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.

Leukaemia – The Peripheral Smear

Differentiating Acute and Chronic Leukaemia

Leucocytosis with anaemia is a feature of acute and chronic leukaemia. It is possible to differentiate acute and chronic leukaemia by looking at the peripheral smear. Patients with acute leukaemia often have thrombocytopenia. Patients with chronic lymphocytic leukaemia may have normal or low platelet counts. Patients with chronic myeloid leukaemia have normal or high platelet counts.

Haemoglobin Platelets Peripheral Smear
Acute Leukaemia Low Low Immature forms other than blasts not seen
Chronic Lymphocytic Leukaemia Low or Normal Low or Normal Normal looking lymphocytes
Chronic Myeloid Leukaemia Low or Normal Normal or High Immature leucocytes, all phases of leucocyte maturation seen

The phases of maturation of myeloid cells (from the least to the msot mature) are blasts, promyelocytes, myelocytes, metamyelocytes, band form and mature granulocyte (see Myeloid Precursors Morphlogy). The peripheral smear from patients with acute leukaemia shows blasts (or promyelocytes) and mature neutrophils. Very few cells, if any, with maturity between the two stages that occupy two ends of the spectrum are seen. Patients with with chronic myeloid leukaemia have cells with all stages of maturity between blasts and mature granulocytes. The peripheral smear in patients with acute leukaemia shows mature lymphocytes.

The explanation for the different peripheral smear findings in acute leukaemia and chronic myeloid leukaemia is in the pathogenesis of the two diseases. Chronic myeloid is a myeloprolferative disease. It is a clonal disease. The stem cells of patients with chronic myeloid leukaemia carry the BCR-ABL mutation. This mutation results in clonal expansion. All blood cells in a patient arise from one clone. The release of cells from the bone marrow of patients with CML is not limited to mature granulocytes. Some cells leave the marrow and result in leucocytosis.

The marrow of a patient of acute leukaemia has two clone one malignant one normal. The malignant clone can not differentiate beyond the stage of a blast (or promyelocyte). It slowly effaces the normal clone. The mature granulocytes seen in the peripheral smear arise from the normal clone and the blasts from the malignant clone. The normal clone releases cells only when they mature to the stage of band cell or beyond. The malignant clone can not mature beyond the stage of a blast. The stages between blasts and band forms/mature granulocytes are not seen in peripheral smear of acute leukaemia.

Platelet Counts and Platelet Indices

Platelet may be performed by manual or automated methods. Automated platelet counts are more accurate.

Blood Collection

Platelet counts are best performed on EDTA-anticoagulated blood obtained by venepuncture. Capillary blood may be used. Counts obtained by capillary blood are lower and show a greater variability. Platelets change shape on coming in contact with EDTA from discoid to spheroid with filamentous extensions. The volume of platelets remains unchanged for about 1-2 hours but increases thereafter by about 7.9% at 30 minutes and 13.4%at 24 hours (Clin Lab Haematol. 2005;27:370-373). It is best to minimize the delay between collection and counting.

Manual Platelet Counts

Manual platelet count is performed by diluting blood by a solution containing 1% ammonium oxalate and counting the number of platelets in a Neubauer chamber.

  1. Blood is diluted 1:20 with 1% ammonium oxalate to lyse the erythrocytes. If the platelet count is performed under natural light then brilliant cresyl blue is used to stain platelets and make them more visible. The diluted blood is mixed for 10-15 minutes.
  2. The Neubauer chambers is filled with diluted blood and left in a moist petri dish for 20 minutes to allow the platelet to settle down.
  3. The platelets may be counted under normal light or under phase contrast. Platelets are visualized better under phase contrast. The condenser needs to be racked down while counting platelets. Platelets appear as small refractile bodies. Dust particles can be confused with platelets and the equipment and reagents used for platelet counting must be dust-free.
  4. The number of platelets is counted in the 1mm squares. At least 200 cells need to get an accurate count (with a coefficient of variation of 8-10%). Each square has a volume of 0.1µl. The platelet count (per liter)  is calculated as follows

[number of platelets counted X dilution X 106]/Volume counted

If accurate erythrocyte count is available the platelet count may be performed by count the proportion of platelets to erythrocytes in the thin part of the peripheral smear and estimating the platelet count as a proportion of the erythrocyte count.

Automated Platelet Counts

Automated counters count platelets by impedance, light scatter, optical fluorescence or by the use of monoclonal antibody to platelet glycoproteins (see Principles of Automated Cell Counts).

Impedance counters Impedance counters count platelets by the change in impedance they produce while passing through an electric field. The degree of change is proportional to the volume of the cell. Platelets are separated from erythrocytes by size, the cut-off being 30fl. Large platelets may (e.g. those seen in May-Hegglin anomaly) may not be counted and erythrocyte fragments may be counted as platelets.

Light Scatter Laser light scattering patterns of cells depending on their shape and reflectivity. Cells are passed under a laser beam and their light scattering patterns determined. Cells are characterized according to these patterns.

Monoclonal Antibody Platelets can be counted by immunofluorescence using the monoclonal antibodies CD41, CD42a or CD61. Usually a combination of CD41 and CD61 us used to eliminate errors that may occur because of the lack of one antigen on the platelet surface membrane.

Newer Platelet Parameters Generated by Automated Platelet Counters

The electronic platelet counters can calculate three new platelet parameters. As of now these have a limited role in diagnosis

Mean Platelet Volume (MPV) MPV is a reflection of megakaryocyte ploidy. MPV is increased in conditions associated with increased platelet turnover. The platelet mass remains constant in normal individuals.. The MPV falls with increasing platelet counts in a non-linear manner. The effect of storage in EDTA on MPV depends on the method used to perform the platelet count. The MPV increases if measured in an impedance counter because of a change in volume of platelets (see above). When measured by optical methods the MPV decreases nearly 10% possible because of a fall in the refractile index of the platelets because of dilution of cytoplasmic contents.


Platelet Distribution Width (PDW) PDW is the variability in the size of platelets. Normally PDW increases with MPV. In patients with megaloblastic anaemia, aplastic anaemia and those on chemotherapy the relationship between MPV and PDW is lost. The patients show a low MPV but a high PDW.

Reticulated Platelets Freshly released platelets have RNA that is lost in 24-36 hours after release. These are known as reticulated platelets and can be counted by counters by using a RNA banding dye. Reticulated platelet count, like reticulocyte count, is an index of platelet production. The normal range varies from <3% to 20%. Reticulated platelet counts are useful in distinguishing thrombocytopenia from peripheral destruction from that due to decreased production. It has a reported sensitivity and specificity of 95%.


 Sources of Error in Platelet Counts

Falsely Low Platelet Counts
  1. Pre-analytic errors: Pre-analytic errors are the common causes of fictitious low platelet counts and include
    1. Partial clotting of specimen with activation and aggregation of platelets during venepuncture.
    2. EDTA induced platelet aggregation:
    3. Platelet Satellitism: Platelet statellitism is phenomena in which platelets encircle neutrophils. It is usually seen in blood collected in EDTA but may be seen with other anticoagulants and is caused by IgG or IgM antibodies that bind the CD16 antigen. Satellitism around other leucocytes may be seen and some platelets may be phagocytosed.
  2. Giant Platelets Impedance counters separate platelets from erythrocytes by size. Large platelets may not be counted as platelets but as erythrocytes. Patients whose peripheral smears a significant number of large platelets should have a manual platelet count.
False High Counts

Severe Microcytosis or Fragmentation of Erythrocytes: Erythrocytes are separated from platelets by size. Small erythrocytes or erythrocyte fragments can be counted as platelets giving a falsely elevated count.

Cryoglobulinaemia: Pseudothrombocytosis and pseudoleucocytosis has been reported in patients with cryoglobulinaemia. Haemoglobin H disease has been associated with pseudothrombocytosis.

Counting Platelets When Counts are Suspected to the Falsely Abnormal

All low counts must be confirmed on a second sample. If the sample is a collected in evacuation tubes the correct order of draw must be followed. If collected by a syringe and needle then it must be a small (preferably only for performing the platelet count) sample, taken from an adequately large vein avoiding applying excessive suction as all these factors can cause platelet aggregation. Measures appropriate for specific causes of thrombocytopenia include

Platelet Aggregation: The counter will often flag the presence of platelet clumping but the peripheral smear must be examined for the presence of platelet clumps, fibrin strands or platelet satellitism. The options available in case of a suspected EDTA induced platelet aggregation include

  1. Adding 20mg kanamycin
  2. Adding excessive EDTA to disrupt the aggregates
  3. Collecting blood in another anticoagulant – occasionally aggregation may be seen with other anticoagulants
  4. Performing a platelet count no-anticoagulated blood collected by finger prick.

Variations in Platelet Size: It is impossible to get an accurate automated platelet count in patients with a large number of giant platelets. Manual platelet counts should be performed in such patients. The patients should be informed of this discrepancy to prevent him/her from undergoing investigations for “thrombocytopenia”. Pseudothrombocytosis due to severe microcytosis or erythrocyte fragments is rare. These patients also need a manual count.