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.

Why Blood Loss From Sites other than Gastrointestinal Tract Rarely Causes Iron Deficiency?


A 55 year old man presented with breathlessness on climbing stairs. He saw his family physician who found the patient to be pale. The patient was advised a complete haemogram. He was found to be anaemic and was asked to see a haematologist.

The haemogram showed an haemoglobin of 3.1 g/dL with an MCV of 63fl. The WBC count was 5600 with 65% neutrophils, 30% lymphocyte, 3% monocytes and 2% eosinophils. The platelet count was 475 X 1009/L. The reticulocyte count was 1%.

The serum iron was 15μg/dL and the total iron binding capacity 450μg/dL and a transferrin saturation of 3.3%. The serum ferritin was 8ng/ml. A diagnosis of iron deficiency anaemia was made he was initiated on oral iron which he responded to. 

Iron deficiency is common in 

  1. Growing children because of dietary deficiency
  2. Women in the reproductive age group because of menstrual blood losses and iron depletion because of foetal transfer of iron during pregnancy.

When iron deficiency occurs in a well nourished man or a well nourished post-menopausal women it is invariably due to a gstrointestinal blood loss. Why is gastrointestinal blood loss different from other forms of blood loss?

Bleeding is an alarming symptom. It is rare for a person to ignore bleeding. Bleeding from the respiratory system, urinary system and skin is apparent and alarming. Such bleeding prompts the patient to promptly seek medical attention. Gastrointestinal bleeding may be of three types. The patient may pass fresh blood, the patient may have malaena or the patient may have occult bleeding. Passage of fresh blood with stools is a symptom of a lower gastrointestinal pathology. Such patients seek attention early and usually do not become anaemia. Haemorrhoids is an exception not because the patient does not realise that there is bleeding but because the patient may ignore the bleeding because he/she attributed it to a known cause. Some patients may not realise the significance of malaena and may present only when anaemic. A patient may loose upto  30ml of blood without a change in the consistency or colour of stools. Such patients present with iron deficiency anaemia. As the anaemia has a gradual onset the it may become severe and yet not cause symptoms. 

The diagnosis of anaemia is complete only if the type of anaemia and the cause of anaemia are determined. A rule of thumb is that unexplained iron deficiency anaemia in a man or a postmenopausal woman should be considered to be due to a occult gastrointestinal blood loss unless proven otherwise. The importance of this practice can not be overemphasised. A colon cancer develops in an adenoma. Completing the evaluation of an iron deficency anaemia provides an opportunity to diagnose a colorectal cancer either in the premalignment stage or when the disease has a limited extent. Not perusing the diagnostic evaluation of an iron deficiency anaemia to completion may close the window of early diagnosis of gastrointestinal cancer.

Case 2 – Thrombocytopenia Due to Large Platelets


A 52 year old patient presented with thrombocytopenia discovered on a haemogram performed about 2 years before presentation. The first haemogram performed as a part of a health checkup showed a platelet count of 81 X 109/L with an MPV of 13.1fl. The haemogram had been performed on multiple occasions since then and the platelet count has been reported by different laboratories to be between 63 X 109/L and 103 X 109/L. The haemoglobin, red cell indices, total and differential WBC count was normal.

The patients had no personal or family history of bleeding. He had undergone an appendicectomy and a tooth extraction without excessive bleeding.

The first step in the evaluation of a patient with thrombocytopenia is to confirm if the platelet count is really low. The causes of pseudothrombocytopenia include

  1. Presence of large platelets
  2. Platelet satellitism
  3. EDTA (or rarely other anticoagulant) induced platelet aggregation

The figure below shows the peripheral smear and the platelet histogram of the patients compared to a normal individual.

The platelet count performed manually was 130 X 109/L. The patients was a native of North India. Mild thrombocytopenia (platelet count between 100 X 109/L and 150 X 109L) is found in 26% of blood donors from eastern India and 18% blood donors from northern india (HVK Naina et al. Journal of Thrombosis and Haemostasis 2005; 11:2581-2).

Automated counters have limitations in evaluation of thrombocytopenia. The peripheral smear must be examined in every patient with thrombocytopenia to exclude giant platelets, platelet aggregation and platelet satellitism that may cause thrombocytopenia.

Case No 1 – Severe Microcytic Anaemia


A 17-year-old woman from a farming community presented with the history of severe weakness, fatigue and dyspnoea on moderate exertion.

She was well till about 4 months before presentation when she complained of fatigue that gradually became worse. Though progressively symptomatic she was able to attend school till 3 days before presentation.

There was no history of fever, weight loss, abnormal bleeding or melena. There was no increase in the menstrual blood loss. The patient was living with her parents and two sisters both of whom were healthy.

Her appetite had been normal through most of the illness though off late it had diminished.

On examination the patient was comfortable but tachypnic  in bed. She had severe pallor of the skin, conjunctiva and mucous membranes.  There was platonychia and glossitis. The jugular venous pressure was elevated. There was no icterus, clubbing, lymphadenopathy. The pulse was 108/min and the blood pressure 110/60. The third heart sound was audible at the apex. She had bilateral fine crepitation in the bases of both lungs. The was no hepatomegaly. There was no splenomegaly. Examination of the neurological system revealed no abnormality.

A haemogram was performed and the results were as follows:

  • Haemoglobin: 1.7g/dL
  • RBC Count: 0.9 X 1012/L
  • Haematocrit: 5.9%
  • WBC Count: 4.1 X 109/L
  • Differential Count:
    • Neutrophils: 35%
    • Lymphocytes 49%
    • Monocytes 10%
    • Eosinophils: 6%
  • Platelet Count: 505 X 109/L
  • RBC Indices
    • MCV: 66.1 fl
    • MCH: 19.4 pg
    • MCHC: 29.3 g/L
    • RDW: 44%
  • Reticulocyte Count: 8%

What are the probable causes of anaemia in this patient?

Presence of platelet and/or leucocyte anomalies in a patients with anaemia suggests that the pathology lies in the bone marrow. Anaemia without leucocyte or platelet anomalies may be caused by increased erythrocyte loss or decreased erythrocyte production. Anaemias caused by decreased production of erythrocytes are further divided according to erythrocyte size into microcytic, normocytic and macrocytic  (see Evaluating anaemia).

This patient does not have platelet or leucocyte anomalies. The anaemia is either a result of a decreased production or increased loss of erythrocytes. Both erythrocyte loss and decreased production are difficult to quantify in clinical practice and are diagnosed by indirect methods. Increased eryrthrocyte production should increase haemoglobin unless there is an equal or greater loss. Increased erythrocyte production result in reticulocytosis. In patients with anemia the reticulocyte production index is a more accurate index of erythrocyte production. A patient with normal or low haemoglobin but an increased reticulocyte production index is presumed to be loosing erythrocytes  (see diagnosis of haemolytic anaemia).  Others are presumed to have decreased erythrocyte production.

Reticulocyte production index is calculated as follows:

RPI = [Reticulocyte count X HCTPatient] / [HCTNormal X CF]

where

RPI is the Reticulocyte Production Index. RPI values less than 2 indicate inadequate erythrocyte production

HCTPatient is the patient’s haematocrit

HCTNormal is the normal haematocrit

CF is the correction factor. The correction factor depends on the haematocrit. It’s values are as follows:

  • Haematocrit 40-45%: 1
  • Haematocrit 35-40%: 1.5
  • Haematocrit 25-35%: 2
  • Haematocrit 15-25%: 2.5

The reticulocyte production index in this patient is as follows

RPI = [8 X 5.9 ] / [45*2.5]  =  0.41

RPI of 2 indicates an adequate marrow response. This patient, despite having a high reticulocyte count, has decreased erythrocyte production.

Anaemias due to inadequate erythrocyte production are classified according to erythrocyte size as microcytic, normocytic and macrocytic (see red cell indices). This patient has microcytosis. The causes of microcytic anaemia include:

  • Iron deficiency
  • Thalassaemia
  • Sideroblastic anaemia
  • Anaemia of chronic disease

Chronic inflammation is associated with mild to moderate anaemia that is proportional to the degree of inflammation. Most of the patients have normochromic anaemia but about 30-50% of the patients may develop mild microcytosis with a normal RDW.  This patient has no evidence of chronic inflammatory process. She has severe anaemia, pronounced microcytosis and a high RDW. She is unlikely to have anaemia of chronic disease.

Iron deficiency and thalassaemia are the commonest causes of microcytic anaemia. β-Thalassaemia has three clinical forms, major, minor and intermedia. Thalassaemia minor does not cause symptoms. Symptomatic β-thalassaemia (thalassaemia major and intermedia) usually present early. The transfusion dependent patients present by 8.4±9.1months and non-transfusion dependent patients by 17± 11.8 months (Cao A Birth Defects 1988;23;219) . This patent has a late presentation making thalasaemia unlikely.

The haemogram of patients with iron deficiency shows anaemia, microcytosis, hypochromia and anisocytosis. Thrombocytois is not uncommon. The platelet counts in patients with iron deficiency are twice those without deficiency (Dan K Intern Med 2005; 10: 1025). The thrombocytosis associated with iron deficiency has an inverse relationship with serum iron but no relationship with TIBC and transferrin saturation. (Park MJ et al Platelets 2012 Jun 27. [Epub ahead of print]). This patients has anaemia, microcytosis, hypochromia and thrombocytosis. The high red cell distribution width (RDW) indicated the presence of anisocytosis. The most likely diagnosis in this patient is iron deficiency anaemia.

Is there a haemoglobin value that should trigger a blood transfusion?

It was common to use the 10/30 (haemoglobin 10g/dL or PCV 30%) trigger for transfusion. Transfusion is associated with morbidity and mortality (Marik et al. Crit Care Med 2008; 36:2667-74). Following the 10/30 rule results in overtransfusion. The case highlights poor relation between haemoglobin levels and symptoms. Anaemia is compensated by increasing cardiac output, redistributing blood from non-critical to critical circulations and increasing oxygen extraction (see Pathophysiology of anaemia). Older individuals may have one or more of these mechanisms compromised hampering their ability to withstand anaemia. Younger individuals can compensate efficiently and can withstand lower haemoglobin values. Compensation takes time and is more complete in anaemias that have a slow onset. Nutritional deficiencies develop slowly. Patients with nutritional anaemia compensate better and become symptomatic at lower haemoglobin values than anaemia with a more acute onset.

She was in cardiac failure and was transfused. But would she have been transfused if she was not in cardiac failure and had a haemoglobin of 7g/dL? The threshold for transfusion according to most current guidelines is 7-8 g/dL. Trials evaluating transfusion threshold have been performed in hospitalized, surgical or intensive care patients. Transfusion guidelines are most appropriate when used for indications the were developed. There are few trials addressing the issue of transfusion in patients with nutritional anaemia. Clinical experience shows that patients without respiratory or cardiovascular co-morbidities are able to tolerate severe nutritional anaemia. One needs to balance the risk of anaemia against risk of transfusion in patients with nutritional anaemias. The transfusion trigger for patients of nutritional is not established. Symptoms guide transfusion in patients with nutritional anaemia.

How should the patient be investigated?

As iron deficiency is the commonest cause of microcytic anaemia, patients should be evaluated for iron deficiency. The serum iron, total iron binding capacity and serum ferritin are estimated and the transferrin saturation calculated . The results of these investigation are diagnostic for iron deficiency and often allow the diagnosis of other causes of microcytic anaemia. The interpretation these tests are as follows:

Serum Iron Total Iron Binding Capacity Transferrin Saturation Serum Ferritin
Iron Deficiency Anaemia  Low  Normal/High  Low (<16%)  Low (<12ng/ml)
Thalassaemia Normal Normal Normal Normal/High
Sideroblastic
Anaemia
High Normal High (>50%) High
Chronic Disease low/Normal Low Normal Normal/High

This patients was diagnosed as iron deficiency anaemia because of a low serum iron, high total iron binding capacity and a low transferrin saturation. Does that complete the diagnosis?

The diagnosis of iron deficiency is incomplete without finding out its cause. The following populations are prone to iron deficiency.

  1. Preterm infants: Most of the iron transfer to the foetus takes place in the third trimester. Premature infants have less time to absorb iron from the mother.
  2. Infants and Toddles: Dietary iron is not able to keep up with the requirements of rapid growth.
  3. Women in the childbearing age group: Losses due to menstruation and pregnancy cause iron deficiency.
Iron deficiency is due to inadequate iron intake or increased iron/blood loss. Blood/iron is lost because of diseases/conditions causing exacerbation of physiological blood/iron loss or diseases causing physiological loss. Multiple pregnancies cause exacerbation of physiological iron loss. The only physiological blood loss is menstrual blood loss.
This patient gave no history of increased menstrual blood loss. Hookworm (Ancyclostoma duodenale and Necator americanus) infestations infestations affect over 500 million individuals in the hot and humid areas of the world. Infestations are common in farming communities. The stool examination of this patients showed hookworm ova.
Finally, does the diagnosis of iron deficiency rule out other causes of microcytic anaemia? No. Iron deficiency is very common that it may co-exit with another cause of microcytic anaemia particularly anaemia of chronic disease and thalassaemia trait. Anaemia of chronic disease is characterized by a low iron, low total iron binding capacity and normal transferrin saturation. When such patients become iron deficient the transferrin saturation falls below 16%. Ferritin is increased in the presence of inflammation/infection. While a low ferritin is diagnostic of iron deficiency in the presence of infection/inflammation, a normal ferritin does not exclude iron deficiency. β-Thalassaemia is diagnosed by an elevated HbA2. HbA2 has been shown to fall with iron deficiency. This may not have any clinical significance (Madan N et al. Ann Hematol 1998; 77; 93-6, Passarello C et al 2012; 97:472). If the haemoglobin and/or microcytosis fail to correct after therapy the patients must be evaluated for thalassaemia.