megaloblastic anemia

MEGALOBLASTIC

ANAEMIA

Muhammad Asif Zeb

Lecturer Hematology

Khaybar Medical University

Pershawar

DEFINITION

The megaloblastic anaemias are a group of disorders characterized

by the presence of distinctive morphological appearances

of the developing red cells in the bone marrow and Pancytopenia in the

peripheral film.

The cause is usually deficiency of either cobalamin (vitamin B 12 ) or folate

but megaloblastic anaemia may arise because of inherited or

acquired abnormalities affecting the metabolism of these vitamins

or because of defects in DNA synthesis not related to

cobalamin or folate1

Megaloblastic anaemia is characterized by the appearance

in the bone marrow of morphologically abnormal

nucleated red cell precursors called megaloblasts and

also by hypersegmented or multi-segmented

neutrophils.

Megaloblasts are abnormal in functional as well as

appearance with the result that the mature red cells

formed from them are abnormal in size and shape; the

most prominent abnormality being macrocytosis.

Megaloblast:

Megaloblast describes a bone marrow cell that is part of the factory making red cells that has become abnormally large and has an abnormal appearance. It is a counterpart of erythroblast. These are usually large oval shaped cells and are fully haemoglobinized due to neuclear cytoplasmic asynchronisim .

CAUSES

Cobalamin deficiency or abnormalities of cobalamin metabolism.

Folate deficiency or abnormalities of folate metabolism.

Therapy with antifolate drugs. (e.g. methotrexate)

Independent of either cobalamin or folate deficiency and refractory to cobalamin and folate therapy.

Therapy with drugs interfering with synthesis of DNA. (e.g. cytosine arabinoside)

TRANSPORT: There are two major vitamin B12 binding proteins

in plasma.

TC 1: it is an α1 globulin which carries from 70-90% of circulating vitamin B12.

It is primarily a storage protein and its absence doesn’t lead to clinical signs of B12 deficiency.

Functionally dead

TC 2: It is a β-globulin and it is essential for transport of vitamin B12 from one organ to the other and in and out of cells. Congenital deficiency of TC2 leads to severe megaloblastic anaemia.

TC 3: similar to TC1, binds only a small quantity of circulating B12.

FUNCTIONS

Vitamin B12 acting as co-enzyme for two important

biochemical reactions in humans:

The conversion of methylmalonyl-CoA to succinyl-CoA.

Synthesis of methionine from homocystine.

Vitamin B12 is required for maintenance of the

integrity of nervous system.

MEGALOBLASTIC ANAEMIA DUE TO

VITAMIN B12 DEFICIENCY MECHANISM DISORDER

Decreased intake Nutritional deficiency

Impaired absorption

i). Gastric causes Pernicious anaemia

Gastrectomy (total or partial)

ii). Intestinal causes Lesions of small intestine

Coeliac disease

Tropical Sprue

Tapeworm Infestation

Zollinger -Ellison Syndrome

Abnormalities of cobalamin metabolism

i). Transport protein defects Inherited TC 2 deficiency

ii). Congenital intrinsic factor Present before the age of two

deficiency years

iii). Congenital methylmalonic acidaemia Infants are ill from birth

and aciduria

METABOLISM OF FOLATE

Folic acid was synthesized as a yellow crystalline powder. Folic acid doesn’t exist as such in nature, but is a parent compound of a large number of derivatives referred to as folates which plays an important role as co-enzymes in cellular metabolism. Reduction to dihydro - and tetrahydrofolate derivatives is necessary to participate in metabolic reactions.

Contents in food green Vegetables: rich

Meat: moderate

Effect of cooking 60-90% loss

Adult daily requirements 200 micro gram

Adult daily intake 100-500 micro gram

Site of absorption Duodenum & jejunum

Body stores 5-20 mg

SOURCES:

Widely distributed in plants and animals tissues. Richest sources are liver, kidney, yeast and fresh green vegetables such as spinach and cabbage.

ABSORPTION:

Normally absorbed from duodenum and upper jejunum and to a lesser extent from lower jejunum and ileum. Absorption of folate is a rapid active process 80% is absorbed unchanged. Synthetic polyglutamates are absorbed as well as monoglutamates. Polyglutamates are cleaved to monoglutamates by the enzyme pteroylpolyglutamate conjugase. Monoglutamates than undergo reduction and enters in circulation as methyltetrahydrofolate.

TRANSPORT:

Folate circulates in plasma as methyltetrahydrofolate monoglutamate, either in a free form or weekly bound to a variety of proteins.

FUNCTIONS

Folate co-enzymes are required for several bio-

chemical reactions in the body involving transfer of

one-carbon units from one compound to another.

Two reactions that are important in the context of

clinical folate deficiency are:

Methylation of homocysteine to methionine.

Synthesis of pyrimidine nucleotide, thymidylate

monophosphate from deoxyuridylate

monophosphate in the DNA synthesis pathway.

MEGALOBLASTIC ANAEMIA DUE TO

FOLATE DEFICIENCY

MECHANISM DISORDER

Decreased intake Nutritional deficiency

Impaired absorption Coeliac disease

Tropical sprue

Increased demand Pregnancy, haemolytic anaemia, myeloproliferative

disorders, leukaemia & lymphoma, carcinoma, Inflammatory disorders

Dihydrofolate reductase Methotrexate

Inhibitors Trimethoprim

Pathophysiology In megaloblastic anaemia, the anaemia results from failure of

the megaloblastic bone marrow to compensate for a moderate reduction in red cell life span. Red cell survival studies have shown the presence of mild haemolysis.

Lack of vitamin B12 or folate causing slowing of DNA synthesis in developing erythroblasts with an accumulation of cells in premitotic phase of cell cycle. Some of these cells die within the marrow.

The neutropenia and thrombocytopenia also appear to result from ineffective production by abnormal precursor cells in the marrow.

PATHOGENESIS Both vitamin B12 and folic acid are required for ordered DNA

synthesis. But due to deficiency of vitmain B12 & folic acid

in megaloblastic anaemia DNA synthesis is impaired or

blocked in rapidly dividing cells.

As a result the cells proliferates abnormally and increases in

size and become megaloblasts which are fully

heamoglobinized cells and abnormal in appearance and

function.

Because of their increased size they occupy much of the space

in marrow and they disturb other cell lines too.

MASKED MEGALOBLASTIC

ANAEMIA In certain megaloblastic anaemias , iron deficiency is sometimes

present at the same time as folate or B12 deficiency.

Associated iron deficiency may partly mask the typical

haematological features of megaloblastic anaemia.

PF showes double population (dimorphic blood picture) some red

cells being oval and well haemoglobinized, and others small

and poorly haemoglobinized.

In other cases masking takes the form of a lesser degree of

macrocytosis, so that most cells are of normal size and

MCV is normal or even mildly reduced. However, careful

scrutiny of blood film shows a small no. of oval

macrocytes and hypersegmented neutrophils.

Other disorders:

Thalassaemia, Infection, chronic renal

disease, rheumatoid arthritis.

CLINICAL MANIFESTATIONS

Features of anaemia: pallor, anorexia, weight loss, diarrhoea.

i). Macrocytic megaloblastic anaemia.

ii). Glossitis.

iii). Peripheral neuropathy and subacute combined degeneration (demyelination) of spinal cord.

NOTE: Deficiency of folate doesn’t produce sub-acute combined degeneration of spinal cord, but peripheral neuropathy is occasionally seen.

DIAGNOSIS

Diagnosis is usually made on:

Patient history

Clinical features

Lab diagnosis

LAB DIAGNOSIS HAEMATOLOGICAL FINDINGS

CBC:

Haemoglobin Decreased

RBC Decreased

WBC Leukopenia Neutropenia

Platelets Mild, usually symptom less thrombocytopenia

Hct Decreased

MCV Increased (125 fl is diagnostic of megaloblastic anaemia)

MCH Increased

MCHC Normal RDW Increased

Reticulocytes usually reduced

Peripheral blood picture:

Macrocytic Normochromic anemia

Anisocytosis: (Increased variation in RBC size)

Oval Macrocytes

Poikilocytosis: (Increased variation in RBC shape)

oval macrocytes, target cells, Tear drop cells

Schistocytes

Inclusions:

Howell-jolly bodies

Cabot rings

WBC: hypersegmented neutrophils

Platelets: Giant platelets

Bone Marrow Examination

Erythropoiesis

Hypercellular

increased erythroid /myeloid ratio

erythroid cell changes (megaloblasts,

RBC precursor a abnormally large with

nuclear- cytoplasmic asynchrony)

Thrombopoiesis

magakaryocytes may

be decreased, normal,

or increased.maturation,

however, is distintly

abnormal.some larger

than normal forms can

be found with separation

of nuclear lobes and

fregments

Grannulopoiesis

is abnormal large as

typical granulocytes giant

metamyelocytes(30µm)

and bands with loose,

open chromatin in the

nuclei are diagnostic.

Myelocytes show poor

granulation more mature

stages

BIOCHEMICAL FINDINGS

Serum vitamin B12 Decreased

Serum folic acid Decreased 2-15micro g/l

Red cell folate level Decreased 160-640 micro g/l

Serum bilirubin Increased 0.1-1.0 mg/dl

Homocysteine level Increased

Methylmalonic acid Increased

LDH Increased

FIGLU Increased

Serum Iron Increased

Serum ferritin Increased

Serum heptoglobin Decreased

Haemosiderinuria

Serum methylmalonic acid and homocysteine Increased

SPECIAL TESTS

Serum vitamin B12 assay:

It is performed by two ways:

Microbiological assay.

Radio-isotope assay.

Radioactive vitamin B12 absorption test:

The ability of body

to absorb vitamin B12 can be assessed by measuring the

absorption of a small dose of Co- labelled vitamin B12.

The test is called Schilling test.

SCHILLING TEST

An oral dose of 1 micro gram radioactive vitamin B12 is administered to the fasting subject followed two hours later by a large parenteral injection of unlabelled B12(1000 micro gm). The injection flushes out about one-third of absorbed radioactive B12 into the urine in the next 24 hours. Normal subject excretes 10% of 1 micro gm dose. Patients with pernicious anaemia excrete less than 5%. If the patient absorbs normal amounts of vitamin B12 no further testing is required.

UNSATURATED B12 BINDING CAPACITY

Measurement of unsaturated B12 binding capacity which in the normal subject reflects the amount of TC2 and to a lesser extent TC1 & TC3, may be diagnostically useful. Normal range is 500-1200 ng/l.

Deoxyuridine Suppression Test

In normal bone marrow, dU considerably suppresses

the uptake of radioactive thymidine into DNA.

This is thought to be due to conversion of dU to

thymidine triphosphate via dUMP, which inhibits

thymidine kinase, on which thymidine uptake

depends. Deoxyuridine suppresses radioactive

thymidine incorporation less effectively in meg.

Anaemia due to folate or cobalamin deficiency

because of the block in dUMP methylation to

dTMP.

Tests for the cause of

cobalamin deficiency

Clinical History :diet, drugs,operation etc.

Cobalamin absorption using radioactive cobalamin: Alone, with food, with intrinsic factor.

Tests for tissue specific antibodies in serum (e.g. IF, parietal cells etc)

Endoscopy with gastric biopsy.

Measurement of intrinsic factor in gastric juice after maximal stimulation. (rarely performed)

Small intestinal studies.

Stool for fish tapeworm ova.

SERUM FOLATE ASSAY

Microbiological Assay.

Radio-isotope Assay.

(Levels below 3micro gm/l suggest clinically significant

folate deficiency).

RED CELL FOLATE ASSAY

Red cells contain 20-50 times as much folate as serum. It is

usually a more reliable indicator of tissue folate stores than

the serum folate. It reflects mean folate that existed in

plasma during maturation of precursors.

Parietal cell antibodies:

Serum antibodies to surface membrane and

cytoplasmic antigens of gastric parietal cells are found in at least

85% of patients with pernicious anaemia.

Intrinsic factor antibodies:

Two types of antibodies are found:

Blocking antibodies.

Binding antibodies.

THANX!