1. APPROACH TO ANEMIA IN PREGNANCY
Sitalakshmi S
Professor and Head
Department of Clinical Pathology
St John’s medical College, Bangalore

2. What is anemia? Sign? Clinical finding? Laboratory finding? Disease?
Pointer to a systemic disease?

3. The first step in any diagnosis:
physical examination
determining its cause
This may be difficult in pregnancy

4. A detailed medical, personal, and dietary history :
Any family or personal history of anemia
A history jaundice, or enlarged spleen
Heavy menstrual bleeding in women
Any signs of internal bleeding.

5. Of all the diagnostic tests available, complete blood count is one of the single most valuable tools in assessing the general health of the body.
Provides a snap shot of the hematopoietic system at a specific point in time.
It is a valuable indicator of disease, either local or systemic

6. Complete Blood Count (CBC)
a panel of tests that measures RBC,WBC and platelets.
For diagnosis of anemia, the CBC provides critical information on:
the size, volume, and shape of RBC
Hemoglobin
Anemia is when Hb is
< 11 g/dL for pregnant women
< 12 g/dL for non-pregnant women

7. Complete blood counts should be included in evaluation of:
As a part of routine examination
every sick patient
every patient with vague signs of disease
every pre-anesthetic evaluation
as a recheck test for patients previously diagnosed with erythrocyte, leukocyte or platelet abnormalities.

8. Results from the CBC test can help:
Provide basic information about one’s health
Detect a disease condition even before the patient is symptomatic
Identify the causes of the disease process
Rule out a disease process
Assess response to therapy
Establish a baseline that can be used for comparison with future test results

9. CBC
The CBC includes
quantitative evaluation of erythrocytes, leukocytes and platelets and
microscopic examination of the blood film to detect morphological abnormalities that provide valuable insight to various disease conditions.

10. Components of CBC RBC parameters Platelet parameters
WBC parameters - Scattergram
Reticulocyte count

11. Parameters… RBC parameters WBC parameters Platelet parameters
RBC count
Hemoglobin
Hematocrit
MCV
MCH
MCHC
RDW
Reticulocyte
Nucleated RBC
WBC parameters
WBC count
Differential
Platelet parameters
Platelet count
MPV
PDW
PCT

12. Hemogram: Hemogram
quantitative data (total cell counts, differential cell counts, hematocrit, hemoglobin, red cell indices ) and
Qualitative data (blood film evaluation).
Proper interpretation depends upon the integration of the two and a systematic approach.
For all cell compartments, interpretation can be guided by answering a series of key questions.

14. All automated hematology analyzers in addition to enumerating the CBC results generate:
red cell histograms
platelet histograms and
white cell histograms or scattergrams

15. The RBC and platelet histograms depict the cell sizing data in graphic form by plotting relative cell number versus size (volume)
whereas scattergrams are two- dimensional or three- dimensional plots of multiparameter data obtained for the DLC.

17. T Red cell Indices help us to classify an anemia as:
mean cell volume (MCV)
mean corpuscular hemoglobin (MCH)
and mean cell hemoglobin concentration (MCHC)
help us to classify an anemia as:
regenerative (blood loss or hemolysis) or
nonregenerative (production defect)
often giving us insight to the etiology

18. Erythrocyte (RBC) HistogramRL RU
PLT
RBC
25-75 fl fl
RBC size: fl
RBC detection: between 25 and 250 fl
Distribution curves are separated by flexible discriminators: RL & RU

19. RBC - Histogram Distribution Width
RDW-CV
100 %
RDW – CV is equivalent to 68,26 % of the distribution curve
68.26%
Reference range less than 16 %
200
250 fl .
RDW-SD
200
250 fl
100%
RDW – SD standerd devation at 20 % of the distrubution curve
Reference range: fl
20%

20. RDW
RDW - coefficient of variation of the red blood cell distribution histogram.
Quantitative measure of variation in RBC size (anisocytosis),
RDW is elevated in:
iron deficiency anemia
RDW is normal in microcytic anemia of thalassemia.

25. The new additions to the CBC profile which have useful clinical application.
Red cell distribution width (RDW) – an indicator of the degree of anisocytosis
Hemoglobin distribution width ( HDW) - is a red cell parameter that measures anisochromia which along with MCV and RDW are useful in the differential diagnosis of microcytic anemia.

26. Red cell indices in Anemia
Anemia associated with thalassemia minor:
low MCV, a normal RDW, and elevated HDW
Iron deficiency anemia:
Low MCV, an increase in both RDW and HDW
Marrow regeneration as in haemolytic anemias :
elevated MCV, RDW and HDW.

27. The severity of anemia is categorized by:
Mild anemia Hb g/dL
Moderate anemia Hb g/dL
Severe anemia Hb below 7.0 g/dL

28. Anemia is a laboratory diagnosis?
Men
Women
Hemoglobin (g/dL)
Hematocrit (%)
42-50%
36-44%
RBC Count (106/mm3)
Reticulocytes
1.6 ± 0.5%
1.4 ± 0.5%
WBC (cells/mm3)
~4,000-11,000
MCV (fL)
80-96
MCH (pg/RBC)
30.4 ± 2.8
MCHC (g/dL of RBC)
34.4 ± 1.1
RDW (%) %

29. Role of Labs Good history and physical examination CBC With Diff
Leukopenia with anemia may suggest aplastic anemia
Increased Neutrophils may suggest infection
Increased Monocytes may suggest Myelodysplasia
Thrombocytopenia may suggest hypersplenism, marrow involvement with malignancy, autoimmune destruction, folate deficiency
Reticulocyte Count
Peripheral Smear

30. Red cell indices MCV (mean corpuscular volume)
The average volume of RBC
Hct
=  10 (fl)
RBC count (m/µL)
e.g. Hct= 40%
RBC=5.0 (m /µL)
MCV= 40/5.0  10 = 80 fl
NR= fl

31. MCH (mean corpuscular hemoglobin)
The average content of Hb in average RBC.
It is directly proportional to the amount of Hb and RBC size. Hb
MCH =
 (pg)
RBC count (m/µL)
e.g. Hb = 14 g/dl
RBC = 4 (m/µL)
MCH= 14/4  10
= 35 pg
NR= pg

32. NR= 32-36% MCHC (mean corpuscular hemoglobin concentration) Hb (g/dl)
Express the average concentration of hemoglobin per unit volume of RBC.
It defined as the ratio of the weight of hemoglobin to volume of RBC.
Hb (g/dl)
MCHC=
 (%)
Hct (%)
e.g. Hb = 14 g/dl
Hct = 45 %
MCHC 14/45  100 = 31%
NR= 32-36%

33. Peripheral smear
Reticulocyte smear

34. All automated hematology analyzers in addition to enumerating the CBC results generate:
red cell histograms
platelet histograms and
white cell histograms or scattergrams

35. Reticulocyte Count
Reticulocyte count is the percent of immature RBCs (released earlier in anemia from the marrow)
Normal levels % for non anemic stages
<1% means Inadequate Production
>/equal to 1 means increased production (hemolysis)
Corrected reticulocyte count compares anemic to non-anemic counterparts to assess response as reticulocyte count may overestimate response
Corrected Reticulocyte Count = % Retic X HCT/45

36. Reticulocyte Correction Factor
RPI = % reticulocytes X HCT/45 X 1/Correction Factor
Normal RPI =1
RPI < 2 Hypoproliferative
RPI greater than/equal 2 Hyperproliferative Disorder
Hematocrit
Correction Factor
40-45 1
35-39
1.5
25-34 2
15-24
2.5

37. CLASSIFICATION OF ANEMIAS
Anemias may also be classified functionally into:
Hypoproliferative (when there is a proliferation defect)
Ineffective (when there is a maturation defect)
Hemolytic (when there is a survival defect)

38. Kinetic Approach Decreased RBC production Increased RBC destruction
Lack of nutrients (B12, folate, iron)
Bone Marrow Disorder
Bone Marrow Suppression
Increased RBC destruction
Inherited and Acquired Hemolytic Anemias
Blood Loss

39. FUNCTIONAL CLASSIFICATION OF ANEMIAS

40. CLASSIFICATION OF ANEMIAS
Anemias may be classified morphologically based on the average size of the cells and the hemoglobin concentration into:
Macrocytic
Normochromic, normocytic
Hypochromic, microcytic

41. Morphological Approach
Microcytic (MCV < 80)
Reduced iron availability
Reduced heme synthesis
Reduced globin production
Normocytic ( 80 < MCV < 100)
Macrocytic (MCV > 100)
Liver disease, B12, folate

42. MORPHOLOGICAL CLASSIFICATION OF ANEMIAS

43. NORMOCYTIC NORMOCHROMIC ANEMIAS

44. MICROCYTIC HYPOCHROMIC ANEMIAS

45. MACROCYTIC ANEMIAS

46. Hemoglobin/Hematocrit
Symptoms of Anemia
Normal or High
Hemoglobin/Hematocrit
Check other
Causes of symptoms
e.g. Cardiac Pulmonary
Low
RBC indices
MCV < 80
MCHC < 32
MCV=80-96
MCV > 98
History of acute blood loss
Auto immune Hemolytic anemia
Anemia of chronic Diseases
Anemia of infection
B12 and folate levels
Serum iron and Total Iron binding capacity of Ferritin
Low Iron
IDA, chronic diseases, Renal diseases
Normal
Hb electrophoresis for Thala.
High Iron
BM exam
For Sideroblastic anemia
Low B12
PA, GI problems
Severe malnutrition.
Low folate
Folate malnutrition
GI problems
Liver disease
High or Normal
MPD
Liver Disease

47. Iron Deficiency Anemia: Peripheral Smear
The arrows show that the RBCs are smaller and paler
Microcytosis &, Hypochromic RBCs

48. What Causes Iron Deficiency?
Blood Loss (occult or overt): PUD, Diverticulosis, Colon Cancer
Decreased Iron Absorption: achlorhydria, atrophic gastritis, celiac disease
Foods and Medications: phytate, calcium, soy protein, polyphenols decrease iron absorption
Uncommon causes: intravascular hemolysis, pulmonary hemosiderosis, EPO, gastric bypass
Decreased Intake (rare)

49. Iron Deficiency Anemia
Low Retic Count
High RDW
Low iron level
High TIBC
Low ferritin

50. Degrees of Iron Deficiency

51. Causes of iron deficiency

52. Causes of Hypochromic Microcytic Anaemias
Blood losses
Inadequate intake
Enterocyte
Erythroid precursor
Defects in heme synthesis
or iron acquisition
Malabsorption
Breastfeeding with inadequate
supplementary food
Preterm, low birth weight
Growth spurt
Inadequate calorie intake
Vegetarian diet
Polymenorrhea
Parasitic infestations
Peptic ulcer
Inflammatory bowel
disease
Meckel diverticulum
Celiac disease
Helicobacter pylori gastritis
Autoimmune atrophic gastritis
IRIDA (TMPRSS6 mutation)
Chronic inflammation
Haemoglobinopathies
Sideroblastic anaemia
Erythropoietic porphyria
DMT1 mutations
Ferroportin disease
Hereditary atransferrinaemia
Hereditary aceruloplasminaemia . 53

53. Tests for Assessing Iron Status
Serum iron
Total iron binding capacity (TIBC)
Transferrin saturation = serum iron/TIBC x 100
Serum ferritin
Serum transferrin receptor (sTfR)/serum ferritin
[R/F ratio]
Reticulocyte haemoglobin content
Stainable iron in bone marrow
Serum iron exhibits diurnal variations, is affected by meat and oral iron supplements, and is assay methodology dependent. Transferrin saturation, which is the ratio of serum iron to total iron binding capacity, is low in iron deficiency. Serum ferritin, a good measure of tissue iron stores, and low in iron deficiency, exhibits a remarkable diurnal variation and increases in infection and liver disease. An approach that uses the ratio of serum transferrin receptor to serum ferritin (R/F ratio), has been shown to be an excellent test for body iron stores. But, again, this ratio has limited value in cases of inflammation or liver disease. CHr is The strongest predictor (better than hemoglobin) of Fe deficiency and Fe deficiency anemia in infants, toddlers and children 54

54. Iron Deficiency Stages
Prelatent
Latent
IDA
Parameter
Reduced iron stores with normal serum iron levels
Exhausted iron stores with normal haemoglobin
Low haemoglobin
Haemoglobin
Normal
Decreased
MCV/MCH
Serum iron
TIBC
Increased
Transferrin saturation
Serum ferritin
Marrow iron
Absent
Iron deficiency represents a spectrum (Table_3) ranging from iron depletion, which causes no physiological impairments, to iron-deficiency anemia, which affects the functioning of several organ systems. In iron depletion, the amount of stored iron (e.g., as measured by serum ferritin concentration) is reduced but the amount of functional iron may not be affected (30,31). Persons who have iron depletion have no iron stores to mobilize if the body requires more iron. In iron-deficient erythropoiesis, stored iron is depleted and transport iron (e.g., as measured by transferrin saturation) is reduced further; the amount of iron absorbed is not sufficient to replace the amount lost or to provide the amount needed for growth and function. In this stage, the shortage of iron limits red blood cell production and results in increased erthryocyte protoporphyrin concentration. In iron-deficiency anemia, the most severe form of iron deficiency, the shortage of iron leads to underproduction of iron-containing functional compounds, including Hb. The red blood cells of persons who have iron-deficiency anemia are microcytic and hypochromic (30,31).
Abbreviations: IDA, iron deficiency anaemia; MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume; TIBC, total iron binding capacity.

55. Case Studies

56. Anemia Case Study #1
A 22 year old lady has the CBC findings shown. Peripheral RBCs are hypochromic & microcytic.

57. Anemia Case Study #1 A-Hemoglobin Electrophoresis B-Retic count
What test would you order for this patient?
A-Hemoglobin Electrophoresis
B-Retic count
C-Stool for occult blood
D-Serum Iron studies
E-Bone marrow biopsy

58. Anemia Case Study #1 Two questions: What is your diagnosis?
What is the next step for this patient?

59. Anemia Case Study #1 Answers Question 1 Question 2
Likely Iron Deficiency Anemia
Question 2
Serum iron studies

60. Anemia Case Study #2
A 28 year old lady G2P1 16 weeks gestation has become progressively more fatigued at the end of the day. This has been going on for months. In the past month she has noted paresthesias with numbness in the feet. A CBC demonstrates the findings shown.

61. Anemia Case Study #2
A peripheral blood smear (the slide is representative of this condition) shows red blood cells displaying macro- ovalocytosis and neutrophils with hypersegmentation.

62. Anemia Case Study #2
Which of the following tests would be most useful to determine the etiology?
A. Hemoglobin electrophoresis
B. Reticulocyte count
C. Stool for occult blood
D. Vitamin B12 assay
E. Bone marrow biopsy

63. Anemia Case Study #2 Questions:
What is the diagnosis from these findings?
How do you explain the neurologic findings?

64. Anemia Case Study #2 Answers: Question 1 Question 2
This is a macrocytic (megaloblastic) anemia. The neurologic findings suggest vitamin B12 deficiency (pernicious anemia).
Question 2
The B12 deficiency leads to degeneration in the spinal cord (posterior and lateral columns).

65. Case 3
27 year old lady at 18 weeks gestation referred to the Emergency because of sudden onset of generalized weakness, severe pallor and low grade fever
During the last week treated with an antibiotic for pharyngitis

66. Case 3 On examination: pallor, febrile, mild jaundice;
spleen mildly enlarged, soft, tender
No lymphadenopathy / hepatomegaly

67. Case 3 Laboratory evaluation - Set I: - Hemoglobin – 7.9 gm/dl
- MCV – 130fl
- WBC – 20,000/cmm DC N 88% L05% Myelo 03% Meta 02% Band 02%
- Platelets – normal
Peripheral blood smear:

68. Approach to Anemia – Case 3
Laboratory evaluation - Set IV:
Blood smear
- Anisocytosis (high RDW)
- Polychromasia (reticulocytosis) -

69. Case 3 Laboratory evaluation - Set II: - Reticulocytes – 14%
- Corrected reticulocyte count:
% Retics X (measured to the expected Hb ratio)/
14% x 7.9/15 = 7.4%
- Reticulocyte Production Index (RPI):
Corrected reticulocyte count/shift correction factor
7.4 / 2 = (>2)

70. Case 3
Interpretation:
Red cell clumping, Polychromasia, nucleated RBCs, spherocytes
Diagnosis:
- Hemolytic anemia? Yes

71. Case 4 Laboratory evaluation - Set III: - LDH – high
- Bilirubin – high (unconjugated) -
Diagnosis confirmed: Hemolytic anemia

72. Approach to Anemia – Case 4
Hemolytic anemia – differential diagnosis:
- Intra-corpuscular
hemoglobinopathy
enzymopathy
Red cell membrane
- Extra-corpuscular
hypersplenism
mechanical hemolysis
immune hemolysis
toxic/infectious agents

73. Approach to Anemia – Case 3
Laboratory evaluation – Set V:
- Direct anti-globulin (Coombs) test – IgG +3

74. Approach to Anemia – Case 3
Summary:
- Mild splenomegaly, fever, jaundice
- Reticulocytosis
- Red cell clumping, few spherocytes, evidence of hemolysis
- Positive direct anti-globulin test
Warm-type immune hemolytic anemia, possibly – drug related

75. Case 4
24 year old lady presented at 34 weeks of gestation with headache, vomiting and epigastric pain of acute onset. On examination she had anemia and jaundice.
Investigations
Hb 5.6g/dl
TC 22000/cmm
DC N 86 L 5 Bd 3 Myelo 2 meta 4 NRBC 7/100 WBC
Platelet count /cmm

76. Case 4
Peripheral smear:

77. Case 4 Biochemical investigations: PT 13 sec C 12 sec INR 1
Serum bilirubin 2 .5 mg
Conjugated 0.8
AST 786 IU/L ALT 1030 IU/L
Alkaline phosphatase: 240
PT 13 sec C 12 sec INR 1

78. Case 4
HELLP syndrome
Acute fatty liver of pregnancy

79. Classification of Hemolytic Anemias
Hereditary
1. Abnormalities of RBC interior
a.Enzyme defects: G-6-PD def,PK def
b.Hemoglobinopathies
2. RBC membrane abnormalities
a. Hereditary spherocytosis etc.
b. PNH
Acquired
c. Spur cell anemia
3. Extrinsic factors
a. Hypersplenism
b. Antibody: immune hemolysis
c. Mechanical trauma: MAHA
d. Infections, toxins, etc
Ref : Harrison’s

80. A Very Simple Classification of Hemolytic Anemias
Intracorpuscular
1- Abnormalities of RBC interior
a. Enzyme defects
b. Hemoglobinopathies & Thalassemia M
2-RBC membrane abnormalities
a. Hereditary spherocytosis, elliptocytosis etc
b. Paroxysmal nocturnal hemoglobinuria
c. Spur cell anemia
3- Extrinsic factors
a. Hypersplenism
b. Antibody : immune hemolysis
c. Traumatic & Microangiopathic hemolysis
d. Infections , toxins , etc
Hereditary
Extracorpuscular
Acquired

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

82. Case 5
A 20 year old lady, 32 weeks gestation presented with fever and anemia. She had received multiple blood transfusions in the past
H/O having undergone splenectomy 3 years ago
On examination:
Pallor
Hepatomegaly

84. Peripheral smear shows many microcytes, target cells, nucleated red cells and Howell Jolly bodies

86. Hb Electrophoresis Hb A: 11.31% Hb F: 88.69%

87. B thalassemia Major

90. B thalassemia Post splenectomy blood picture with malarial parasite

91. Sickle cell anemia
Thalassemia

92. IDA
Target cells
Stomatocytes
Sickle cells
Poikiolocytosis
Acanthocytes
Spherocytes
Ovalocyte
Nucleated RBC

93. spherocytes Hypochromic
Macrocytic Microcytic

94. Normal red cell morphology

95. Anemia of Chronic Disease
EPO production inadequate for the degree of anemia observed or erythroid marrow responds inadequately to stimulation
Causes: inflammation, infection, tissue injury, cancer
Low serum iron, increased red cell porphyrin, transferrin 15-20%, normal to increased ferritin

96. Anemia of Chronic Disease

97. Approach to Anemia An approach to anemia is based on asking 3
main questions:
1. MCV?
2. Reticulocyte count? (Bone marrow function)
3. Involvement of additional lineages?

98. Approach to Anemia MCV: - Low: Iron deficiency Thalassemia trait
Chronic disease
Sideroblastic anemia
- Normal: Iron deficiency (early)
Renal failure

99. Approach to Anemia - High: Megaloblastic anemia Hemolytic anemia
MDS / Aplastic anemia
Drug-related
Hypothyroidism
Liver disease

100. Approach to Anemia Reticulocyte count:
- Adequte (RPI>2): Hemolytic anemias (most)
Acute blood loss
Liver disease
- Inadequate (RPI<2): Iron deficiency
Megaloblastic anemia
Anemia of chronic disease
Bone marrow failure

101. Approach to Anemia Additional lineage(s) involvement:
- Leukopenia: Malignancy
Chemotherapy
Hypersplenism
Drug-related
Megaloblastic anemia
Aplastic anemia

102. Approach to Anemia - Thrombocytopenia Drug-related
Megaloblastic anemia
Aplastic anemia
HELLP, DIC

103. Approach to Anemia
In addition, blood smear always needs to be carefully assessed:
Schistocytes? Spherocytes?
Target cells? Normoblasts?
Spur cells (acanthocytes)? Burr cells? Sickle forms?
abnormal cells (leukemia, lymphoma)?
Malaria parasites?

104. 26 year old lady 14 weeks pregnancy presents with weakness and bleeding gums
Hb 7g/dl
TC 1200/ul
DC N 2 L 98
Platelet count 20 thousand /ul

105. Hypoplastic anemia

106. Acute promyelocytic leukemia

107. Hb 8 g/dl
TC 1200/ul
DC N 40 L 30 Blast 10 Abnomal promyelocytes 20
Platelet count 20,000/ul

108. Anemia in pregnancy Iron Deficiency Anemia
Vit B 12 and Folate deficiency
HELLP syndrome
Sickle cell anemia
B Thalassemia
Autoimmune Haemolytic Anemia
Acute myeloblastic Leukemia - APML
Aplastic anemia

109. A logical stepwise approach in the context of clinical presentation
selection of appropriate laboratory tests to support the clinical suspicion
Use of laboratory automation
Arrive at the correct diagnosis