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Definition
• These are the anaemias in
which the RBC have an MCV of
greater than 100 fl.
• There are two groups of
macrocytic anaemias
- Megaloblastic anemia
- Non megaloblastic macrocytic
anemia
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Macrocytosis Without Anemia
Neonatal RBCs (at birth 100-130 fL)
Genetic predisposition
Early cases of vitamin B12 deficiency. (Macrocytosis characteristically
precedes the development of anemia and may even do so by several years)
Hence raise in MCV should not be ignored and correlated clinically
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Case 1
45 year old female
Hb of 7.1 g/dL
RBC 0.8 million/cumm
MCV – 111.9 fL
MCH – 86.4 pg
MCHC – 77.8 g/dL
Agglutination
of RBCs result
in grossly
abnormal
indices
RBC count,
MCV, MCHC
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Case 2
32 yr female with menorrhagia
Hb- 8.8 g/dL
RBC – 2.7 million
MCV – 101 fL
MCH – 27.6 pg
MCHC – 31.8 g/dL
Polychromatophils
20% larger in size
As a very rough rule
of thumb, for every
1% increase in the
reticulocyte
percentage, there
will be an increase of
1 fL in the MCV.
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Case 3
54 yr male
Hb- 9.8 g/dL
RBC – 3.5 million
MCV – 109 fL
MCH – 27.6 pg
MCHC – 28.8 g/dL
HbA1c- 9%
RBCs are glucose
loaded and swell
when they are
mixed with
isotonic diluent
MCV is elevated,
PCV is increased
and MCHC is
abnormally low.
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Case 4
EDTA induced changes
• Increase in red cell size with time, due to
degenerative changes that permits ingress
of water into the cells
• artefactual increase in RBC size could mask
microcytosis and lead to erroneous
diagnoses of macrocytosis.
• Other morphological changes such as
spherocytosis, echinocytosis &
spheroechinocytosis were observed from
Day 2 onwards
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Factitious macrocytosis
Hyperosmolar conditions
• Uncontrolled diabetes where RBCs are glucose loaded and swell when they are mixed with
isotonic diluent
• MCV is elevated, PCV is increased and MCHC is abnormally low.
Prolonged storage especially in EDTA
Extreme leucocytosis – leucocytes may enter RBC channel
Methanol poisoning – increasing MCV value.
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CAUSES OF MACROCYTIC ANEMIA
Macrocytic anemia
Megaloblastic Non megaloblastic
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Megaloblast
The term ‘megaloblast’ is a designation that was first applied by
Ehrlich to the abnormal erythrocyte precursors found in the bone
marrow of patients with pernicious anemia
Megaloblasts are characterized by their large size and by specific
alterations in the appearance of their nuclear chromatin – Sieve like,
nucleo-cytoplasmic asynchrony
Normoblastic erythroid
precursor
Megaloblastic erythroid
precursor
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B12 deficiency
Methyl malonyl coA Succinyl coA
Methyl malonic acid
Myelin
destabilizer
Deoxyadenosyl B12
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Vitamin B12 (Cobalamin)
Present in animal protein predominantly.
Stored in liver
Co factor for one carbon metabolism
RDA is 2.4 μg in adults
The total amount of vitamin B12 stored in body is about 2–5 mg in
adults.
2 important forms in body are methyl cobalamin and deoxyadenosyl
cobalamin
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Mechanism of absorption
In stomach, pepsin cleaves protein bound B12, Free B12 binds to
R-binder secreted from saliva
In duodenum pancreatic enzymes lyse the R-binder and B12
attaches to IF which is synthesized from parietal cells
B12 with IF is absorbed in terminal ileum by cubulin receptors
and IF is disintegrated and B12 binds with transcobalamin II
enters portal circulation and partly stored in liver.
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Causes of B12 deficiency
Dietary deficiency (Vegans)
Lack of intrinsic factor
Pernicious anemia
Gastric surgery – Bariatric
surgery
Prolonged use of PPI
Biologic competition for vitamin
B12
Small bowel bacterial overgrowth
Fish tapeworm disease
Familial selective vitamin B12
malabsorption
Chronic pancreatic disease
Zollinger-Ellison syndrome
Diseases of the ileum
Ileal resection
Regional enteritis/ intestinal TB
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Clinical features
Malaise, irritability, nausea, vomiting, abdominal
pain, diarrhea, anorexia, cognitive impairment,
forgetfulness.
Mild jaundice
Atrophy of tongue papillae gives rise to a beefy
red tongue.
Aphthous stomatitis and oral soreness in some
patients even without anemia.
Transient darkening of nails and skin (knuckle
hyperpigmentation)
Beefy red tongue
Aphthous ulcer
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Neurological manifestations
Neurologic changes can precede anemia
General symmetricalness of manifestations is typical
The earliest manifestations are loss of vibratory sense in the feet and
numbness, tingling, and loss of fine sensation
Posterior and lateral column degeneration
Ataxia, spasticity, gait disturbances, impotence, and loss of bladder and bowel
control, gait disturbances, neuropsychiatric manifestations are common signs
of advanced involvement
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Folate
RDA - 400 μg daily for adults
600 μg for pregnant women
500 μg for lactating women
Food folate, mostly methylTHF and
formylTHF, is labile
Storage of Folate – upto 7.5 mg in liver,
depleted within 100 days
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PERNICIOUS ANAEMIA
• Immunological mediated-Gastric mucosa
• Chronic atrophic gastritis-loss of parietal cells, infiltrate
by lymphocytes and plasma cells, megaloblastic changes
of mucosal cells.
Three types of antibodies
• Type I- 75%- Ab that blocks binding of B12 to IF- plasma
and gastric juice
• Type II- Ab blocks binding of IF-B12 complex to Ileal
receptor.
• Type III- Ab 85%-recognize α and β subunit of gastric
proton pump.
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Lab parameters
Hb values <7 or 8 g/dl are not unusual
The MCV usually between 110 and 130 fl
RDW is substantially increased, and the increase may precede the
development of anemia
Reticulocyte count is reduced due to ineffective erythropoiesis
As megaloblastic anemia progresses, neutropenia and
thrombocytopenia develop
The pancytopenia may mimic aplastic anemia
Serum lactate dehydrogenase and indirect bilirubin levels – elevated
reflecting the intramedullary destruction of RBCs as well as shortened
life span
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PS
The main products
of megaloblastic
erythropoiesis are
oval macrocytes
with reduced or
absent central
pallor.
Hypersegmentation
(5% with 5 lobes or 1
with 6 lobes) is among
the first hematologic
abnormalities to
appear
It persists for an
average of 14 days
after institution of
specific therapy
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Macrocytic anemia: CBC report from a case of
macrocytic anemia shows increased MCV
(111.7 /L) and MCH (37.7 pg).
•However MCHC is 33.7 g/dL in the normal
range.
• RDW as increased to 30.3%.
• Histogram curve (green) is shitted to right
suggesting increased MCV.
•The green curve is widened indicating
moderate degree of anisopoikilocytosis and
therefore RDW-CV is increased to
30.3%.
• Red curve is of normocytic normochromic red
cells (Normal}
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Bone marrow examination
• The initial marrow change is
megaloblastosis, even before
macrocytosis appears in the
peripheral blood.
• Bone marrow is markedly
hypercellular with erythroid
hyperplasia
• Thus reversal of M : E ratio to even
1 : 8.
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• The hallmark in the marrow is the
nuclear- cytoplasmic maturation
dissociation which is best seen in the
erythroid precursors resulting in
megaloblastosis
• Megaloblasts have open sieve like
nuclear chromatin and basophilic
cytoplasm.
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• There is preponderance of early megaloblasts which are more than
intermediate and late megaloblasts
• Intermediate and late megaloblast die in the marrow because of
unbalanced, nuclear and cytoplasmic maturation.
• There is widespread intra-medullary apoptosis of defective erythroid
cell precursors resulting in rise of unconjugated bilirubin.
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• Though marrow is very hypercellular and full of megaloblasts, only
10—20% of the potential progeny of proerythroblasts survive to
become the macrocytic red cells and this ineffective erythropoiesis
results in anemia.
• Howell Jolly bodies and stippling of cytoplasm is seen in some
megaloblasts. Both abnormal and normal mitoses are observed
frequently .
• Nuclei of megaloblasts demonstrate features of dyserythropoiesis
like nuclear budding, irregular nuclei, dumb-bell shaped nuclei, and
nuclear fragments etc,
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• D) Marrow aspirate shows
a giant megaloblast {*)
(at the bottom of the
picture)with megaloblasts
showing features of
dyserythropoiesis in the
form of irregular nuclei .
E) Bone marrow shows
erythroid hyperplasia.
Mitosis is frequent .
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• There is abnormal hemoglobinization of cytoplasm of megaloblasts.,
Megaloblastosis changes to normoblastic erythropoiesis within 12
hours of giving Vit. B12/FA and marrow morphology reverts to normal
within 2-3 days.
• Therefore, bone marrow should be carried out pretreatment if
required.
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• The misshapen neutrophils fray and the cells disintegrate resulting in
neutropenia— ineffective myelopoiesis, Giant metamyelocytes show
bizzare shapes of the nuclei (misshapen nuclei), and abnormal chromatin
and staining character
38. •Megakaryopoiesis Is less disturbed as compared to erythropoiesis and myelopoiesis,
Megakaryocytes of variable maturity display nuclei with open chromatin pattern and
complex nuclear lobular hypersegmentation .
• Megakaryocytes show larger size, fragmentation and very few platelets are formed— some
of them being megathrombocytes. This "ineffective thrombopoiesis accounts for reduced
platelet count.
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Trephine biopsy
• Bone marrow trephine biopsy displays preponderance of early
megaloblasts with uniform size and shape, having open nuclear
chromatin with linear 1-4 nucleoli and frequent mitoses.
• It is essential to differentiate these cells from blast cells which are
seen as small clusters (ALIP-Abnormal localization of miniature
precursors) in MDS.
• Morphologic aberrations are also apparent in myeloid cells and‘giant
metamyelocytes and band forms' are pathognomonic of
megaloblastosis,
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• Bone marrow trephine
biopsy shows eryihroid
hyperplasia with
preponderance of early
magaloblasts which are of
uniform size with delicate
nuclear membrane,
vesicular nuclei and 1—3
linear nucleoli which touch
the nuclear membrane,
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Vitamin B12 Levels
Standard initial routine diagnostic test
Microbiological assay replaced by automated radioimmunoassay,
chemiluminescence
In cobalamin deficiency, serum cobalamin levels fall below the
generally accepted cutpoint of 200 to 250 ng/L (<125 pmol/L)
Measures total Cobalamin ( Tc I, Tc II, Tc III) – hence may be elevated
in CML
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Methyl Malonic acid
Increases in serum and urine in cobalamin deficiency but not folate deficiency
Serum levels are elevated in 98.4% of patients with symptomatic cobalamin
deficiency
Levels often exceed >0.75 µmol/l in symptomatic cobalamin deficiency but are
usually only mildly elevated in subclinical deficiency
Falsely elevated in hemoconcentration, renal disease, small bowel bacterial
overgrowth
MMA is a more specific test for cobalamin deficiency than homocysteine
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Total homocysteine
Homocysteine rises with either folate or cobalamin deficiency
A normal result helps to exclude clinically relevant deficiency
Levels above 12 to 14 μmol/L in women and 14 to 15 μmol/L in men are generally
regarded as elevated.
Homocysteine rises in many acquired and genetic conditions
Folate intake and serum creatinine levels are major determinants of
homocysteine status
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Holotranscobalamin
Holotranscobalamin (HoloTC) – measures TC II, the ‘active’ fraction of plasma
cobalamin
Relatively new test
Immunoassay for this fraction is now available
Values for HoloTC in healthy individuals are 35–171 pmol/l
Better sensitivity and specificity characteristics than B12 levels
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Deoxyuridine suppression test
Abnormal Deoxyuridine suppression serves as a sensitive index of the presence
of megaloblastosis due to vitamin B12 or folate deficiency.
Test reflects impaired conversion of deoxyuridylate to thymidilate
done for research purposes
Can be done even in subclinical deficiency
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Cobalamin
(156-672 pmol/L)
>250 pmol/L
No deficiency
<125 pmol/L
B12 deficiency
125-250 pmol/L
Grey area
0.29-0.75 µmol/L
Grey area (Rpt after 1 yr)
<0.29 µmol/L
No deficiency
>0.75 µmol/L
B12 deficiency
MMA(0.08-0.28
µmol/L)
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TEST TO DIAGNOSIS FOR B12 DEFICIENCY:
• The Schilling test is a 3 stage test.
• Stage 1 - Give radioactive labeled vitamin B12 orally. Then screen the
patient's urine for the radio labeled B12.
• *If the radioactive B12 appears in the urine, the patient has successfully
absorbed the B12 and the abnormality is a dietary deficiency of Vitamin
B12. End of test.
• *If no or scant B12 appears in the urine, then the ingested B12 was not
absorbed. The defect in absorption is not due to dietary deficiency.
Proceed to Stage 2.
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• Stage 2 - Give oral radio labeled B12 with oral intrinsic
factor and again screen the patient's urine for B12.
• *If B12 is found in the urine, then the defect is due to a
lack of intrinsic factor. End of test.
• *If no or scant B12 is present in the urine, then the
defect is not due to a lack of intrinsic factor (because
absorption is not corrected by giving extrinsic IF). The
defect is at the intestinal level. Proceed to stage 3
SCHILLING TEST (Contd..)
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• Stage 3 - Treat patient with 7-10 days of antibiotics.
Then give radio labeled B12.
• *If decreased Ileal absorption is due to bacterial
overgrowth, antibiotics will correct this absorptive
defect and B12 will be detected in the urine.
• *If decreased Ileal absorption is secondary to an
anatomic defect, antibiotics will have no effect on B12
absorption and no B12 will be found in the urine.
SCHILLING TEST (Contd..)
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Tests to diagnose cause of B12 deficiency
• Nothing further if diet, resection history, very elderly patients
• Anti IF antibodies – 100% specific, 70% sensitive for Pernicious anemia
• All patients with anaemia, neuropathy or glossitis, and suspected of having
pernicious anaemia, should be tested for anti-IFAB regardless of cobalamin
levels (Grade 1A)
• Gastric anti parietal cell antibodies – May be +ve, very low specificity
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Other tests
• Cobasorb – Functional measure of Vit B12 absorption
• Baseline holoTC measured
• Oral B12 given
• Increased holoTC – normal absorption
• Unchanged HoloTC – No absorption
• Needs further evaluation before introduction into routine clinical practice
• Endoscopy for gastric malignancy every 5 years in case of atrophic
gastritis
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Tests to diagnose Folate deficiency
Serum Folate
The definition of folate deficiency in patients with megaloblastic
anemia, is a serum folate level below 3 μg/L.
Fluctuates with folate intake, falsely elevated in hemolysis
RBC folate
MethylTHF (60%) and formylTHF (26%) are the major folates in red
cells.
Red cell folate content is constant and is viewed as a truer indicator
of tissue folate
Red cell levels below 160 μg/L are usually considered low
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Investigation for folate
FIGLU test
Histidine -----------------→ FIGLU -------------------------→ Glutamate
↑
THF
• Oral challenge of Histidine Increased urinary excretion of FIGLU in folate deficiency.
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• Gastric biopsy: Cases with,
pernicious anemia demonstrate
gastric atrophy with loss of gastric
glands and parietal cells. There is
infiltration of lamina propria by
lymphocytes and plasma cells.
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Indications for bone marrow
Generally no role except
Lack of facilities for serum B12 and folate levels
Atypical clinical presentation
Splenomegaly
Bleeding
Sternal tenderness
Presence of circulating blasts
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Treatment
Treatment with folate alone in B12 deficiency cases may result in
masking of peripheral blood picture and neurological symptoms may
continue worsening.
Standard initial therapy for patients without neurological involvement is
1000 µg hydroxocobalamin intramuscularly (I.M) three times a week for 2
weeks
Patients with neurological symptoms should receive 1000 µg
hydroxocobalamin I.M. on alternate days until there is no further
improvement
Folate is also empirically administered 400μg to 1mg/day
• Folate deficiency : 5 mg of folic acid daily is taken for 4 months.
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Response to treatment
Often response to empirical treatment is used as a diagnostic test
Reticulocyte level starts by 2-3 days and peaks by 6-8 days
Marrow is completely normoblastic after 3-4 days. Giant metamyelocytes
persists for 12 days
A complete blood cell count 10-14 days after starting the treatment should
reveal a rise in hemoglobin (0.1g/dl per day) and a decrease in MCV
Full hematologic response should occur within 8 weeks.
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Masked megaloblastosis
Deficiency of vitamins without classical blood picture and bone marrow
picture.
Coexisting Iron deficiency or thalassemia
PS findings :
Dimorphic population of macroovalocytes and microcytic
hypochromic RBCs
Increased RDW
MCV may be normal
Hypochromic ovalocytes is a clue
Megaloblastic anemia (crisis) may be the initial presentation in
congenital hemolytic anemia
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Non-megaloblastic macrocytic anaemia
Represent macrocytic anemias in which the RBC precursors appear
normal without the characteristic nuclear and cytoplasmic findings of
megaloblasts
DNA synthesis is unimpaired.
Macrocytosis is usually mild; the MCV usually ranges from 100 to 110 fl
and rarely exceeds 120 fL
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Causes
Aplastic anemia
Myelodysplastic syndrome
Acquired sideroblastic anemia
Congenital dyserythropoietic
anemia types I and III
Diamond-Blackfan anemia
Erythroleukemia
Alcoholism
Liver disease
Hypothyroidism
Copper deficiency
HIV
Drugs – (independent of DNA
synthesis )
Valproic acid, Recombinant EPO,
Sulfonamides
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Case 5
• 55 male with ascites,
splenomegaly
• Hb 8.5 g/dl
• TLC 3800/cmm
• Platelet 1,25,000/cmm
• N 70, L 26, E2, M2
• MCV 103 fl
• MCH 28 pg
• MCHC 29 gm/dl
• Retic 2.5%
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Alcoholism and liver disease
Associated with thin macrocytes, defined as cells with increased surface area
but without a corresponding increase in volume
Consequence of excessive membrane lipids, especially cholesterol , but also
phospholipids.
The MCV rarely exceeds 115 fl
Cause
Faulty lipid metabolism
Associated Folate deficiency
Alcoholics may be associated with spur-cell hemolytic anemia
Poorly defined direct effects of alcohol on the bone marrow
Serum and erythrocyte folate levels are usually normal, and the macrocytosis
does not respond to folate treatment – abstaining from alcohol brings down
the MCV
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Case 6
• 16 year male
• Severe anemia and bleeding
gums – 2 months
• Hb 4 gm/dl
• TLC 1600/cmm N 10 L 90
• Platelet count 20,000/cmm
• Reticulocyte 0.1%
• MCV 108 fl
10-20 % aplastic anemia have
macrocytosis less than 110 fl
Much severe pancytopenia
No oval macrocytes or hypersegmented neutrophils
Lymphocytic preponderance
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Case 7
• 4 month old child
Severe pallor and
supernumerary
thumb
• Hb 5 gm/dl
• TLC 5600/cmm
• Platelet
2,15,000/cmm
• Retic < 0.2%
Congenital red cell hypoplasia or Diamond-Blackfan anemia is a pure RBC aplasia in
children.
Congenital abnormalities, including short stature and skeletal defects, specially of
thumbs
The diagnosis of DBA is suggested by a macrocytic (although sometimes normocytic)
anemia with reticulocytopenia presenting in the first 6 months of life.
Bone marrow examination reveals decreased erythroid precursors.
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Case 8
• 14 year old male
• Refractory anemia since early
childhood
• Multiple transfusions
• Hb 6 gm/dl MCV 110 fl
• TLC 8700/cmm
• Platelet 3,50,000/cmm
• DLC – normal
• Retic 1%
Peripheral blood in CDA-I
Bone marrow in CDA-I
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Congenital dyserythopoietic anemia
CDA - characterized by
ineffective erythropoiesis and
dyserythropoiesis.
Morphologically abnormal
erythroblasts in the bone marrow
with multinuclearity, nuclear
bridges, karyorrhexis, or
megaloblastic changes.
Morphologically, the anemia is
generally mildly macrocytic in
CDA types I and III and is
normocytic in CDA type II.
Bone marrow in CDA-III
Polychromatophilic
erythroblasts with
multinucleation
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Case 9
• 70 year male
• Progressive pallor
• Hb 6 gm/dl
• TLC 2900/cmm
• Platelet 45,000/cmm
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Myelodysplastic syndrome
Erythrocyte morphology is usually macrocytic
Oval macrocytosis is the best-recognized erythrocyte abnormality.
In extreme cases, elliptocytes , teardrops, schistocytes,
stomatocytes, or acanthocytes
Basophilic stippling, Howell-Jolly bodies, and megaloblastoid
nucleated red cells
Circulating blasts
5 q- syndrome – classically has macrocytosis
Dyspoietic megaloblastic morphology is also a feature of
Erythroleukemia
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CASE 10
• A 69-year-old man
• presented with a 2-year history of progressive thrombocytosis,
referred to exclude essential thrombocythemia.
• no palpable splenomegaly.
• Hemoglobin- 10.9 g/dl;
• mean corpuscular volume 100 fL;
• platelets 9.3lacs;
The peripheral blood smear showed marked thrombocytosis, giant platelets, and red
cell dysplastic changes including oval cells, target cells, numerous red cell stippling,
and Pappenheimer bodies (panel A). These findings suggested acquired sideroblastic
anemia and an associated myeloproliferative neoplasm.
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Case 11
• A 57-year-old African American woman with
shortness of breath and chest pain
• complete blood count profile
showed pancytopenia,
• white blood cell count of 3.3 × 103/μL,
• a hemoglobin level of 4.4 g/dL,
• platelet count of 29000/cumm
• Numerous nucleated red blood cells,
• erythroblasts, and a rare
circulating megakaryoblast were identified on
peripheral smear examination.