1.

3 Macrocytic anaemia
Megaloblastic and Non megaloblastic

Dr Niki Vyrides MD PhD

Haematology Course Lead MD6
Clinical associate Professor 01/09/2021
Vyrides Clinic
 Learning Objectives
3
►The importance of medical history and the main signs and symptoms which
would lead to the correct diagnosis of anaemia
►The common causes of macrocytic anaemia and macrocytosis without anaemia
►Nutritional and metabolic aspects of vitamin B12 and folate (dietary aspects,
absorption, body distribution and transport)
► The concept of megaloblastic anaemia and the effect of vitamin B12 and folate
deficiency on inhibition of DNA synthesis.
►Comparison of megaloblastic and non-megaloblastic anaemia.
►Differences between B12 and folate deficiency with respect to underlying causes,
time of development of clinical deficiency state and clinical manifestations.
►Signs, symptoms and laboratory diagnosis of macrocytic anaemia
►Investigation and management of vitamin B12 and folate deficiency
 Medical History
4
Family history Personal history
• Thalassaemia • Physiological conditions: pregnancy, infancy
• Dietary preferences/ habits :vegan or vegetarian,
• G6PD deficiency alcohol use
• Cancer for ex in GI tract • Dietary conditions :anorexia nervosa, bulimia
• Blood loss :menstruations, hemorrhoids,
• Blood diseases: Inflammatory Bowel disease, peptic ulcer,
diverticulosis
◦ leukemia,
• Change in stools habits: malabsorption
◦ lymphoma • Occupation and hobbies : solvents, chemicals
• Gastrointestinal tract surgery, gastric coil
• Recent illness or immunization
 Physical examination
►Evaluate conjunctiva and mucous membranes for paleness 5
►Cardiovascular system for murmur, tachycardia and
arrhythmia, palpitations or chest pain during exercise
►Liver and / or spleen enlargement
►Lymph nodes
►Look for jaundice , purpura or ecchymosis
Classification of Anemia based on RBC size
1fL=10-15 L 6
 ➢ Macrocytic
Megaloblastic and Non-Megaloblastic anemia
Hypersegmented 7
neutrophils
 Macrocytosis
Megaloblastic
8
Non- Megaloblastic

Hypersegmented Normal Hyposegmented

Neutrophils Neutrophils Neutrophils
▪ Vitamin B12 deficiency ▪ Alcoholism “ Pseudopelger Huet
▪ Folic acid deficiency ▪ Hypothyroidism Anomaly”
▪ Drug related ▪ Liver disease ▪ Myelodysplastic
▪ Haemolysis syndromes
▪ Drugs
 Vitamin B12 (Cobalamin)
9

• An essential nutrient
➔must be obtained from the diet
• Found in meat, fish and dairy products
• Needed for producing red blood cells
and for a healthy nervous system

C63H88CoN14O14P
Sources and daily requirement of VitB12 & Folic acid
Vitamin Β12 Folic acid (Β9)
Daily intake : 10-20 μg Daily intake : 100 – 500μg
10
Daily requirement: 1μg Daily requirements:100-200μg
Loss by cooking: 10-30% Loss by cooking: 60-90%
Absorption of hematinic substances
11
The importance of intrinsic factor
12
 Vitamin Β12 Deficiency
13
Malabsorption

Decreased
Dietary intake Intrinsic factor Small Intestine Small Intestine
Parasites and Drugs
deficiency disease
bacteria

pernicious Resection Of Diphyllobothrium -Nitrous

anaemia Small Latum - Fish oxide
intestine Tapeworm -Omeprazole
Poor diet gastrectomy
Crohn’s -Metformin
Disease Βlind loop syndrome -Colchicine
Congenital IF
deficiency Small Intestinal Bacterial -Neomycin
Tropical Overgrowth
sprue

Diverticulosis
 Signs in megaloblastic anaemia
B12 Deficiency Folate Deficiency/B9 14
►Glossitis, reduced sense of taste
►Glossitis
►Jaundice
►Jaundice
►Neuropathy
►Tiredness, dizziness.

The exact mechanism of neurological damage in B-12 deficiency is still not fully
understood ... (degeneration of dorsal and lateral columns, demyelination)
▪ The prevalence is higher in the elderly
▪ may take decades to develop and patients may be asymptomatic or
▪ may present with a wide spectrum of neuropsychiatric manifestations
 Drug-induced Macrocytosis
15
With
Low Vit. B12 Megaloblastosis
◼ Omeprazole
◼ Metformin
◼ N2O Low Folic
Low Folic acid
acid
◼ Methotrexate, Hydrea
◼ Trimethoprim
◼ Phenytoin

◼ Zidovudine
Without ◼ Azathioprine
Megaloblastosis ◼ Cytotoxics

N2O: Nitrous oxide

 Function of Vit B12 and Folic acid
16

Folate

A) Methylation of Synthesis of DNA by conversion of:

Homocysteine ➔ Methionine deoxy Uridine deoxy Thymidine
B) Conversion of Monophosphate Monophosphate
Methylmalonyl CoA➔ Succinyl CoA
Biochemistry 6th edition Lippincott’s
Macrocytic megaloblastic anaemias
Aetiopathogenesis 17

Disturbance in DNA synthesis

Disturbance in synthesis of pairing bases of DNA

(Adenosine – Thymine and Cytosine - Guanine )

Disturbance in cell
division
Ineffective erythropoiesis
 Basic characteristic 18
Asynchrony between nucleus and cytoplasm

► Ineffective DNA synthesis

in nucleus ➔ immature

► Normal RNA synthesis

in cytoplasm ➔ mature
 Result…….
LARGE / RBC in B12 deficiency:
19
• The cell is stuck in the G1 phase of the cell cycle and during this stage the cell
accumulates all the nutrients needed to divide.
• Since there is inhibition of DNA synthesis the cell can not advance to S phase,
so it becomes enlarged but does not divide.
• Long time at G1 phase forces the cell to expel it’s nucleus with out dividing
which leads to enlarged red blood cells in the periphery.
 Case Nr. 1
►Male 65 years old 20
►Symptoms: weakness, fatigue, dizziness,
numbness of lower extremities begun a year ago
with exacerbation in the last few months
►Personal History:
• gastrectomy for gastric cancer 7 years ago
• on thyroxine due to hypothyroidism for 10 years
►Upon clinical examination: pale with jaundice
and glossitis. Spleen and lymph node not palpable
► FBC: What test would you
• Hb:5.4g/dl (RV: > 13g/dl) order next and why?
• MCV: 110 fL (RV: 80-100 fL)
• WBC: 4.000/μL ( RV: 4.000-10.000/μL)
• PLT: 180.000 μL ( RV: 150.000 – 400.000/μL)
 Answer
21
►Vitamin B12: 40pg/ml (RV: 240 – 900 pg/ml)
►Folic acid: 10.5 ng/ml (RV: 4.20 – 19.9 ng/ml)
►Reticulocytes: 0.1 % (RV: 0.2- 2.0 %)
►Blood film: macrocytes + hyper segmentation
of the neutrophils
➔ diagnosis: macrocytic megaloblastic anaemia
due to malabsorption of Vit B12 What is the diagnosis
and why?
Intrinsic factor is produced by Parietal cells in stomach
and transports Vit B12 to the terminal ileum where it is
absorbed.
➔Intrinsic Factor deficiency due to gastrectomy
 Case Nr. 2
22
►Female 58 years old suffering from Essential
thrombocytaemia (increased platelet count)
►She has been on Hydroxyurea treatment for 4 years
►No other medication
►Symptoms: weakness, fatigue, headache and
inability to concentrate
Which test would you order first?
►Upon clinical examination: no abnormal findings
 FBC and blood film
23

Why does this patient have macrocytic megaloblastic anaemia?

 Answer
24
►Because she is on Hydroxyurea
►Which is an Antifolate drug

Antifolate drugs

• Methotrexate
• Hydroxyurea
• Pyrimethamine
• Trimethoprim
• Anticonvulsant
drugs
 Folic acid deficiency
25
Decreased Increased
intake
Malabsorption Antifolate drugs Demand

Jejunum • Methotrexate
resection • Hydroxyurea Pregnancy
• Pyrimethamine
Poor diet Jejunum Neonates
• Trimethoprim
pathologies
• Anticonvulsant drugs
Steatorhea . Myelo-
hyperplastic
syndromes
Coeliac disease

Tropic sprue

Alcohol
 Antifolate drugs
▪ Methotrexate
26
▪ Hydroxyurea
▪ Pyrimethamine
▪ Trimethoprim
▪ Anticonvulsant drugs

➢ Inhibit the enzyme Dihydrofolate reductase DHFR

➢ responsible for the production of Tetrahydrofolic THF and Methyl-THF
➢ which is a donor of Methyl (CH3) necessary for synthesis of Thymidine from Uracil and
Thymidine monophosphate from its precursor deoxyuridine monophosphate
➢ Thymidine Monophosphate is important for DNA synthesis and cell division

Essential Haematology Hoffbrand Biochemistry 6th edition Lippincott’s

 Treatment
►Based on the specific vitamin deficiency 27
►Vitamin B12 deficiency
• In vegetarian and vegans ➔ vit B12 Per oral
• in pernicious anaemia or malabsorption➔ Vit B12 Intramuscular
• In weekly injections of 1mg at the beginning
• and afterwards monthly lifelong
►Folic acid can be administered orally
►Inappropriate treatment of Vitamin B12 deficiency with folic acid
• improves anaemia but does not correct or stop the progress
of the neurologic damage which may advance to an irreversible state.
►When proper treatment is initiated the body’s respond is prompt:
• In a few hours after the 1st injection of Vit B12 the bone marrow reverts to normoblastic
• In a week a reticulocyte respond is apparent
• In 2 weeks hypersegmented neutrophils disappear from the peripheral blood
• In 3 weeks the haemoglobin becomes within normal range
 Case Nr. 3
►Female 83 years old
28
►Symptoms: weakness, fatigue, Headache, dyspnea during
exercise over the last 3 months
►Personal history: dyslipidemia, hyperuricemia and
hypertension, treated with statins, allopurinol, Ramipril
and aspirin
►Clinical examination: slight splenomegaly, no
lymphadenopathy, no other pathological findings What is the first diagnosis?
►FBC:
• Hb: 9.4g/dl (RV >12g /dl) ➔ Macrocytic anaemia
• MCV: 103 fL ( RV: 76-96)
• MCH: 32.1pg (RV:27 – 32) What test would you order next and why?
 Blood film
Normal 29
Macrocytosis
Hyposegmented neutrophil
“Pseudo Pelger-Huet anomaly”

Suspected
Myelodysplastic Syndrome

Bone marrow smear & trephine biopsy

 Myelodysplastic syndromes
►Heterogeneous group of clonal hematopoietic stem cell disorders present with : 30
• peripheral cytopenias
• hypercellular bone marrow and
• characteristic morphological disturbances in one or more than one cell line.
►Are preleukaemic conditions ➔ progress to acute leukaemia.
►Based on the cell line involved and the stage of the disease are classified in:
LOW GRADE: Sideroblastic anaemia Blue arrow:
• Refractory anaemia Ring Sideroblast➔ Iron
• Refractory anaemia with ring Sideroblast as a ring around nuclear
• Myelodysplastic syndrome with del 5q erythroblasts >15%
HIGH GRADE: Red arrow:
• Refractory anaemia with excess of blasts Normal siderocyte ➔ few
• Refractory anaemia with excess of blasts granules of hemosiderin
in transformation
scattered through the
• Multilineage myelodysplastic syndrome
cytoplasm
 Morphological characteristics of MDS 31
►Neutrophils and granulocytic precursors : 1 2 Normal neutrophil
• 1) Hypo granulation of cytoplasm(decreased granules)
• 2) Hypo segmentation of the nucleus (decreased number
of segments of the nucleus) with bilobed appearance:
“Pseudo Pelger-Huet anomaly” nucleus as “sunglasses”

►RBC:
• 3) Macrocytosis: Large red blood cells
• Anisocytosis and poikilocytosis 4 Normal Erythroblasts

►Erythroblasts in bone marrow

• 4) Megaloblastoid changes : asynchrony of maturation
between nucleus and cytoplasm.
• Bridges between erythroblasts Normal
5
►Megakaryocytes
• 5) Micro megakaryocytes: small nucleus and small
cytoplasm poor in platelets
 Case Nr. 4
►28 years old female
32
►Symptoms: suddenly begun having severe headaches,
abdominal pain and fever
►Personal and family history: negative
►Upon clinical examination: Petechiae in lower limbs and
abdomen
►Laboratory investigation:
What test would do you order next ?
• Hb: 8g/dl ( RV: > 12)➔ anaemia
• MCV: 98 (RV: 76-96)➔ macrocytosis
• Platelets: 12 x 10 9 ( RV: 150-400)➔ thrombocytopenia
• Reticulocytes: 15% (RV:0.2- 2.0)➔ reticulocytosis
 Blood Film
33

Fragmented cells > 2/ HPF*

Macrocytes (Young RBC/Retics)
Absence of Platelets
Nuclear erythroblast

What test would you order next ?

*High-Power Field (HPF), when used in relation to microscopy, references the area visible under the maximum magnification
 Microangiopathic Haemolytic anaemia (MAHA)

34
5) Negative Direct Coombs
6) Fragmented cells=Schistocytes > 2 /HPF 1) Hb ~8-10 gr/dl
7) Low platelet

2) ↑ Reticulocytes
3) ↑ LDH
4) ↑ Bilirubin

** PT and aPTT: normal

DD from DIC
HPF: high-power field = area visible under the microscope
 ADAMTS-13
A Disintegrin And Metalloprotease with ThromboSpodin type 1 repeats

35

ADAMS -13 is Decreased due to :

1. Gene Mutation on chromosome 9q34
2. Development of neutralizing antibodies
VWF, ADAMTS13 and Platelet Adhesion
With ADAMTS13 Without ADAMTS13

Normal VWF Multimers Ultra large VWF Multimers

Normal Hemostasis Microvascular Thrombosis
TTP: Thrombotic, Thrombocytopenic Purpura
 TTP Thrombotic Thrombocytopenic Purpura
►Primary 1) Congenital
37
2) Acquired (Idiopathic)
►Secondary to other pathological or physiological conditions:
▪ Pregnancy
▪ Infections(HIV)
▪ Drugs:clopidogreli, INF-a, cyclosporine, statins
▪ Chemotherapy: cisplatin, gemcitabine
▪ Allogenic Bone Marrow Transplantation
▪ Systemic Lupus Erythematosus - Scleroderma
▪ Cardio surgery

BJH 2008, 142

 Conclusions - 1 38
►Vitamin B12 and folate (B9) are needed for the production of thymidine
nucleotides for DNA synthesis.
►Deficiencies of either vitamin impair DNA replication, halt cell division,
and increase apoptosis , which results in ineffective haemopoiesis and
megaloblastic morphology or erythrocyte precursors
►Vitamin B12 deficiency is associated with peripheral neuropathies and
neuropsychiatric abnormalities as a result of demyelinization of the nerves
in peripheral and central nervous system
►Folate and vitamin B12 deficiency leads to the elevation in homocysteine
that represents a thrombotic risk factor for coronary artery disease
 Conclusions - 2
►Absorption of Vitamin B12 depends on production of intrinsic factor 39
by parietal cells of the stomach
►Impaired absorption of Vitamin B12 can be due to
• Pernicious anaemia , an autoimmune disease that causes destruction
of gastric parietal cells (anti- parietal cell antibodies) or antibodies to
intrinsic factor
• Inherited Intrinsic factor deficiency
►Treatment for megaloblastic anaemia is directed at correcting the
cause of the deficiency and supplementing the missing vitamin
►For pernicious anaemia and malabsorption lifelong supplementation
with parenteral vitamin B12 is needed
 Conclusion – 3 Macrocytic anaemia 40
Megaloblastic Non - Megaloblastic
• Megaloblastic bone marrow • Megaloblastoid bone marrow • Normal bone marrow
• Hypersegmented neutrophils • Hyposegmented neutrophils • Normal neutrophils

►Alcoholism
►Vitamin B12 deficiency ►Myelodysplastic ►Hypothyroidism
►Folic acid deficiency syndromes ►Liver disease
►Drugs ►Haemolysis, drugs
Do you have any questions?

vyrides.n @unic.ac.cy
 Bibliography
43

►Essential Haematology 7th Edition A.V Hoffbrand

►Kumar & Klarks clinical Medicine 8th Edition
►Kaplan USMLE Step 1 Lecture Notes 2016 Pathology
►Rodaks Haematology 5th edition 2016
►Junqueira’s Basic Histology, by Antony L Mescher
►BJH 2008, 142 Thrombotic Thrombocytopenic Purpura