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.

 

 

Evaluating Anaemia


Anaemia is not disease but a manifestation of an underlying disease. The list of diseases causing anaemia is exhaustive. The list includes disease as innocuous as a nutritional iron deficiency or as serious as a leukaemia or iron deficiency from a colonic cancer. Every patient of anaemia must have a complete evaluation. Failure to do so may may delay the diagnosis of serious diseases like cancer. Figure 1 gives the outline for evaluation of a patients with anaemia. The details of evaluation are given below.

 

Approach to Anaemia

Approach to anaemia: Given above is a flowchart for the approach to a patients with anaemia. The diagnosis depends on whether the anaemia occurs with changes in platelets and/or leucocytes and whether the bone marrow is responding normally to anaemia. Bone marrow response to anaemia manifest as reticulocytosis. Abbreviations used AA: Aplastic anaemia, BM: Bone Marrow, CML: Chronic Myeloid Leukaemia, MDS: Myelodysplastic syndrome, MDS/MPN: Myelodysplastic/myeloproliferative neoplasms, MPD: Myeloproliferative disease (click for a larger image)

Definition of Anaemia

Anaemia is defined and severity classified as per the WHO guidelines given in table 1. These definitions are for non-smokers individuals living at sea level (<1000 meters above seas level). Definitions for smokers and those living ≥1000 meters above sea level can be found at Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. World Health Organization (WHO/NMH/NHD/MNM/11.1)

Population Normal haemoglobin Mild Anaemia Moderate Anaemia Severe Anaemia
6-59 months ≥11 g/dL 10-10.9 g/dL 7-9.9 g/dL 7 g/dL
5-11 years  ≥11.5 g/dL 11-11.4 g/dL  8-10.9 g/dL  <8 g/dL
12-14 years  ≥12 g/dL 11-11.9 g/dL  8-10.9 g/dL  <8 g/dL
Pregnant Woman  ≥12 g/dL 11-11.9 g/dL 8-10.9 g/dL  <8 g/dL
Non-Pregnant Woman  ≥11 g/dL 10-10.9 g/dL 7-9.9 g/dL  <7 g/dL
Men (≥15 years)  ≥13 g/dL 10-12.9 g/dL 8.8-10.9 g/dL  <8 g/dL

Initial Evaluation of a Patient of Anaemia

  1. The diagnostic workup of a patient of anaemia should establish the
    1. type of anaemia,
    2. the cause of anaemia and
    3. complications caused by anaemia.
  2. Red cell transfusions in anaemic patients are for relieving symptoms due to a low haemoglobin. Patients without symptoms should not be transfused. A pre-transfusion serum sample should be stored to perform tests likely to be affected by blood transfusion (e.g. serum ferritin, serum B12, serum folic acid and haemoglobin electrophoresis). Apart from risks associated with transfusion the relief of symptoms following transfusion reduces the patient’s and the doctor’s zeal to pursue a complete diagnosis.
  3. The history, examination, haemogram and red cell indices are available in all anaemic patients. This information gives a reasonable idea about the cause of anaemia. A diagnosis can be made with a few additional investigations. No patient should be treated without a complete diagnosis.
  4. Iron deficiency is the commonest cause of anaemia. It may be a result of an innocuous cause like nutritional iron deficiency or may be a manifestation of a serious underlying disease like a cancer of the gastrointestinal tract. Most patient of anaemia will respond to iron supplementation. Iron supplementation without complete investigation of anaemia is a common but dangerous practice as it may delay the diagnosis of dangerous underlying illnesses like colorectal carcinoma.

Clinical manifestations of anaemia result from manifestations of  impaired oxygen deliver, manifestations of the underlying disorder and manifestations due to compensation to anaemia.

Manifestations of of anaemia: The symptoms of anaemia depend on the

  1. Degree of anaemia
  2. Rate of fall of haemoglobin
  3. Conditions of the vessels.

The body adapts to anaemia by increasing cardiac output, redistribution of blood from non-critical to the critical circulations and the and increased oxygen extraction (see Pathophysiology of anaemia). These adaptation occurs slowly. Patients with a gradual fall in haemoglobin can tolerate a more severe anaemia than a patient who become  anaemic rapidly because they get time to adapt to anaemia. Vascular disease results in the patient becoming symptomatic at a higher haemoglobin level. These patients manifest with ischaemic symptoms of the region supplied by the vessel. A patient with a gradually developing anaemia and without coronary heart disease becomes symptomatic at a lower haemoglobin than a patient who develops anaemia rapidly or one having a coronary artery disease or one having both.  Nutritional anaemias have a slow onset and thus present with a lower haemoglobin than anaemia or blood loss or acute haemolysis. If one sees a patients who is asymptomatic with severe anaemia one can be fairly certain that the patients suffers from nutritional deficiency. If the haemogram shows microcytosis the anaemia is likely to be due to iron deficiency. If there is macrocytosis the anaemia is likely to be due to B12 or folate deficiency.
Manifestations of anaemia are a result of impaired oxygen delivery. In patients without a significant coronary compromise the symptoms of anaemia include fatigue, lightheadedness and breathlessness. Frank cardiac failure sets in as the severity increases. Ischaemic symptoms, typically angina pectoris, may predominate in patients with significant coronary narrowing. Pallor, the most prominent sign of anaemia is of less diagnostic use than it is percieved to be. It has been reported to have a sensitivity of 19% to 70% and a specificity of 70% to 100%. There is inter-observer variation and clinical examination can not exclude mild anaemia. There have been suggestions that the tongue is the best site for diagnosing anaemia others have differed (PLoS ONE 5(1): e8545. doi:10.1371/journal.pone.0008545). Lack of pallor does not exclude mild anaemia and a haemoglobin assessment must be done is every patient with suspected anaemia.

Clinical features may give a clue to the nature of the disease causing anaemia:

The table below gives clinical features that may give a clue to the nature of the disease causing anaemia.

Clinical Finding Significance
Painful Crisis Sudden onset of pain is a feature of sickle cell disease. Painful crisis commonly affects bone, chest, abdominal and joints
Jaundice
  1. Achloruric Januduce: Achloruric jaundice is a result of increased haemoglobin catabolism (see haemoglobin catabolism) It is seen in patients with haemolytic anaemia and megaloblastic anaemia. In haemolytic anaemia the source of haemoglobin is erythrocytes and in megaloblastic anaemia the it the haemoglobinized erythroid precursors that are destroyed as a part ineffective erythropoiesis. The presence of reticulocytosis in haemolytic anaemias (see diagnosis of haemolytic anaemia) as opposed to reticulocytopenia megaloblastic anaemia differentiates the two conditions. Every anaemia with achloruric jaundice may not be a haemolytic anaemia or megaloblastic anaemia. Gilbert’s syndrome is a common asymptomatic defect of bilirubin metabolism that may co-exist of anemia of another cause. Rarely achloruric jaundice with reticulocytosis may be seen in large occult haematomas. Haematomas of the retroperitoneal region may present in this manner.
  2. Chloruric Jaundice: Patinets with haemolytic anaemia may develop chloruric jaundice because of biliary obstruction from pigment stones.
Nail Changes Platonychia (flat nails) and koilonychia (spoon shaped nails) are features of iron deficiency. These changes may be congenital and the duration of the changes must be enquired into.
Clubbing Clubbing is seen in infectious endocarditis, chronic liver diseases, bronchiectasis, inflammatory bowel disease and lung cancer
Leg ulcers Leg ulcers are a complication typically seen in patients with sickle cell anaemia. They are not specific to sickle cell disease. They may be seen in severe α and β thalassaemia, hereditary spherocytosis and pyruvate kinase deficiency. The incidence of leg ulcers increases with age. They are more common in the tropics where footwear is not worn. The are less common in Sβ0 and not seen in Sβ+ and SC disease. Co-inheritance of α thalassaemia with sickle cell disease decreases the incidence of ulcers. They are typically seen on the medial aspect of the tibia of behind the medial malleolus, are persistent and are a major cause of morbidity in sickle cell disease
Bleeding Manifestations Bleeding due to thrombocytopenia is characterized by petichiae and purpura. Anaemia with thrombocytopenia may be seen in

  1. Bone marrow pathology: Aplastic anaemia, acute leukaemia, bone marrow infiltrations and myelodysplastic syndromes
  2. Increased periphreal distruction/sequestration of platelets: Thrombotic thrombocytopenic purpura/Haemolytic ureaemic syndrome, Evan’s syndrome and hypersplenism
  3. Anaemia caused by bleeding associated with thrombocytopenia
Mouth ulcers Mouth ulcers are a feature of aplastic anaemia
Lymphadenopathy Generalized lymphadenopathy is seen in patients with acute or chronic lymphoid leukaemia and sometimes in autoimmune diseases
Splenomegaly Splenomegaly may be seen in patients with lymphoid neoplasia, chronic myeloid leukemia, idiopathic myelofibrosis, extra-hepatic portal hypertension and hairy cell leukemia
Neurological changes
  1. Subacute Combined degeneration of the Spinal Cord: Subacute combined degeneration is a myelopathy caused by B12 deficiency. It presents with parasthesiae of the upper and lower limbs that progress to weakness and ataxia in untreated patients. Vibratory and joint position sense is lost early in disease and progresses to impaired pinprick, light touch and temperature sensations. Upper motor neuron involvement results in hyperreflexia of the knees but the ankle reflexes are depressed due to a co-existing peripheral neuropathy. Plantars reflexes give a extensor response. Rarely features of autonomic involvements may be seen including orthostatic hypotension and bladder and bowel incontinence.
  2. Neuropathy: Chronic lead poisoning is characterized by a peripheral neuropathy that manifests with motor disturbances with few sensory symptoms.
  3. Other Neurological Manifestations: B12 deficiency can rarely cause cognitive or psychiatric manifestations (3%) or bilateral optic neuropathy and blindness (0.5%). Exposure to lead can cause encephalopathy.

Manifestations due to compensatory changes:

  1. Cardiovascular: Tachycardia, wide pulse pressure and decreased exercise tolerance result from cardiovascular compensation to anaemia.
  2. Musculoskeletal: Chronic haemolytic anaemias result in marrow hyperplasia. Marrow hyperplasia causes typical facies and increased risk of fracture. Facial features include bossing of the skull hypertrophy of the maxilla resulting in exposure of upper teeth, prominent malar eminences with depression of the bridge of the nose, puffiness of the eyelids and a mongoloid slant of the eyes.

Initial Investigations in a Patient of Anaemia

The starting point in evaluation of an anaemic patient is a haemogram and a reticulocyte count. The aim of initial investigation is determine

  1. If the pathology involves only the red cells or is there involvement of the white cells and /or platelets.
  2. If the bone marrow responding to anaemia by producing erythrocytes

Alterations in more than one cell lines of blood cells occurs because blood cells share precursors, developmental microenvironment and antigens. Diseases involving more than one series of blood cell include.

  1. Diseases of haemopoietic precursors: Aplastic anaemia, Acute leukaemia, myeloproliferative diseases, Myelodysplastic syndrome, Myelodysplastic syndrome/myeloproliferative neoplasm.
  2. Pathology of bone marrow microenviorment: Bone marrow infiltration, myelofibrosis
  3. Antibodies mediates diseases
    1. Antibody to antigen of precursors: Aplastic Anaemia
    2. Shared antigen Aplastic anaemia: Autoimmune pancytopenia, Evan’s syndrome
  4. Complement Mediated Damage: Paroxysmal nocturnal haemoglobinuria
  5. Other diseases: Thrombotic thrombocytopenia purpura/haemolytic uraemic syndrome (TTP/HUS)

All the above diseases other the TTP/HUS need a bone marrow examination for diagnosis. TTP/HUS is characterized by microangiopthic haemolytic anaemia in a patients with characteristic clinical presentation.

Anaemia induces erythropoietin production. Erythropoietin stimulated red cell production. Increased erythrocyte production manifests as reticulocytosis (see reticulocyte count). Reticulocytosis in an anaemic patient indicates that the anaemia is because of erythrocyte loss and the bone marrow is normally responding to anaemia. Patients with erythrocyte loss may fall into three catagories.

  1. Acute blood loss: These patients give history of blood loss and present with anemia with reticulocytosis without evidence of haemolysis.
  2. Chronic Blood loss: Blood loss is a alarming symptom that prompts patients to immediately seek medical advise. Patients with occult blood loss, those with altered blood loss or those with not capable of recognizing a blood loss may present with manifestations of chronic blood loss. Chronic blood loss causes iron deficiency. Unlike acute blood loss the manifestations of chronic blood loss are those of iron deficiency anaemia (microcytic anaemia with reticulocytopenia).
  3. Haemolytic anaemia: Patinets who have anaemia with reticulocytosis but have no demonstrable blood loss should be considered to have a haemolytic anaemia.
Microcytic_Anaemia_New_2016-800px

Evaluation of Microcytic Anaemia

Evaluation of Microcytic Anaemia

As haemoglobin forms about 97% of the erythrocyte proteins. Disorders impairing haemoglobin synthesis cause microcytic anaemia. Haemoglobin synthesis needs an functional genetic code, normal heme synthesis, adequate supply of iron and proper incorporation of heme in haemoglobin. If any of these are defective a microcytic anaemia develops. The commonest cause of microcytic anaemia is iron deficiency. The first investigation in a patients of microcytic anaemia is assessment of serum iron, total iron binding capacity and serum ferritin. The results of iron studies microcytic anaemia are depicted in figure 3. Further evaluation of patients depends on the result of iron studies. If the irons studies are normal then an study for abnormal haemoglobin should be performed either by HPLC or cellulose acetate electrophoresis in alkaline pH. Patients with HbA2 between 3.5-7% have heterozygous β-thalassaemia. Higher HbA2 values should be considered to be due to an abnormal haemoglobin that separates with HbA(e.g. HbE). Patients with homozygous and heterozygous HbE disease may be diagnosed in this manner. Those with a normal hemoglobin electrophoresis are likely to have α-thalassaemia trait. α-Thalassaemia needs estimation of globin chains synthesis. This investigation may not be readily available. If HbH disease is prevalent in the ethnic group the patients belongs to, it is essential to compete evaluation for α-thalassaemia to prevent Bart’s hydrops fetalis. Patients with transferrin saturation more than 50% need to have  bone marrow aspiration for diagnosis of sideroblastic anaemia.

 

 

Evaluation of Normocytic Anaemia

Evaluation of Normocytic Anaemia

Patinets with normocytic anaemia need to be evaluated for liver disease, renal failure or endocrine disease. The endocrine disease associated with anaemia include hypothyroidism, hyperthyroidism, adrenal insufficiency and hypopituitarism. If these disorders are not found the iron status should be assessed. Patinets may have iron deficiency (transferrin saturation <16%), anaemia of chronic disease (transferrin saturation ≥16% with a low total iron binding capacity). A bone marrow aspiration should be performed in whom diagnosis can not be made by the above investigations.

 

 

Evaluation of Microcytic Anaemia

Evaluation of Macrocytic Anaemias

Macrocytic anaemias may be megaloblastic or non-megaloblastic. Features of megaloblastic anaemia include hypersegmentation of neutrophils, macroovalocytosis, indirect hyperbilirubinaemia and markedly elevated lactate dehydrogenase levels. Macrocytosis is pronounced and almost all patients with an MCV > 110fl have nutritional megaloblastic anaemia, are of antiviral therapy or on chemotherapy. The converse is not true. Patients of megaloblastic anaemia may have a MCV <110fl in early disease or in the presence of co-existing iron deficiency. Serum iron levels are increase in nutritional megaloblastic anaemia. They  fall rapidly within 24-48 hours after therapy. Iron studies should be performed after a few days after administration of vitamins when the iron levels have stabilized. Nonmegaloblastic macrocytic anaemias are seen with alcoholism, hypothyroidism, liver disease and myelodysplastic syndromes.

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.

Diagnosis of Haemolytic Anaemia


Haemolysis is decreased erythrocytes lifespan (normal about 100-120 days). The bone marrow responds to haemolysis by increasing erythrocyte production. Anaemia occurs only when the erythrocyte life span is reduced to about 15-20 days. A compensated haemolytic state is when a patient has a shorted erythrocyte lifespan that is adequately compensated by increased erythrocyte production. These patients may have features of haemolysis except anaemia.


Figure 1. Manifestations of Haemolysis

Clinical Manifestations of Haemolysis

The site of haemolysis, intravascular or extravascular, determines the clinical manifestations of haemolysis (figure 1). Extravascular haemolysis is an exacerbation of a physiological catabolic process. The end product of extravascular haemolysis, like that of normal erythrocyte breakdown, is unconjugated bilirubin. Unconjugated bilirubin can cause brain damage in the forms of kernicterus. The conjugation of bilirubin is so efficient that the serum bilirubin rarely increase to more than 5mg/dL only due to bilirubin overproduction. The blood brain barrier prevents entry of bilirubin in the brain. Both the mechanisms are compromised in a neonate making them prone to kernicterus in case of haemolysis as is seen in haemolytic disease of the newborn. The only consequences of extravascular haemolysis occurring in patients beyond the neonatal period are anaemia, unconjugated hyperbilirubinaemia, mild to moderate splenomegaly and pigment gallstones.

Intravascular haemolysis is lysis of cells within the blood vessels. Cell free haemoglobin is oxidizing and pro-inflammatory. Haptoglobin binds and detoxifies cell free haemoglobin but it has a limited capacity. Haemoglobinaemia resulting from intravascular haemolysis causes serious clinical manifestations including renal failure, hypertension, smooth muscle spasm and a prothrombotic state. Many of these result from the nitric oxide scavenging capacity of haemoglobin (Rother et al JAMA 293:1653;2005). Persistent haemoglobinuria is a feature of chronic intravascular haemolysis, e.g. those with haemolysis due to prosthetic valves and paroxysmal nocturnal haemoglobinuria. Iron deficiency may accompany chronic intravascular haemolysis because of haemoglobinuria. Extravascular haemolysis is not associated with haemoglobin loss and does not cause iron deficiency.

Intravascular haemolysis often presents with acute severe haemolysis as may be seen with drug induced immune haemolysis, haemolysis in G6PD deficient patients, severe malaria and mismatched transfusion reactions. The diseases causing extravascular haemolysis have a less acute presentation.

The Diagnosis of Haemolytic Anaemia

Estimation of erythrocyte lifespan is too cumbersome to be used routinely in clinical practice. The diagnosis of haemolytic anaemia instead relies on indirect evidence of increased erythrocyte destruction. This includes:

  1. Increased production of erythrocytes in an anaemic patient with no evidence of blood loss
  2. Increase in products of erythrocyte destruction – Lactate dehydrogenase (LDH) and haemoglobin
  3. Consequences of haemoglobinaemia – low haptoglobin, haemoglobinuria, renal failure and hemosiderinuria

Increased erythrocyte production manifests as reticulocytosis (see The Reticulocyte Count for performing and interpreting reticulocyte count). If polycythaemia is not seen despite reticulocytosis, an equivalent erythrocyte loss is presumed to be occurring. Erythrocytes loss may internal, e.g., haemolysis, or external, e.g., bleeding. Reticulocytosis is seen when a large amount of blood is lost over a short period. Such losses are rarely occult. If there is no obvious blood loss, reticulocytosis in a patient whose haemoglobin is not increasing indicates haemolysis. Large occult haematomas, typically occurring retroperitoneally, may mimic haemolysis.

Lactate dehydrogenase (LDH) is abundant in brain, erythrocyte, liver and lung. Injury to any of these organs increases LDH. LDH is also increased in diseases characterized by increased cell proliferation e.g. acute leukaemia, lymphomas (particularly high grade non-Hodgkin lymphoma), myeloproliferative diseases and myelodysplastic syndrome. The clinical picture, radiology and laboratory investigations can exclude non-erythrocyte sources of LDH.

Haemolytic anaemias are characterized by an increase in LDH. The increase in LDH is more pronounced in patients with intravascular haemolysis and is matched by that seen in patients with nutritional megaloblastic anaemia. Unlike megaloblastic anaemia, intravascular haemolysis is characterized by reticulocytosis, haemoglobinaemia and haemoglobinuria.

The alterations caused by catabolism of haemoglobin released during haemolysis depend on the site of haemolysis (see haemoglobin catabolism). Haemoglobin released during extravascular haemolysis is metabolized to unconjugated bilirubin. The liver responds by increasing conjugation and excretion of bilirubin which increases urinary urobilinogen. Unconjugated hyperbilirubinaemia (unconjugated bilirubin >85% of total bilirubin) with increased urobilinogen is a characteristic of haemolytic anaemia. It differentiates haemolytic anaemia from Gilbert’s Syndrome, a common disorder of bilirubin conjugation that is seen in 3-7% of population. The urobilinogen levels in Gilbert’s syndrome are low. Crigler-Najjar syndrome type I and II are present with a more pronounced increase in bilirubin (>5mg/dL and >20mg/dL respectively) and are rarely considered as a differential diagnosis of an uncomplicated haemolytic anaemia. Bilirubin congugation defects do not show reticulocytosis. The bilirubin levels in patients with haemolytic anaemia rarely exceed 5mg/dL. Higher levels indicate a co-existing illness. If the bilirubin is unconjugated then an incidental co-inheritance of Gilbert’s syndrome should be suspected. Haemolytic anaemia increases the risk of pigment stones. These may cause obstructive jaundice. These patients, unlike those with haemolysis have a higher proportion of conjugated bilirubin with an elevated alkaline phosphatase. Donor screening for hepatitis B and C have made transfusion induced hepatitis a thing of the past.

Haemolysis releases haemoglobin. Cell free haemoglobin has oxidizing and pro-inflammatory properties. Haptoglobin is a haemoglobin scavenger that protects the body from the toxic effects of cell free haemoglobin. Haemolysis decreases serum haptoglobin (see haemoglobin catabolism). Extravascular haemolysis does not cause haemoglobinaemia, but contrary to what may be expected, it does cause low haptoglobin levels.  Low haptoglobin is of no value in differentiating intravascular and extravascular haemolysis. It appears that there is some haemoglobinaemia during extravascular haemolysis. It is possible that there is some intravascular component to extravascular haemolysis or there is regurgitation of haemoglobin from the macrophage during phagocytosis. Haptoglobin is synthesized by the liver. Decrease synthesis limits the usefulness of haptoglobin in the diagnosis of haemolysis in the presence eof chronic liver disease. Haptoglobin is an acute phase reactant that in increased by corticosteroid administration. Patients with acute inflammation and those on corticosteroid therapy may have a normal haptoglobin despite haemolysis.

Haemoglobin filtered into the glomerular fluid results in haemoglobinuria when the absorptive capacity of renal tubules (5g/day, Turgeon, ML. Clinical Hematology: Theory and procedures 4th ed. Lippincott Williams and Wilkins, 2005:89) is exceeded. Haemoglobin can precipitate in the tubules and causes renal failure. The haemoglobin absorbed by the renal tubules in metabolized by haemoglobin oxygenase to iron, bilirubin and amino acids. The iron is stored in the renal tubular cells as hemosiderin. These cells desquamate and cause hemosiderinuria. Hemosiderinuria appears a few days after hemolysis and may persist for a week or more after hemolysis has abated. Iron deficiency anaemia caused by hemosiderinuria may complicate chronic low grade intravascular haemolysis as is seen in prosthetic valve haemolysis, paroxysmal nocturnal haemoglobinuria or march haemoglobinuria.

Diagnosis of haemolysis by transfusion requirements

Even with complete cessation of erythropoiesis only about one unit of blood is needed every week in an adult to maintain haemoglobin of about 10g/dL. A greater requirement in the absence of evidence of blood loss suggests haemolysis. Transfused blood will only be lysed if there is an extrinsic defect e.g. autoimmune haemolytic anaemia. Cells transfused to patients with an intrinsic defect e.g. hereditary spherocytosis will have a normal life span. A patient who needs more than one unit of blood per week to maintain haemoglobin of about 10g/dL is likely to have a haemolytic anaemia if blood loss can be excluded. This haemolysis is likely to be due to an extrinsic cause.

Differential Diagnosis of Haemolysis

Haemolysis may be confused with conditions associated with unconjugated hyperbilirubinaemia, anaemia and reticulocytosis. These include bilirubin conjugation defects, acute blood loss and megaloblastic anaemia.

Bilirubin Conjugation Defects Bilirubin conjugation defects include Gilbert’s syndrome and Crigler-Najjar syndrome types I and II. The bilirubin levels in Crigler-Najjar syndrome type I and II are more than 5mg/dL so these entities are rarely confused with haemolysis. Gilbert’s syndrome is characterized by bilirubin levels which can be seen in haemolysis but there is no associated reticulocytosis. Bilirubin conjugation defects are characterized by low urobilinogen levels.

Acute blood loss: Acute blood loss, like haemolysis, causes anaemia and reticulocytosis but these are not the presenting complains. Blood loss needed to cause reticulocytosis is rarely ignored by the patient and is in fact the reason for seeing the doctor. When severe, it may be accompanied by signs of hypovolemia. The presenting complains of patients with haemolytic anaemia are related to anaemia and hyperbilirubinaemia. Chronic occult blood loss has a different presentation that acute blood loss or haemolysis. The gradual fall in haemoglobin associated with occult blood loss makes the patients tolerant to anaemia unless a cardiovascular or respiratory co-morbidity exist. These patients present with a clinical picture of iron deficiency namely, fatigue, malaise and hypochromic microcytic anaemia with anisocytosis. There is no jaundice and there is reticulocytopenia. Large occult haematomas cause anaemia and reticulocytosis. As the haematoma resolves jaundice may be seen.

Megaloblastic anaemia Megaloblastic anaemia, like haemolytic anaemia, presents with macrocytic anaemia, indirect hyperbilirubinaemia, and a low serum haptoglobin. These patients have reticulocytopenia. The macrocytosis is more pronounced. An MCV of >110 fl usually indicates a megaloblastic anaemia. Patients with nutritional megaloblastic anaemia respond to treatment with pronounced reticulocytosis, usually seen by 5-7 days after initiating therapy and lasting for up to 2 weeks.  Reticulocyte counts may be as high as 20-25%. A history of treatment with vitamin B12 and/or folic acid must be sought in every patient of haemolytic anaemia to avoid confusion with megaloblastic anaemia.

 

Related Pages

  1. Reticulocyte Count
  2. Evaluating Anaemia
  3. Haemoglobin Catabolism
  4. Sickle Haemoglobins and Variants
  5. Red Cell Indices

Haematology Atlas Pages

  1. Sickle Cells
  2. Pappenheimer Bodies

The Reticulocyte Count


Anaemia induces production of erythropoietin which promotes differentiation of the hemopoietic stem cell along the erythroid lineage. With maturation, the erythroid precursors shrink in size, loose nucleus and haemoglobinize (see Morphology of Erythroid Precurssors). A newly released erythrocyte contains ribosomal RNA that it looses over 24-36 hours. These young erythrocytes are called reticulocytes because the ribosomal RNA gives a reticular appearance when stained by certain supravital stains like new methylene blue.

Anaemia may result from an increased destruction or impaired production of erythrocytes. The former is characterised by an increased erythrocyte production and the latter by an inappropriately low erythrocyte production. The normal life span of the erythrocyte is about 120 days. About 0.8% of the erythrocytes are destroyed everyday. If erythrocyte production ceases completely, a 10% fall in erythrocyte count (also in haemoglobin) would take about two weeks. Though a greater fall would indicate a loss of erythrocytes in the form of haemolysis or acute blood loss, the erythrocyte count is of little in diagnostic value in assessing bone marrow activity accompanying anaemia. The number of the reticulocytes indicates the bone marrow activity in a short period preceding assessment (a day or two) and allows classification anaemia into those resulting from decreased production and increased destruction.

The reticulocyte

On Romanowsky staining, other than polychromasia and a slightly larger size, there are no morphological features to differentiate reticulocytes from other erythrocytes. Differentiation on the basis of size is difficult and all reticulocytes do not show polychromasia. When reticulocytes are stained with new methylene blue or brilliant cresyl blue the ribosomes precipitate as a blue staining reticulum (hence the name reticulocytes). As the staining is done on cells that are not fixed (are live) it is knows as supravital staining. New methylene blue stains the cytoplasm blue-green obviating the need for a cytoplasmic counterstain. The density of the reticulum falls with age of the reticulocyte. The most mature reticulocytes may show only one or two dots. The majority of reticulocytes have a few dots and the precise definition of a reticulocyte has a bearing on the reticulocyte count. The definition of a reticulocyte is any non-nucleated erythroid cell containing two or more bluish–stained material corresponding to ribosomal RNA. The reticulocyte needs to be differentiated from other intracellular inclusions viz. Pappenheimer bodies, Heinz bodies, Howell-Jolly Bodies and Hb H inclusions.

Performing the Reticulocyte Count

The count is performed by mixing 2-3 drops of new methylene blue (or brilliant cresyl blue) with 2-4 volumes of EDTA-anticoagulated blood, allowing the mixture to stand for 15-20mins, making films on a glass slide and examining these when dry under 100X oil immersion. The number of erythrocytes and reticulocytes is counted till at least 100 reticulocytes and a total of 10 oil immersion fields are counted. The reticulocyte count is expressed as follows:

Reticulocyte count = [number of reticulocytes counted]/[number of erythrocytes counted]

The reticulocyte count may also be expressed as absolute reticulocyte count as follows:

Absolute reticulocyte count = [RBC count X Reticulocyte count]/100

The normal reticulocyte count is 0.5-1.5% and the normal absolute reticulocyte count is 50-100X109/L

Correcting the reticulocyte count for anaemia

Anaemia decreases the amount of time the reticulocyte spends in the marrow. The reticulocytes of patients with haematocrits in the range of 45% are estimmated to spend 3.5 days in the marrow and about 1 day in the peripheral blood. At a haematocrit 15% these times are 1.5 and 2.5 days respectively. Decreases RBC count gives a false elevation in reticulocyte count. The haematological parameters of four patients given in the table below highlight the limitations of a reticulocyte count.

Patient A

Patient B

Patient C

Patients D

Haemoglobin

13g/dL

5 g/dL

7.5 g/dL

7.2 g/dL

Erythrocyte Count

4.4 Million/mm3

1.2 Million/mm3

2.5 Million/mm3

3.5 Million/mm3

Haematocrit

40.48%

16.2%

23.25%

25.2%

MCH

29.55 pg

41.67 pg

30 pg

20.57 pg

MCV

92 fl

135 fl

93 fl

72 fl

MCHC

32 g/dL

31 g/dL

32 g/dL

29 g/dL

Reticulocyte Count

1.3%

3%

15%

2%

Absolute Reticulocyte count

57200 /mm3

36000/mm3

375000/mm3

70000/mm3

Corrected Reticulocyte Count

1.17

1.08

7.75

1.12

Reticulocyte Production Index

0.78

0.43

3.1

0.56

  • Low erythrocyte count can cause an false impression of reticulocytosis: The patient B has an erythrocyte count of 1.2 million/mm3, a hematocrit of 16.2% and a reticulocyte count of 3%. The absolute reticulocyte count is 36000/mm3. Patient A has a erythrocyte count of 4.4 million/mm3, a haematocrit of 40.48% and a reticulocyte count of 1.3%. The absolute reticulocyte count is 57200/mm3. Despite having a higher reticulocyte count patient B actually has a lower absolute reticulocyte count and a less active marrow than patient A. Corrected reticulocyte count corrects reticulocyte count for low erythrocyte count and is calculated as follows:

Corrected Reticulocyte Count = Reticulocyte count X (Patinets Haemotocrit/Normal Haemotocrit)

(Haemoglobin or erythrocyte count may be used for the correction instead of haematocrit)

The corrected reticulocyte count in both patients is almost identical.

  • In addition to correcting for erythrocyte count one must correct for a premature release of erythrocytes: Patinet A and patient B have almost identical corrected reticulocyte count. Does this mean they are producing the same number of erythrocytes per day? The reticulocyte spends about 2.5 days in peripheral blood in the patient A and about 1.5 days in the patient B. Only 14,400/mm3 (36,000/number of days reticulocyte spends in peripheral blood) reticulocytes are produced every day in patient B and about 38,000/mm3 produced in patient A. Despite having an almost identical corrected reticulocyte count as patient B, patients A is producing twice as many reticulocytes as patient B. The corrected reticulocyte needs to be further corrected for an early release of erythrocyte in anaemia. This gives the reticulocyte production index as follows

    Reticulocyte Production Index = Corrected Reticulocyte Count X Correction Factor

    The correction factor is 1 for haemotocrit of 40-45%, 1.5 for haemotocrit of 35-40%, 2 for haematocrit of 25-35% and 2.5 for haematocrit of 15-25%.

    A reticulocyte production index of <2 in the presence of anaemia indicates a bone marrow pathology. Patient C, a patient of haemolytic anaemia, and has a RPI of 3.1 indicating a normally responding bone marrow. Patient D is a patients with a hypochromic n=microcytic anaemia with a low RPI. This indicates a impaired erythropoiesis which in the case is most likely to be an iron deficiency anaemia.

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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.