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.


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



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







29.55 pg

41.67 pg

30 pg

20.57 pg


92 fl

135 fl

93 fl

72 fl


32 g/dL

31 g/dL

32 g/dL

29 g/dL

Reticulocyte Count





Absolute Reticulocyte count

57200 /mm3




Corrected Reticulocyte Count





Reticulocyte Production Index





  • 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.