hematopoeisis
Bone marow examination
Normal bone marrow
1. Haemopoietic cells. Morphology of haemopoietic cells
 Granulocytes & their precursors – 60%
 Erythroid precursors – 20 %
 Lymphocytes & Monocytes & their precursors – 10%
 Unidentified or disintegrating cells – 10%
2. Myeloid : erythroid ratio – 3-4 :1
Anaemia
 Anaemia may be defined as a state in which the blood Hg is below the
normal range fro the age & sex of the patient
Normal count/Range
Hemoglobin
 Male – 13 –18 gm/dl
 Female -11.5 – 16.5 gm/dl
Red cell count –
 Male - 4.5–6.5 million/cu mm of blood
 Female – 3.8–4.8
 Reticulocyte count - 0.2–2.0%
Hemoglobin
(Hb)
Red cell count
Reticulocyte
count
g/dL
13.6-17.2
12.0-15.0
×106/mm3
%
4.3-5.9
0.5-1.5
3.5-5.0
0.5-1.5
Factors necessary for Erythropoiesis.
A] General Factors
 1] Diet-Protein is essential for synthesis of Globin in of Haemoglobin
 Iron-essential for heam production
 Others-Cu, Mg, Cobalt, Ca etc.
 2] Hypoxia-hypoxia causes Liberation of hormone Erythropoietin
which stimulate bone marrow for Erythropoiesis
 3] Erythropoietin
 4] Endocrine gland- Adrenal Gland, Pituitary Gland, Thyroid Gland.
B] Maturation factors
 1] Vit. B12
 2] Folic Acid
 3] Intrinsic Factor of Castle-It helps in absorption of Vit. B12 from gut
and thus indirectly help in maturation.
Classification of Anemia
Morphological & Aetiological Classification
Morphological classification
 Based mainly on MCV & MCHC. MCH may be included
1.Microcytic Hypochromic aneamia
 MCV, MCHC & MCH are below normal
 E.g. – IDA, thalassaemia, anaemia of chronic diseases
2. Macrocytic anaemia
 MCV is above normal . MCHC is normal
 E.g. megaloblastic anaemia
3. Normocytic Normochromic anaemia
 MCV, MCHC MCH are within normal range
 E.g. acute blood loss, haemolytic anemia

Aetiological Classification
This classification is based on pathophysiology & cause
 Impaired RBC production
 Hemolytic anemia
 Blood Loss
Impaired RBC production
A. Deficiency of essential nutrients (Deficiency anaemia)
 Iron deficiency – most common cause
 Vitamin B12 deficiency
 Folic acid deficiency
 Combined deficiency
Others – protein calorie malnutrition , vitamin C deficiency

B. Depression of erythropoiesis
 Anemia of chronic disease – Chronic renal failure . Liver diseases,
Malignancy
 Invasion of bone marrow – leukaemia , secondary carcinoma
 Aplastic anemia
B. Haemolytic anemia
 Intracorpuscular defect
 Extracorpuscular defect
C. Blood Loss
 Acute Blood loss – loss of large volume of blood over a
short period
 Chronic blood loss e.g hookworm infestation, bleeding
peptic ulcer, piles, menorrhagia
Clinical features of anemia
 Tiredness , Fatigue
 Lethargy
 Palpitaion
 Pallor is the most important physical sign
Laboratory diagnosis of anemia
 Hb estimation
 Diagnosis of Morphological type of anaemia
 Diagnosis of aetiological type of anemia – discussed
under individual type of anemia
Packed cell volume (PCV)
 Is the volume of red cells in relation to that of whole blood
PCV = MCV X red cell count
Normal values
 40 – 45 % male
 37- 47 % female
MCV (Mean corpuscular volumes )
 Indicates the average volume red cells
 Normal 76 - 96 fl (femolitre)
MCH (mean corpuscular hemoglobin )
 Indicates average weight of Hb contained in each cell
 Does not take into count cell size
 Obtained by dividing Hb by red cell /liter & multiplied by 1013
If Hb 15g/dl
Red cell – 5X1012/L
MCH = 15X1013 / 5X1012 = 30 pg
 Normal 27-32 pg

MCHC (mean corpuscular haemoglobin concentration)
 Indicates the average concentration of Hb within average red cells
 Usually 3 times higher that the whole blood Hb conc.
 Obtained by dividing Hb by PCV /liter
If Hb 15g/dl
PCV – 0.45l/l
MCHC = 15/0.45 = 33.3g/dl
 Normal – 31-35 g/dl

Diagnosis of morphological type of anemia
1. Examination of peripheral blood film
2. Determination of Red cell absolute value
 MCV & MCHC are below normal values – microcytic hypochromic
anemia
 MCV is above normal & MCHC is normal – macrocytic anemia
 MCV & MCHC are normal – normocytic normochromic aneamia
Iron deficiency anemia
Most common type of anemia
Total body iron content
 Male – upto 6gm
 Female – 2gm
80% of functional body iron is found in hemoglobin, rest found in
myoglobin and iron-containing enzymes (e.g.catalase & cytochromes).
The iron storage pool, represented by hemosiderin and ferritin-bound
iron (15% to 20% of total body iron.)
 Stored iron is found mainly in the liver, spleen, bone marrow, and
skeletal muscle.
Male Fem
Absorption
 Most absorbed in Duodenum
Causes /pathogenesi of iron deficiency
1. Inadequate iron intake
 Nutritional deficiency – deficient diet
2. Impaired absorbtion – coeliac disease, tropical sprue, gastrectomy or
gastro-enterostomy
Causes /pathogenesi of iron deficiency
3. Increased physiological demand
 During period of growth in children
 During reproductive life in female – menstruation, pregnancy,
parturation & lactation increase the physiological requirement fro iron
4.Chronic blood loss – the gastrointestinal tract (e.g., peptic ulcers,
colonic cancer, hemorrhoids, hookworm disease) or the female
genital tract (e.g., menorrhagia, cancers)
Laboratory diagnosis of IDA
Investigation & findings
1. Blood Picture
 Hb – Variably reduced
 Blood film – hypochromic, microcytic, anisocytosis. Poikilocytosis
in severe cases target, elliptical, oval & pencil cells
 Haematocrit – reduced
 MCV – reduced(microcytosis) MCHC – reduced (hypochromic) & MCH
is reduced
2. biochemical findings (confirm IDA)
 Serum iron - reduced
 Serum ferritin - reduced
 Total iron binding capacity – increased
 Percentage saturation of iron binding protein – decreased
3. Bone marrow
 Although erythropoietin levels are increased, the marrow response is
blunted by the iron deficiency, and thus the marrow cellularity is
usually only slightly increased
Diagnostic criteria include
 Anemia, hypochromic and microcytic red cell indices, low serum
ferritin and serum iron levels, low transferrin saturation, increased
total iron-binding capacity, and, ultimately, response to iron therapy
4. Further Investigation
 Stool examination for
 Ova of hookworm &
 Occult blood
 Urine examination for haematuria
 Other investigations depending on clinical findings
Megaloblastic aneamia
 MA are characterized by formation of morphologically abnormal
(enlarged) nucleated red cell precursors called megaloblasts in the
bone marrow.
 The change occurs due to deficiency of vitamin B12 or folate
Vitamin B12
 Abundant in all animal foods, including eggs and dairy products, and
is resistant to cooking and boiling.
 Even bacterial contamination of water and non animal foods can
provide adequate amounts.
 It is stored in the liver, which normally contains reserves that are
sufficient to support bodily needs for 5 to 20 years
 As a result, deficiencies due to diet are rare and are virtually
confined to strict vegans
Metabolism of vitamin B12
 Peptic digestion release dietary vitamin B12,
 Binds to salivary B12-binding proteins called
R binders.
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R-B12 complexes transported to duodenum
Processed by pancreatic proteases to
releases B12,
Attaches to intrinsic factor (gastric juice)
The intrinsic factor-B12 complex passes to
the distal ileum
Attaches to the epithelial intrinsic factor
receptors - absorption of vitamin B12.
B12 bound to transport proteins called
transcobalamins, which then deliver it to
the liver and other cells of the body.
Causes of Megaloblastic Anemia
Vitamin B12 Deficiency
 Decreased intake
 Inadequate diet, vegetarianism
 Impaired absorption
 Intrinsic factor deficiency - Pernicious anemia, Gastrectomy
 Malabsorption states
 Diffuse intestinal disease e.g. lymphoma
 Ileal resection, ileitis
 Competitive parasitic uptake - Fish tapeworm infestation
 Bacterial overgrowth in blind loops and diverticula of bowel
 Increased requirement
 Pregnancy, hyperthyroidism, disseminated cancer
Folate metabolism
 Best sources - fresh uncooked vegetables and fruits.
 The principal site of intestinal absorption is the upper third of the
small intestine
 Conversion from dihydrofolate to tetrahydrofolate by the enzyme
dihydrofolate reductase is particularly important.
 Tetrahydrofolate involved in the synthesis of purines and
thymidylate, the building blocks of DNA,
Megaloblastic anemia
Folic Acid Deficiency
 Decreased intake
 Inadequate diet—alcoholism, infancy
 Impaired absorption
 Malabsorption states
 Intrinsic intestinal disease
 Increased metabolism
 Anticonvulsants, oral contraceptives
 Increased loss
 Hemodialysis
 Increased requirement
 Pregnancy, infancy, disseminated cancer, markedly increased
hematopoiesis
 Impaired use
 Folic acid antagonists – e.g.Methotrxate
Pathogenesis of Megaloblastic anemia
 Deficiency of Vit B12/folic acid
 Impairment of DNA synthesis,
 Results in a delay in nuclear maturation and cell division.
 Synthesis of RNA and cytoplasmic elements proceeds at a normal
rate
 Hematopoietic precursors show nuclear-cytoplasmic asynchrony
 Undergo apoptosis in the marrow (ineffective hematopoiesis)
Laboratory diagnosis of MA
Blood
 Hb– reduced
 Blood film
 RBC- many oval macrocytes, Anisocytosis, poikilocytosis,
Polychromatic & stippled cells, howell-jolly bodies
 White cells – hypersegmented neutrophils are always present (49 lobes)
Megaloblastic blood compared to normal blood
hypersegmented neutrophil with
a six-lobed nucleus
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Haematocrit (PCV)– reduced
Red cell count – reduced
MCV is high, MCH is high, MCHC is normal
WBC – leucopenia (neutropenia)
Platelets – thrombocytopenia
Reticulocyte count – increased
2. Biochemical findings
 Serum iron & ferritin – increased
 Serum bilirubin – may be slightly increased in Vit B12 deficiency
 Lactate dehydrogenase – increased
Male Fem
3. Bone marrow
 Cellularity – Hypercellular (increased numbers of megaloblasts)
 M:E ratio is reduced or even reversed
 Erythropoiesis - Megaloblastic erythropoiesis
 Granulopoiesis is active – giant metamyelocyte with u shaped
nucleus
 Iron in large amount. Sideroblast – increased
A to C, Megaloblasts-various
stages of differentiation
4. Special tests for vitamin B12 deficiency
 Serum vitamin B12 assay
 Schilling test – detects ability of the body to absorb vit B12 after
correction.
Radioactive cobalt labeled vit B12 is used for the test . A small oral
dose is given . Radioactivity in the urine is measured
 Methylmelonic acid excretion in urine - increased
 Therapeutic trail - Response to vit B12 administration
5. Special Tests for folate deficiency
 Serum folate assay
 Red cell folate assay
 Theurapeutic trail - Response to folic acid administration
Pernicious anemia
PA is a vitamin B 12 deficiency megaloblastic anaemia
Pathogenesis
 PA occurs due to failure of secretion of Intrinsic factor by the
stomach due to permanent gastric atrophy
 Vit B12 in food not absorbed (gastrectomy, resection of ileum )
Special tests
 Pentagastrin fast achlohydria
 Anti-intrinsic factor & anti-parietal cell antibodies in serum

Diagnosis is made by
 (1) low serum vitamin B12 levels,
 (2) normal or elevated serum folate levels,
 (3) serum antibodies to intrinsic factor,
 (4) moderate to severe megaloblastic anemia,
 (5) leukopenia with hypersegmented granulocytes, and
 (6) a dramatic reticulocytic response (within 2-3 days) to parenteral
administration of vitamin B12.