Clinical Features of Megaloblastic Anaemia


Megaloblastic anaemia is a macrocytic anaemia resulting from the deficiency of vitamin B12 or folic acid characterised by the presence of megaloblasts in the bone marrow. It has haematological and neurological manifestations. The haematological manifestations are seen with folate as well as vitamin B12 deficiency. Folate deficiency in adults does not affect the nervous system.

Cobalamin deficiency is slow and “pure”. Folate deficiency is rapid and “impure”. Deficiecy of vitamin B12 occurs because of loss of intrinsic factor resulting in an isolated defect of B12 absorption. No other nutrients are affected. The body stores of B12 can last months. This results in B12 deficiency being a slow and “pure” deficiency. Symptoms come on slowly, over months. Folate deficiency evolves relatively quickly and is most commonly because of alcoholism or malabsorption. It is associated with other deficiencies and is rapid and “not pure”.

 

Manifestationf o megaloblastic anaemia

Figure 1. Clinical Manifestations of Megaloblastic Anaemia

Haematological Manifestations

Haematological changes resulting from vitamin B12 deficiency and folate deficiency are indistinguishable. Megaloblastic anaemias are macrocytic anaemia but macrocytosis is not specific to megaloblastic anaemia. It is however exceptional for other diseases characterised by macrocytosis to have an mean capsular volume (MCV) > 110fl.  This value can considered the threshold above which an anaemia is unlikely to be anything other than megaloblastic anaemia.

The earliest change in a megaloblastic anaemia is macrocytosis. This precedes changes in erythrocyte indices. Changes in mean capsular haemoglobin (MCH) follow and then the MCV rises. Haemoglobin usually falls after the MCV increases to >97 fl. As the severity of anaemia increases the peripheral smear shows aniscytosis and poikilocytosis, nucleated cells, Howell-Jolly bodies and Cabot’s ring. Microcytes and erythrocyte fragments that represent dyserythropoiesis may be seen. Polychromasia is absent and this distinguishes megaloblastic anaemia from haemolytic anaemia.

The term megaloblatic anaemia is a misnomer. The disease is actually a panmyelosis.  Erythroid, myeloid and megakaryocytic series are affected. Thrombocytopenia and leucopenia (neutropenia and to a lesser extent lymphopenia) usually occur late in the course. It is uncommon for patients with mild anaemia to have platelets and neutrophils but occasionally changes in leucocytes and/or platelets may dominate.

Iron deficiency or β-thalassaemia trait result in microcytosis and hypochromia and may incidentally co-exist with megaloblastic anaemia. Co-existence of either of these diseases with megaloblastic anaemia may mask macrocytosis of megaloblastic anaemia. Presence of hypersegmented neutrophils in a patients with normocytic normochromic anaemia should raise the suspicion of a megaloblastic anaemia co-existing with Iron deficiency or β-thalassaemia trait.

Neurological Manifestations

Cobalamine deficiceny causes neurological dysfunction. Folate deficiency causes symptoms only in children. Children with inborn errors of folate metabolism may have myelopathy, brain dysfunction and seizures.

The neurological manifestations of B12 deficiency are a result of a combination of upper motor neuron manifestations from subacute combined degeneration of the spinal cord, sensory and lower motor neuron manifestations from peripheral neuropathy and neurophychiatratic manifestations. Subacute combined degeneration of the spinal cord (SACD) is a degerative disease of the spinal cord involving the posterior and lateral column (corticospinal and spinoceribellar tracts) that starts in the cervical and the thoracic region.

The earliest neurological manifestations are impaired sense of vibration and position and symmetric dysesthasia that involve the lower limb. This is frequently associated with sensory ataxia. With progression spastic paraparesis develops. The patients have brisk knee reflexes, reflecting an upper motor neuron involvement and depressed ankle reflex, reflecting a peripheral neuropathy. Bladder involvement is unusual. Some patients may have optic atrophy.

Neuropsychiatric manifestation include memory loss, depression, hypomania, paranoid psychosis with auditory and visual hallucinations.

Other manifestations

Skin and nails can show pigmentations. Mucosa of the villi undergoes megalobkastic change resulting in temporary malabsorption.

Response to therapy

Haematological Recovery

  • Day 1: Feeling better
  • Day2-3: Reticulocytosis appears
  • Day 7-10: Peak retuculocytosis
  • Day 15 onwards: Neutrophilic hypersegmentation disappears
  • Day 56 (8 weeks): Blood counts become fully .normal

Neurological Recovery

Neurologic improvement begins within the first week also and is typically complete in 6 weeks to 3 months. Its course is not as predictable as hematologic response and may not be complete.

 

 

Anaemia with Hyperbilirubinaemia


A 49-year-old female presented with dyspnoea on exertion of 1 month duration. Examination reviled pallor and icterus. There was no lymphadenopathy, clubbing, koilonychia, platonychia, petechiae or purpura. There was no oedema of feet. The pulse was 90/min and the blood pressure 130/70 mm of Hg. Examination of the respiratory, cardiac and nervous systems did not show any abnormality. There was no organomegaly.

The haemoglobin was 4.9 g/dL with an erythrocyte count 1.37 x 1012/L, haematocrit of 16%, MCV of 116.78 fL, MCH of 35.77 pg and MCHC 30.63 of g/L.  The leucocytes count was 2800 with 35% neutrophils and 65% lymphocytes. The platelet count was 90 x 109/L. The peripheral smear showed macrocytosis and anisocytosis. Hypersegmented neutrophils were seen. The reticulocyte count was 3%.

The bilirubin was 2.1 mg/dL with a direct bilirubin of 1.8mg/dL and an indirect bilirubin of 0.3mg/dL. The Lactate dehydrogenase was 1417IU (normal 105 – 333 IU/L).

Anaemia and unconjugated hyperbilirubinaemia are characteristic of haemolysis. Does this patient have haemolytic anaemia?

Haemolysis shortens erythrocyte lifespan and results in increases haemoglobin breakdown. Haemoglobin is made of heme and globin. Heme consists of porphyrin ring at the centre of which is iron in the ferrous state. Iron released from catabolism of heme is reused. The porphyrin ring is catabolised to bilirubin. The bilirubin is transported to the liver for conjugation and excretion (see haemoglobin catabolism). Patients of haemolytic anaemia have unconjugated hyperbilirubinaemia because the increased bilirubin production overwhelms the hepatic bilirubin conjugation capacity.

One of the characteristics of megaloblastic anaemia is ineffective erythropoiesis. Ineffective erythropoiesis is defined as a sub-optimal (fewer) production of mature erythrocytes from a proliferating pool of immature erythroblasts. Each immature erythroblast produces less than the optimal number of erythrocytes because of premature death of erythroid precursors including haemoglobinized precursors. The haemoglobin released from haemoglobinized erythroid precursors is catabolised in the same manner as haemoglobin released from lysed erythrocytes (see haemoglobin catabolism). Megaloblastic anaemias are associated with unconjugated hyperbilirubinaemia because of death of haemoglobinized erythroid precursors.

The treatment of haemolytic anaemia and megaloblastic anaemia are different? How does one differentiate megaloblastic anaemia from that because of haemolytic anaemia? Does this patients have a haemolytic anaemia or megaloblastic anaemia?

Haemolytic anaemia is characterised by shortened erythrocyte survival. Erythrocytes survival is estimated by the use of radionucleotides something that is not possible at most centres. In clinical practice, a shortened erythrocyte survival is inferred from a high reticulocyte count. Reticulocytes are erythrocytes that have been produced in the preceding 24 hours. The erythrocytes survival is about 120 days and about 1% of erythrocytes are produced every day. Consistent with this the normal reticulocyte count is 0.5-1.5%.In patients of haemolytic anaemia, ddestruction of erythrocytes is matched by an increased production by the bone marrow. This manifests as reticulocytosis (see reticulocyte count). Megaloblastic anaemia occurs because of decreased production of erythrocytes and this manifests as reticulocytopenia. The difference between haemolytic anaemia and megaloblastic anaemia is the reticulocytosis in the former reticulocytopenia in the latter. This patient had a high reticulcoyte count but after correction both the reticulocyte production index [0.43] and corrected reticulocyte count [1.07%] were low excluding haemolysis. This patient was evaluated for megaloblastic anaemia.

The haemogram has clues to differentiate between haemolytic anaemia and megaloblastic anaemia. These include

  1. A very high MCV: The MCV is very high. Patients with haemolytic anaemia have a mild elevation in MCV. An MCV value >110fL is almost exclusively found in megaloblastic anaemias because of folate and/or B12 deficiency.
  2. Pancytopenia: B12 and folate deficiency impair DNA synthesis impairing erythrpoieis, myelopoiesis and megakaryopoiesis. Nutritional megaloblastic anaemias because of vitamin B12 and/or folate deficiency may show pancytopenia.
  3. Hypersegmented neutrophils (>5% neutrophils with >5lobes) is a feature of megaloblastic anaemia

Other features of megaloblastic anaemia include rise serum transferrin receptor, increased serum iron, serum ferritin and methemalbumin levels. Like haemolytic anaemia the serum haptoglobin is low and the LDH high. LDH levels in megaloblastic anaemia can ve very high.

This patients had a low serum B12 and was treated with parental B12 (1mg alternate day for 5 doses) and was evaluated for cause of vitamin B12 deficiency. As Schilling’s test was not available a diagnosis of pernicious anaemia was made by documenting gastric atrophy and anti-parietal cell antibodies.

Megaloblasts


Development of erythrocytes involves coordinated changes in the nucleus and the cytoplasm of erythroid processors (see Morphology of Erythroid Precursors) . The proerythroblast is a large cell with a fine chromatin, the earliest forms not being different from other blasts. As the cell matures the chromatin becomes more clumped and the nucleus reduces in size and the cytoplasm becomes acidophilic. Finally a dark pyknotic nucleus is extruded from the orthchromatophilic normoblast to give a reticulocyte.

Figure 1. Basophilic Normoblast

A group of basophilic normoblast are shown above. The cytoplasm is basophilic and the chromatin more clumped than a proerythroblast. The two cells on the right are less mature than the two on the left.

Figure 2. A group of orthochromatophilic normoblasts

The figure above show a group of  orthochromatophilic normoblasts. The cells of the left are more mature. The nucleus is reduced to a dense body in the more mature forms. The cytoplasm still has a blue tinge. which contrasts from the megaloblasts shown below.

Figure 3. Basophilic, polychromatophilic and orthochromatophilic normoblasts

The figure above shows three stages of erythroid maturation. The cell on the top right is a basophilic normoblast, bottom right is a polychromatophilic normoblast and the bottom left is an orthochromatophlic normoblast. Note the evolution of nuclear and cytoplasmic changes.

Figure 4. Megaloblasts

The figure above shows a group of megaloblasts. Cells in the right lower corner have a cytoplasm which is fully haemoglobinized and resembles the mature erythrocyte. These cell still have a nucleus. The cell on the left upper corner has a cytoplasm resembling a orthochromatophilc normoblast (see figure 2 and 3) but nuclear features resembling a basophilic normoblast (figures 1 and 2). Megaloblastic anaemia results from conditions that hamper DNA synthesis (B12 deficiency, folate deficiency, Chemotherapy drugs). The nucleus of a megaloblast thus matures slower than the cytoplasm resulting in cells having a nuclear morphology resembling a previous stage. This, known as nucleo-cytoplasmic dissociation, is the characteristic feature of megaloblastic anaemia.

Haemoglobin Catabolism


Figure 1. Synthesis and breakdown of haemoglobin

Haemoglobin is made up of heme, an iron containing porphyrin and globin, a protein. Normally the erythrocyte lives about 120 days. Ageing or damaged erythrocytes are destroyed by the macrophages of the reticuloendothelial system of the spleen. Other sites notably the, liver and the bone marrow, are also capable of destroying erythrocytes. As the life span of erythrocytes is not increased in splenectomized patients, these sites can completely take over the function in the absence of the spleen. The spleen, unlike other reticuloendothelial sites, is sensitive to subtle damage to the erythrocytes.

Heme splits into globin and hemin and globin. The amino acids released from the catabolism of globin chain are reused for protein synthesis. Hemin is acted upon by heme oxygenase to give biliverdin and iron. The iron is reused for haemoglobin synthesis. Biliverdin, released from the catabolism of protoporphyrin, is finally excreted as conjugated bilirubin in the bile (Figure 1).

Biliverdin is converted to bilirubin by biliverdin reductase. Bilirubin is water insoluble and needs to be conjugated with glycuronic acid in the liver to make it water soluble and make excretion in bile possible. Unconjugated bilirubin binds to albumin and is carried to the liver. The hepatocyte takes up the unconjugated bilirubin by both simple and facilitated diffusion and converts it to bilirubin diglucuronide in two steps. Some bilirubin monoglucuronide is also formed. The enzyme uridine diphophate glucuronyl transferase (UDPGT) facilitated bilirubin conjugation.

Bilirubin is secreted into bile against a concentration gradient. MRP-2 (multidrug resistance like protein 2) is one of the proteins involved in bilirubin secretion.

In the intestine bilirubin is converted to urobilinogen, a colourless compound, by the intestinal flora. Urobilinogen is converted to a pigment responsible for the colour of faeces, urobilin. Some urobilinogen is absorbed and excreted in urine.

Bilirubin metabolism is affected in conjugation defects, secretion defects and in states of increased haemoglobin catabolism.

  1. Bilirubin conjugation defects: Mutations in the UDGPT result in three syndromes. These are, in decreasing severity, Crigler-Najjar Syndrome I (CN-I), Crigler-Najjar Syndrome II (CN-II) and Gilbert’s syndromes. While CN-I is fatal except in those who undergo liver transplantation, Gilbert’s syndrome causes no symptoms otherthan jaundice. Patients with all three diseases have unconjugated hyperbilirubinemia which may range from usually >20mg/dL in CN-I, usually <20mg/dL in CN-II and <4mg/dL in Gilbert’s syndrome. Gilbert’s syndrome can mimic haemolysis. The absence of other evidence of red cell destruction, viz. increase in the LDH and decrease in the haptoglobin, and the absence reticulocytosis differentiates it from haemolysis.
  2. Bilirubin secretion defects: Defects in secretion of conjugated bilirubin include Dubin-Johnson syndrome, Rotor syndrome, benign recurrent intrahepatic cholestasis (types 1 and 2) and progressive familial intrahepatic cholestasis (types 1, 2 and 3). (links take you to the OMIM page for the disease)

Increased haemoglobin catabolism: Increased haemoglobin catabolism, seen in patients with haemolytic anaemia and states associated with ineffective erythropoiesis, increases bilirubin production and overwhelms the hepatic uptake/conjugation capacity. This increases the unconjugated bilirubin. Increased bilirubin production in patients with haemolytic anaemia is a result of increased erythrocyte destruction by the spleen. Typically, hyperbilirubinemia is associated with extravascular haemolysis. Splenomegaly may accompany unconjugated hyperbilirubinemia due to haemolysis because of the increased workload. Patients of haemolytic anaemia show elevated LDH levels and reticulocytosis. Patients with ineffective erythropoiesis, e.g. megaloblastic anaemia due to folate/B12 deficiency, have increased haemoglobin catabolism due to destruction of haemoglobinized precursors in the bone marrow. There is reticulocytopenia, the increase in LDH is more pronounced than haemolytic anaemia, there is no splenomegaly. Haemolysis and ineffective erythropoiesis may co-exist in megaloblastic crises of haemolytic anaemia. Unconjugated hyperbilirubinemia due to increased bilirubin production is associated with increased urinary urobilinogen, a features not seen in inherited syndromes of bilirubin conjugation (CN-I, CN-II and Gilbert’s syndrome). It is unusual for the bilirubin to increase beyond 4mg/dL only from increased haemoglobin breakdown. When higher values are encountered other reasons for an increased bilirubin must be sought. All patients with increased haemoglobin breakdown do not show hyperbilirubinemia.

Neutrophil Segmentation and Projections


The neutrophil nucleus is segmented. The nucleus of the most immature neutrophil, band neutrophil lacks segmentation. It differs from a metamyelocyte in that the concave and convex surfaces of the nucleus are parallel. It may be coiled as is the case of the cell next to the basophil in figure 1.

The Arneth believed that the number of lobes increase as the neutrophil ages. This is not entirely true. The number of lobes in a neutrophil nucleus is decided at the band stage or earlier. After release the neutrophil nuclear segmentation may continue till the cell achieves the number of lobes it is programmed to reach. A cell that is programmed to become a three lobed neutrophil and a cell that is programmed to become a five lobed neutrophil both start as band forms but segmentation in the former stops at three lobes. The average number of neutophil lobes in a normal peripheral smear is 2.5-3.3. The definition of lobes differs from observer to observer (figure 2 and 3)

Infection and inflammation result in increased neutrophil production. Young neutrophils have fewer lobes and the average neutrophil lobulation decreases (shift to left, smaller numbers occupy a left position on the X axis of a graph). Other inflammatory changes including leucocytosis, toxic granules, Döhle bodies and neutrophil vacuolation may also be seen in patients showing “shift to left” because of infection/inflammation. Patients with chronic myeloid leukaemia (CML) and other myeloproliferative diseases or myelodysplastic/myeloproliferative disease (MDS/MPD) also show a shift to left but it is usually possible to differentiate these conditions from infection/inflammation. Peripheral smear of patients of CML (typical and atypical) and chronic myelomonocytic leukaemia show the entire spectrum of myeloid cells (myeloblasts, promyelocytes, myelocytes, metamyelocytes, band forms and segmented neutrophils). CML shows more than 10% immature forms and without monocytosis. Chronic myelomonocytic leukaemia usually shows less than 10% immature forms and shows an absolute monocyte count more than 1 X 109/L. The BCR-ABL translocation can be detected in patients with CML, but not in atypical CML. Band forms and cells with few nuclear segments dominate the peripheral smear in infection/inflammation. An occasional myelocyte or metamyelocyte may be seen. A promyelocyte or myeloblast is almost never seen. When these cells are seen in seen in substantial numbers a diagnosis of MDS/MPD should be considered.

In women one of the X chromosomes is inactivated and appears as the drumstick appendage. It is seen in 0.5-2.6% of the neutrophils. The prevalence of the drumstick increases with segmentation of the nucleus.

The exact mechanism and purpose of segmentation of the granulocyte nucleus is not known. It may aid in making the cell more deformable and allow it to squeeze from the vessels into the tissue. It is rare to find a neutrophil with more than five nuclear lobes. Increased nuclear lobulation is a feature of megaloblastic anaemia, iron deficiency anaemia (Br J Haematol 107:512;1999), uraemia, infection, myelodysplastic syndromes and hereditary neutrophil hypersegmentations. Hypersegmentation increases the average neutrophil lobe count and hence the term shift to right. A ratio of five lobed to four lobed neutrophil of 17% or more is the most sensitive indicator of shift to right. Hypersegmentation due to megaloblastic anaemia recovers in about two weeks after initiation of therapy (Ann Intern Med 90:757). Hypersegmentation can be used for diagnosis in patients who have been empirically treated with vitamin B12 and/or folate as other disease related changes rapidly revert to normal. Macropolycytes are large neutrophils often with a hypersegmented nucleus. They are tetraploid (have 96 chromosomes instead of 48 chromosomes). Hypersegmentation in macropolycytes reflects increased DNA.