duodenum.
 Only Fe2+ (ferrous) form (reduced form) is absorbed. Fe3+
(ferric) form is not.
 Fe3+ (ferric) ions need to be reduced.

25. Regulation of Absorption
 Hepicidin protein plays important role in Iron Absorption .
 It decreases iron absorption by binding to ferroportin.
 So, when Plasma Levels of iron are High hepicidin
expression increases and when plasma levels of iron are
low Hepicidin expression is decreased .

26. Transport and delivery to target sites
 Fe3+ is carried as transferrin in Blood to various tissues.
 Transferrin is a β-Glycoprotein it is circulated in Blood
 Bone Marrow & Liver are the Major sites of Iron
Utilization.
 Liver uses it for Heme proteins and Enzymes Synthesis

27. Plasma Transferrin & TIBC
 Plasma Iron is transferrin Bound .
 Average Levels of Transferrin are 300 mg %
 Normally this transferrin can bound a maximum of 300 ug/dl of
iron.
 This therefore becomes Total Iron Binding capacity
 However under normal Physiological conditions ,transferrin
remains only 30 % saturated with Bound Iron.
 Hence Normal Transferrin saturation is 30 %

28. Storage of Iron -Ferritin
 Ferritin is indicator of Body Iron stores.
 In deficiency state Iron is released from ferritin by the
macrophages mainly in the Liver and spleen

29. Iron Deficiency Anemia
 Most Common Nutritional Deficiency in the world
 Etiology :
1) Dietary Deficiency
2) Blood loss – causes of Gastrointestinal lesion leading to
Blood loss are as follows :

30. Etiology of Iron deficiency
anemia
 Peptic ulcer
 Hiatal Hernia
 Alcoholic Gastritis
 Excessive Salicyclate ingestion
 Carcinoma Colon
 Hookworm infestation
 Hemorrhoids
 Menstruation in women of Child bearing age

31. Etiology of Iron deficiency
anemia
3) Malabsorption
 After gastrectomy
 Atrophic gastritis
 Achlorhydria
4) Increased requirements:
 In pregnancy
 Children less than 5 years
 Elderly
 Women of reproductive age group and Adolescents

32. Patho-physiology of IDA
Stage 1 : During Iron Depletion (No Functional effect )
 Iron Stores are Exhausted indicated by decrease in
S.ferritin
 No Anemia
 Erythrocyte Morphology is normal
 Red cell distribution width is Elevated
 Abnormal RDW can be the first Hematologic
indication of a developing Iron deficiency in non
anemic patients.

33. Patho-physiology of IDA
Stage 2 :
 The second stage of Iron deficiency is characterized by
iron deficient erythropoeisis
 There is insufficient iron to insert into the
protoporphyrin ring to form heme.
 As a result protoporphyrin accumulates in the cell and
complexes with zinc to form ZPP.
 Bone marrow sideroblasts are absent
 Macrophage iron is not seen
 Anemia and Hypochromia are still not detectable but
RBC’s can become slightly microcytic

34. Patho-physiology of IDA
Stage 3 :
 Long standing negative iron flow eventually leads to the
last stage of Iron deficiency.
 Blood loss can shorten the time for this stage to develop
 All laboratory tests for Iron status becomes markedly
abnormal.
 The most significant finding is classic microcytic
hypochromic anemia

36. Clinical findings of IDA
 Insidious onset
 Clinical symptoms of anemia
Fatigue
Dyspnea
Tachycardia
Other symptoms :
Koilonychia
Glossitis
Pharyngeal webs
Pica syndrome : Unusual craving for ingesting
unnatural items

37. Lab findings in Iron Deficiency
anemia
 Peripheral smear shows
 Anisocytosis and poikilocytosis
 Anisocytosis – important early sign Leading to
Raised Red cell distribution width.
 Red blood cells are microcytic smaller than the nucleus
of small lymphocyte
 Erythrocytes are Hypochromic with a Increased central
Pallor
 Elliptocytic and pencil shaped forms can be present

38. Peripheral blood picture of IDA

39. Iron Deficiency Anemia
 Leukocyte count –Usually normal
 Mild Granulocytopenia can be seen in long standing cases
 Recent large Volume hemorrhage leads to neutrophilic
leukocytosis.
 Due to parasitic infestation , Eosinophilia can be seen
 Thrombocytes : Thrombocytosis is frequently seen

40. Transferrin
 The principal source of Iron for hemoglobin
production is carried by the transferrin, Iron
transport protein in plasma.
 When transferrin saturation with Iron is less than
15% ,RBC Production rate decreases and
Hypochromic, microcytic cells are manufactured.
 This stage is known as Iron Deficient
erythropoeisis

41. Serum transferrin receptor assay
 STfR assay is useful in differentiating Iron deficiency
anemia and anemia of chronic disease
 STfR-F index
Serum transferrin receptor = STfR/Log of ferritin
 Patients with Iron deficiency have a mean STfR over twice
that of normal individuals .
 STfR in Anemia of chronic disease is similar to normal
individuals.

42. Iron Profile in Iron deficiency anemia
 S.Iron decreased <30ug/dl
 S.ferritin decreased <15 ug/l
 TIBC Increased
 Pecrentage transferrin saturation <15 %

43. Bone Marrow Features of Iron
Deficiency Anemia
 Cellularity – Increased
 Erythroid Hyperplasia
 Micronormoblastic reaction
 Normoblast are smaller
 Late Micronormoblast demonstrates persistent
basophilia and fraying of cytoplasmic borders
indicating lack of Hemoglobinization
 Myelopoeisis –Normal
 Megakaryopoeisis –Normal
 Depleted Bone marrow iron

44. BONE MARROW FEATURES OF
IRON DEFICIENCY ANEMIA

45. Bone Marrow Features of Iron
Deficiency Anemia

46. Bone Marrow Features of Iron
Deficiency Anemia

49.  Case presentation 2
A 25 year old lady on treatment for Rheumatoid arthritis
Has the following Laboratory Findings:
Hb-9g/dl
MCV-50fl
Ferritin -220 ug/dl
TIBC -240 ug/dl.
S.Iron -30 ug/dl
What is the likely diagnosis ?

50. Anemia of chronic disease
 Also called as Anemia of inflammation or anemia of
infection.
 Most frequent form of anemia in Hospitalized patients.
 Definition : Impaired red cell production associated
with chronic diseases that produces systemic
inflammation.

51. Anemia of chronic disease
 Conditions associated with Anemia of Chronic
Disorders
Category Conditions
Chronic infection (after 1-2
months ) of sustained
infection
Pulmonary infections
Subacute Bacterial
endocarditis
Pelvic inflammatory disease
Osteomyelitis
Chronic urinary tract
infection
Chronic fungal disease
Tuberculosis

52. Anemia of chronic disease
 Conditions associated with Anemia of Chronic
Disorders
Category Conditions
Chronic Non infectious
inflammation
Rheumatoid arthritis
Rheumatic Fever
Systemic Lupus
erythematosus
Sterile abscess
Regional enteritis
Ulcertaive colitis

53. Anemia of chronic disease
 Other causes
Malignant Neoplasm
Alcoholic Liver Disease
Congestive Heart failure
Thrombophlebitis
Ischaemic Heart disease
Severe tissue trauma
Thermal Injury
Fractures

54. Pathophysiology of ACD
 Causes are Multifactorial.
 Block in release of iron from macrophages due to
increased Hepcidin.
 Cytokine inhibition of Erythropoetin production.
 Direct Cytokine inhibition of Erythropoeisis.
 Shortened erythrocyte survival.

55. Pathophysiology of ACD
 Block in iron release from macrophages is mediated by
hepcidin which is produced in response to IL-6 and other
inflammatory cytokines released in anemia of chronic
disease.
 Decreased Erythropoetin production and direct suppression
of erythropoiesis by cytokines.
 Shortened erythrocyte survival :due to non specific
macrophage activation.

56. PathoPhysiology of ACD
 Disorders associated with Anemia of chronic disease
enhance phagocytic activity of macrophages
 Activated macrophages are sensitive to slight alteration
in red cells and causes their premature destruction.

57. PathoPhysiology of ACD
 Release of Iron from macrophages is inhibited leads to
hypoferremia (Low serum Iron) and less iron is
available to erythroid precursors.
 This causes iron deficient erythropoiesis despite
sufficient storage iron

58. Clinical Findings of Anemia of
Chronic Disease
 Signs and symptoms are those of underlying disorders
 Anemia develops during first 1-2 months of illness
 Clinical manifestation may vary widely in anemia of
chronic disease because of its association with so many
diseases.

59. Laboratory Findings of Anemia of
chronic disease
 Peripheral Blood
Erythrocytes are usually Normocytic Normochromic
MCV<85 fl
But long standing cases can present as microcytic
Hypochromic
White blood cell and platelet count are normal.

60. Iron studies
 S.iron –Normal to decreased
 TIBC –Normal to decreased
 Transferrin saturation – normal to decreased
 Increased S.ferritin

61. Bone Marrow Examination
 Increased myeloid: erythroid ratio because of decrease in
erythroid precursors
 Proportion of younger erythroblast increases.
 Sideroblasts <30%
 Macrophages appears to have increased hemosiderin in the
form of coarse iron aggregates

62. BONE MARROW FEATURES OF
ANEMIA OF CHRONIC DISEASE
Bone Marrow aspirate demonstrate
an increased iron staining in a
fragment representing Increased
marrow iron stores.
Normal Iron staining is shown
in histiocytes for comparison.

63. CRITERIA FOR GRADING IRON
STORES
Grade Criteria
0 No Iron Granules observed
1+ Small granules, seen only in oil
immersion lens
2+ Few small granules seen with low
power lens
3+ Numerous small granules in all
marrow particles
4+ Large granules in small clumps
5+ Dense, large clumps of granules
6+ Very large granules obscuring
marrow details
Normal Marrow = 1+
to 3+

64. For Diagnosis of Anemia of chronic disease
 Evidence of chronic disease (Raised ESR),Raised CRP
 Mild to moderate anemia, red cells normocytic
normochromic, in late stages presents as microcytic
hypochromic
 Low S.Iron, Low TIBC , Low transferrin saturation
 S.ferritin Normal or Increased
 S.Hepicidin High

65. Case 3
 A 33 yr old patient on ATT with following laboratory
investigations
 Increased S.iron
 Increased transferrin saturation
 Increased ferritin
 Normal TIBC
 What is the most likely diagnosis ?

66. Sideroblastic Anemia
 Mutations that affect the first enzyme step in Heme
synthesis, formation of δ-aminolevulinic acid result in
sideroblastic anemia

67. Causes of Sideroblastic anemia
 Hereditary
 Acquired  primary refractory anemia with ringed
sideroblasts
Secondary 
Hematological malignancies – Myeloproliferative disorders
, multiple myeloma ,Acute Myeloid leukemia M6 type
Drugs-Isoniazid ,pyrazinamide ,cycloserine .
Alcoholism
Lead poisoning
Others – Collagen disorders ,Carcinoma

68. Pathogenesis of sideroblastic anemia
 Impaired Heme synthesis in Mitochondria
 Defective heme synthesis is associated with increased Iron
uptake
 Protoporphyrin link up with iron fails to occur and excess
iron is deposited in mitochondria

69. Peripheral Blood picture of
Sideroblastic anemia
 Poikilocytosis.
 Moderate to severe anemia
 A Dimorphic picture –Normocytic and hypochromic
 Dual population of normocytes and microcytes
 Hypochromic microcytes more common in hereditary
form of Sideroblastic anemia
 Erythrocytes can contain pappenheimer bodies (Iron
deposits)-Basophilic granules of iron inside RBC.
 Basophilic stippling can be seen in any of
Sideroblastic anemia resulting from aggregated
ribosomes

70. Iron profile in sideroblastic anemia
 S.Iron Increased
 Normal or Increased TIBC
 Increased S.ferritin
 Increased Transferrin saturation levels

71. Bone Marrow findings in Sideroblastic
anemia
 Erythroid hyperplasia
Erythroblast –poorly hemoglobinized with scanty
irregular cytoplasm
 Macrophages contain increased amount of storage iron
 Ringed sideroblasts >40% are erythroblast with iron
granules that encircle one third or more of the nucleus

72. Ringed Sideroblast

73. Case 4
 A 5 yr old child with chronic anemia not responding to
Iron therapy ?
Diagnosis ?

74. Thalassemia
 Definition :
 Thalassemias are heterogeneous group of Inherited
disorders of Hemoglobin characterised by reduced or
absent production of one of the globin chains
 Thalassemia is a quantitaive disorder
 Thalassa Greek word means Great sea
 First observed in Mediterranean sea

75. Classification of Thalassemia
 1)Based on type of globin chain that is deficiently
synthesized
 2) Clinical expression of disease
 Classification according to the type of globin chain which
is deficiently synthesised : The two most common types are
α and β Thalassemia
 Classification According to Clinical severity : β
Thalassemia classified on the basis of severity of anemia
into three types : thalassemia major ,thalassemia minor and
thalassemia intermedia

76.  α Thalassemia are Hb Bart’s ,Hydrops fetalis
syndrome ,HbH disease ,Thalassemia trait and
Silent carrier.
Classification of Thalassemia

77. Molecular Basis of Thalassemia
 β Thalassemia are caused by point mutations.
while most of the α thalassemias result from gene
deletions.

78. Clinical features of Alpha Thalassemia
 Pallor
 Weakness
 Fatigue
 Hepatosplenomegaly
 Heart defects
 Hb barts syndrome can cause
complications in pregnancy

79. Clinical Features of β-Thalassemia
Thalassemia Major
 Age of presentation : 6months -9 months
 Progressive pallor and Jaundice
 Cardiac failure
 Hepatosplenomegaly
 Bouts of Fever and Diarrhoea
 Feeding problems

80. Clinical Features of β- Thalassemia
By childhood
Growth retardation
Transfusion dependent
Changes in skeletal system

81. Skeletal Changes
 CHIPMUNK FACIES(HEMOLYTIC FACIES)
Frontal bossing,maxillary hypertrophy,depression of
nasal bridge, malocclusion of teeth
 PARAVERTEBRAL MASSES
Broad expansion of Ribs at vertebral attachment
 PATHOLOGICAL FRACTURES
Cortical thinning
Premature fusion of epiphysis –Short stature

82. Clinical features of thalassemia
Intermedia
 Moderate pallor ,usually maintains Hb>6gm%
 Less transfusion dependent
 Skeletal changes
 Progressive Splenomegaly
 Growth Retardation
 Longer survival than Thalassemia Major

83.  Usually asymptomatic
 Mild Pallor ,No jaundice
 No Growth retardation
 No Skeletal abnormalities ,No Hepatomegaly
 May Present as Iron deficiency Anemia
 Unresponsive/refractory to Iron therapy
 Normal life expectancy
Clinical features of thalassemia Minor

84. LABORATORY DIAGNOSIS
 Hb-Reduced (3-9 g/dl)
 RBC count Increased
 WBC, Platelets –Normal
 RBC Indices –MCV,MCH and MCHC Reduced
 RDW Normal

85. Laboratory Findings
 Peripheral Blood examination :
 Microcytic hypochromic RBC’s
 Anisocytosis
 Poikilocytosis
 Plenty of target cells
 Howell jolly bodies
 Basophilic stippling
 Nucleated red cells.

86.  Target cells
Peripheral picture of Thalassemia minor

87.  Basophilic
stippling
Peripheral picture of Thalassemia minor

88. Bone marrow Findings in thalassemia
 Bone marrow examination shows Erythroid hyperplasia
 Myeloid : Erythroid ratio reversed (Normal 3:1)
 Increased Bone marrow iron

89. S.IRON studies
 S.Iron Normal
 S.Ferritin Normal
 Percentage Saturation Normal
 Normal TIBC

90. SCREENING TEST
 NESTROFT TEST
 Naked eye single tube red cell osmotic fragility test
 Most common screening test
 Blood sample is collected in test tube and normal saline
is added (0.35%)
 A plain white paper with a black line is placed behind the
test tubes
 After 30 minutes interpretation will be :
A Black line is seen in normal individual.

91.  No Black line seen is indicator of Thalassemia

92. Other Parameters
 Total Bilirubin and Indirect Bilrubin is Increased
 Haptoglobin and Hemopexin depleted
 Free erythrocyte Protoporphyrin Normal

93. Special test
 Diagnosis is confirmed by Hemoglobin electrophoresis
 This characteristically shows elevated HbF in
Thalassemia Major.
Increased HbA2 in β-Thalassemia minor

94. Hb electrophoresis

96. Thalassemia minor vs iron deficiency
anemia
Findings Thalassemia minor Iron deficiency
anemia
Anisocytosis Mild or absent Early and
prominent
finding
Microcytosis More severe Less severe

97. Mentzer index
 Mentzer index=Mean cell volume/Red cell count
 Value greater than 13 is found in iron deficiency anemia
whereas value less than 13 is seen in thalassemia trait
disorder.

98. Differential diagnosis of Microcytic
anemia
Iron deficiency
Anemia
Anemia of
Chronic
Disease
Thalassemia
(α or β)
Sideroblastic
Anemia
S.Iron Reduced Reduced Normal Raised
S.TIBC Raised Reduced Normal Normal
S.Ferritin Decreased Raised Normal Raised
Iron in Bone
Marrow
Absent Present Present Present
RDW Increased Normal Normal Normal
Percentage
Transferrin
saturation
Reduced Reduced Normal Raised
Free
Erythrocyte
Protoporphyrin
Raised Raised Normal Raised

100. Summary
 Iron Deficiency Anemia is the most common
nutritional deficiency
 In Iron Deficiency Anemia TIBC is Increased.
 In Anemia of Chronic Disease TIBC is Decreased.
 In sideroblastic anemia TIBC Normal
 In Thalassemia Iron studies will be Normal

101. References
 Mckenzie S.Clinical Laboratory Hematology 3rd
Edition.Pearson Education : New Jersey;2015.p200-268
 Wintrobe’s clinical Hematology 11th Edition.Lippincott and
Wilkins: USA;2004,p.972-976
 Kumar V.Robbins and Cotran Pathologic Basis of disease
9th Edition.RELX Pvt.Ltd;2015.p629-650
 Kawathalkar S.Essentials of Clinical Pathology 2nd ed.New
Delhi:Jaypee Brothers Medical publishers (p)
Ltd;2018.p213-216.
 Google Images
 www.ncbi.nilm.nih.

102.  THANK YOU