MACROCYTIC ANAEMIA

INTRODUCTION Macrocytic anaemias are

characterized by large erythrocytes (average mean corpuscular volume more than 100fl ) that usually have normal haemoglobin content in relation to their size.

Macrocytosis is frequently a sign of disease process that can result in significant morbidity if left untreated .

In 60% cases macrocytosis is not accompanied by anaemia ; however isolated macrocytosis should be always investigated.

Macrocytosis without anaemia may be an indication of early folate or cobalamine deficiency ,as macrocytosis precedes development of anaemia.

• Causes of artifactual macrocytosis when blood is analyzed on on automated instruments -

cold agglutinins hyperglycemia marked leukocytosis

MOST COMMON CAUSE OF MACROCYTOSIS is

alcoholism followed by folate & cobalamin deficiency .

CLASSIFICATION BASED ON MORPHOLOGICAL CHARACTERISTIC OF

ERYTHROID PRECURSORS IN BONEMARROW

MEGALOBLASTIC NONMEGALOBLASTIC

Abnormal DNA synthesis( nuclear maturation defect)

not well defined : may be due to increase in memberane lipids

Oval macrocytes Round macrocytes

Causes-Vitamin B12 & folate deficiency(95% cases)

Causes-Alcoholism

Chemotherapy ,myelodysplasia & other primary marrow tumors

Liver disease, haemolysis or bleeding Hypothyroidism.

MEGALOBLASTIC ANAEMIA

• Characterised by distinctive cytological and functional abnormalities in peripheral blood and bone marrow cells due to nuclear maturation defect with anaemia primarily attributed to a large degree of ineffective erythropoiesis.

• The anaemia was called megaloblastic so as to describe the giant abnormal appearing erythroid precursors( megaloblasts) in the bone marrow.

• Paul Ehrlich is credited with coining the term megaloblasts used to describe the large abnormal precursors.

VITAMIN B 12

VITAMIN B12 MOLECULE

Structurally classified as a corrinoid.

Molecule is composed of three portions

1. A corrin ring with four pyrrole group and a cobalt at the centre.

2. A nucleotide which lies perpendicular to the ring & attached to the ring and cobalt.

3. A beta group attached to cobalamine on the opposite side of the ring from the nucleotide.

METABOLISM..ABSORPTION

TRANSPORT

FUNCTION

Pathophysiology of cobalamine deficiency

• Impaired DNA synthesis – lack of methionine synthetase >>>> defect in thymidylate synthesis>>>> defect in DNA synthesis.• All dividing cells are

affected>>> megaloblastic anaemia & columnar and squamous epithelial cells abnormalities.

• Defective fatty acid degradation- Demyelination of nerves due to defect in degradation

• PROPIONYL coA Primer for FA synthesis•

Adenosylcobalamine FA with odd number of C>>

incorporated into neuronal

membranes>> disruption

of membranes• SUCCINYL co A

Neurologic disease

REQUIREMENTS

Diet: 5 – 7 ug / day

Needed: 3 – 5 ug / day

STORAGEStorage form; adenosylcobalamine

Max in liver and then in heart and kidney

• Vitamin B12 deficiency symptoms include:• Pallor, weakness, fatigue and tachycardia. • Palpitations. • Loss of appetite.• Difficulty in concentration and irritability. • In severe cases, symptoms of what is called

"Sub-acute Combined Degeneration" can occur and they include:

• Numbness and tingling in the peripheries. • Tingling and pain in the palm of the hands

and the tongue. • Impaired sensations and maybe impaired

equilibrium. • Abnormal reflexes. • Atrophy in the peripheries

CAUSES OF VITAMIN B12 DEFICIENCY

• Inadequate intake – strict vegetarians • Malabsorption- pernicious anaemia,

gastrectomy, coeliac dis. ,tropical sprue, crohns dis. , tuberculosis,ileal resection, drugs , fish tapeworm infestation

• Impaired utilization-transcobalamin ll deficiency nitrous oxide inhalation, inborn error of metabolism

• Increased demand- pregnancy, neoplasms,chronic haemolytic anaemia

• Myeloproliferative disorders, hyperthyroidism

PERNICIOUS ANAEMIA

• Autoimmune disease

• Decreased absorption of vitamin B-12 from the gastrointestinal tract in pernicious anemia results from the presence of an autoantibody against intrinsic factor (IF), a protein made in the stomach that is necessary for the absorption of vitamin B-12

• It is associated with other autoimmune diseases like addison disease , thyroiditis, vitiligo

FOLIC ACID

STRUCTURE OF FOLATE

Chemical name- PTEROYL GLUTAMIC ACID

Structurally- 3 parts:

1. Pteridin, a nitrogen containing ring.

2. A ring of para-aminobenzoic acid.

3. A chain of glutamic acid residues.

It is the inert form of folate.

THF active form of folate is formed by a four hydrogen reduction of pteridine ring.

Function of THF is to transfer carbon units from donors to acceptors.

• Most Folic Acid in food is in the conjugated polyglutamate form.

• It is deconjugated in intestine to the monoglutamate form.

• Absorption takes place through out small intestine but esply in proximal jejunum.

• Once absorbed into the intestinal epithelial cells the folate is reduced to N5 Methyl THF (Primary circulating form of THF in blood stream)>>>> attaches to cells by means of specific receptor.

• Once inside the cell it must be demethylated and conjugated to keep it from leaking out again.

• Demethylation reaction requires cobalamine.• Deficiency of Vitamin B 12 >>>> TRAP

FOLATE IN METHYLATED FORM blocking the formation of conjugated THF.>>>> The cells are unable to retain their folate leading to tissue folate depletion.

FUNCTION

vital role in metabolism of nucleotides & aminoacids

Pathophysiology of folate deficiency

REQUIREMENT FOLATE DEFICIENCY

• Irritability or Depression• Tongue changes - glossitis• High Homocysteine Level-People

with high homocysteine levels have been shown to have a higher increase in cardiovascular events such as heart attacks.

• Birth Defects and Miscarriage- Folic acid is critical in the development of your fetus.

• Cancers• Diarrhea and loss of appetite

•Recommended daily dietary allowance for adults -200 micrograms•Minimum daily requirement-50microgram/day•Storage in liver -5 – 10 mg of folate .

CAUSES OF FOLATE DEFICIENCY• Inadequate dietary intake (90% cases)-

nutritional deficiency• Malabsorption- coeliac disease ,tropical

sprue• Drug induced- methotrexate, OCP,

anticonvulsants, cholestyramine• Inherited enzyme deficiency-DHFR , N-

MTHFT,FIT• Increased requirement-

pregnancy ,lactation ,haemolytic anaemia, prematurity ,myeloproliferative disorder, neoplasms, chronic inflammation, hyperthyroidism

MISCELLANEOUS CAUSES OF MEGALOBLASTIC ANAEMIA

• Congenital disorders of DNA synthesis-

orotic aciduria

methionine synthase deficiency

congenital dyserthropoietic anaemia

homocystinuria,methylmalonic aciduria

• Acquired disorders of DNA synthesis-

myelodysplastic syndrome

acute erythroblastic leukaemia

metabolic inhibitor drugs- methotrexate, hydroxyurea ,cytosine arabinoside

refractory sideroblastic anaemia

Diagnosis of megaloblastic anaemia

LABORATORY FINDINGS:- Haematological parameters vary considerably. Megaloblastic anaemia involves all 3 blood cell

lines : erythrocytes, leukocytes, platelets. Haemoglobin- normal to very low Erythrocyte count - normal to very low Leukocyte count - decreased (absolute

neutropenia) Platelet count - decreased Relative reticulocyte count – normal RPI less than 2 Indices- MCV- increased , MCH-increased, MCHC-

normal

PERIPHERAL BLOOD FILM

TRAID OF FEATURES

• OVAL MACROCYTES

• HOWELL-JOLLY BODIES

• HYPERSEGMENTED NEUTROPHILS

• Anisocytosis- moderate to severe with normocytes & few microcytes in addition to macrocytes.

• Poikilocytosis- severe with severe anaemia

• Polychromatophillia & nucleated RBCs

• RBCs may show – basophillic stippling , cabot ring

• WBCs- neutrophil show hypersegmentation & mild shift to left may be noted

• Platelets may be enlarged

Oval

Macrocyte

Howell Jolly Body

Hypersegmented neutrophils

Basophillic stippling

Cabot ring

Nucleated RBCs

Hypersegmented neutrophil

Polychromasia

BONE MARROW MORPHOLOGY CELLULARITY:- hypercellular with an increase in

erythroid precursors

M:E ratio:- decreased ERYTHROPOIESIS:- megaloblastic changes occur at

all stages of red cell development.

MEGALOBLASTS ARE LARGE NUCLEATED ERYTHROID PRECURSORS WITH NUCLEAR MATURATION LAGGING BEHIND CYTOPLASMIC MATURATION

MATURATION:- maturation arrest – promegaloblasts & basophillic megaloblasts constitue more than 50% of erythroblasts

NUCLEUS:-

-chromatin network is more open , being arranged in a fine reticular fashion to give a stippled appearance

- well marked in polychromatic cells , & is sometimes seen in orthochromatic cells .

-nucleus of orthochromatic cell is indented or lobulated with one/more howell- jolly bodies.

DISSOCIATION OF CYTOPLASMIC & NUCLEAR MATURATION :- the nucleus is younger than normal appearance for the apparent , more mature stage of the cytoplasm . Cytoplasm development (haemoglobinization) occurs at normal rate ( NUCLEUS- CYTOPLASMIC ASYNCHRONY)

MITOSIS:- common

Erythroid hyperplasia megaloblasts

• Prussion blue staining of marrow shows – increase in number & size of iron granules in erythroid precursors .

..

LEUCOPOIESIS:-

-absolute number of developing granulocytes is increased but their % is decreased.

- presence of large atypical granulocytes , occur at all stages of development especially – metamyelocyte stage – GIANT STAB / giant metamyelocye – 30micron in diameter & large U-shaped nucleus .

BAND forms – with loose open chromatin.

MEGAKARYOPOIESIS:- -present in normal/ increased / decreased number.

-Some are atypical & have deep basophillic granular cytoplasm or hypersegmented nucleus.

giant metamyelocyte

OTHER LABORATORY FINDINGS Increase in plasma iron turnover

serum iron

indirect bilirubin

urobilinogen

LDH1 & 2

FIGLU excretion (after administration of histidine) Decrease in haptoglobin

uric acid

alkaline phosphatase

Specific tests for vitamin B12 & folate – include assays of serum levels.

• Special tests to diagnose vitamin B12& folate deficiency-

• SERUM vitamin B12 assay•

microbiological radio-isotope

• MICROBIOLOGICAL ASSAY – • Principle –the serum to be assayed is added as a source

of vitamin B12 to a medium containing all other essential growth factors for vitamin B12 dependent microorganism (lactobacillus leishmanii & Euglena gracilis )

• The medium is then inoculated with the organism , the

amount in serum is determined by comparing the growth as estimated turbimetrically with the growth produced by standard amount of vitamin B12.

• RADIO-ISOTOPE ASSAY – isotope dilution of non-radioactive serum vitaminB12 by adding co57- labelled B12. & a vit. B12 binding protein .

• Normal serum B-12 levels – 200-1000pg/ml False increase- • CML, liver disease , polycythemia rubra vera,

transcobalamine 2 deficiency, nitrous oxide anaesthesia

False decrease-• severe folate deficiency ,pregnancy, multiple myeloma,

veg. diet , R-protein deficiency , trancobalamin 1 deficiency

• Serum & erythrocyte folate assay • Microbiological radio-isotope

• MICROBIOLOGICAL – lactobacillus casei ( reference range is 3-25ng/ml

• RADIO-ISOTOPE – uses pteroylglutamic acid or methyltetrahydofolate & a folate binding protein (R.r-2-10ng/ml)

Erythrocyte folate level – gives better indication of folate stores.

• The diagnosis of folate and vitamin B12 deficiencies may be confusing.

• With cobalamindeficiency, serum cobalamin levels are usually low, but many are normal.

• The serum folate level is very sensitive to folate intake, and a recent folate-rich meal can normalize it.

• Red cell folate measurements, which can better reflect tissue levels, have several problems ( false increase & decrease).

• measure homocysteine, which increases in both disorders because methionine synthesis is impaired by deficiency of either.

• This laboratory finding typically precedes decreases in serum levels of folate and cobalamin.

• A cobalamin-dependent, but folate-independent, enzymatic reaction leads to increased serum levels of methylmalonic acid (MMA) with cobalamin deficiency.

• Accordingly, measurement of both homocysteine and MMA reliably detect, and distinguish between, folate and cobalamin deficiencies.

• When both are elevated, cobalamin deficiency is confirmed, although concurrent folate deficiency is possible.

• If homocysteine is elevated and MMA is normal, folate deficiency is likely.

• If both are normal, deficiency of either is highly improbable.

Specific tests for pernicious anaemia• Gastric analysis – if there is no free

HCl after histamine stimulation, this may indicate PA since the same cells that secrete HCl, also secrete intrinsic factor (IF)

• Test for antibodies –• antibodies to parietal cells(not specific)• Antibodies against IF(specific)

Schilling test – is the definitive test for the diagnosis of PERNICIOUS ANAEMIA

• The test measures the amount of an oral dose of

radioactively labeled B12 that is absorbed in the gut and excreted in the urine.

• This is followed by an injection of unlabeled vitamin B12 to saturate all vitamin B12 receptors in the tissue and plasma. Thus any amount absorbed in the gut will be in excess, and will be filtered in the kidneys to appear in the urine.

• If there is no radioactivity in the urine, this means that there is either malabsorption or pernicious anaemia

• The test is repeated, but this time the radioactively labeled B12 is accompanied by a dose of IF.

• If absorption is now normal, this means that the patient has PA if not this means malabsorption .

NON MEGALOBLASTIC MACROCYTIC ANAEMIA

Macrocytic RBCs are not oval, but are round. No hypersegmented neutrophils or Howell-Jolly bodies

LAB RESULTS OF MEGALOBLASTIC VS NON-MEGALOBLASTIC MACROCYTOSIS

DIFFERENTIAL DIAGNOSIS WITH INCREASED MCV

TREATMENT• To treat the underlying cause eg:- To determine which deficiency exists

&treat the patient with the specific vitamin.

pernicious anaemia- lifelong monthly parentral doses of hydroxycobalamin

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