Tag Archives: anemia

Microcytic Hypochromic Anaemia

13 Mar hypochromic Micro Anaemia

hypochromic Micro Anaemia

Hypochromic Microcytic Anaemia

Shown above is an image of hypochromic microcytic anaemia. This patient had iron deficiency anaemia. Microcytosis is presence of small erythrocytes and hypochromia presence of erythrocytes that are poorly haemoglobinized. The nucleus of a small lymphocyte, the cell in the centre of the image, is a good guide to the size of erythrocytes on a peripheral smear. The nucleus has a diameter of 8.5 µm and a normal erythrocyte a diameter of 7.5 µm. The erythrocytes in in the image are substantially smaller than the lymphocyte nucleus. Erythrocytes have a central pale staining area which occupies about one third of erythrocyte diameter. As the cells get less haemoglobinized the central pale staining area increases. Many of the erythrocytes in the image above have a pale staining area occupies all but a thin rim at the periphery. In others, the pale staining area is increased. The erythrocyte sizes vary. Anisocytosis, increased variation in erythrocyte size, a feature of iron deficiency anemia, is evident in the image.

 

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Iron Studies in Microcytic Anaemia

8 Mar

Diagnosis Serum Iron Total Iron Binding Capacity Transferrin1 Serum Ferritin2
Iron deficiency anaemis Low High or Normal <16% <12ng/mL
Anaemia of Chronic Disease Normal Low or Normal ≥16% High or normal
β-Thalasaemia Trait, HbE, HbC Normal Normal ≥16% Normal
Sideroblastic Anaemia High Normal High High
  1. Two causes of microcytic anaemia may co-exist e.g. thalassaemia trait with iron deficiency anaemia or anaemia of chronic disease with iron deficiency anaemia. When iron deficiency exists with other forms of microcytic anaemia the transferrin saturation is <16%
  2. Patients with low ferritin (<12ng/ml) always have iron deficiency. Higher values of ferritin do not exclude iron deficiency particularly in patients with anaemia of chronic disease. There are no guidelines about the ferritin levels that exclude iron deficiency in patients of anaemia of chronic disease. The reported values vary between 60-100ng/ml.

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Laboratory diagnosis of Iron Deficiency

7 Mar

The investigation to diagnose iron deficiency include:

  1. Haemoglobin and red cell parameters
  2. Bone marrow iron staining
  3. Serum Iron, total iron binding capacity and transferrin saturation
  4. Serum ferritin
  5. Zinc Protoporphyrin
  6. Soluble transferrin receptor

Haemoglobin and Red cell Parameters

Patients undergo evaluation for iron deficiency because they are anaemic. Latent iron deficiency is characterized by a progressive decrease in bone marrow iron stores in patients who have yet not developed symptoms. These patients are asymptomatic and latent iron deficiency is not a clinical problem. Iron deficiency causes microcytic hypochromic anaemia. There are no reliable test to differentiate iron deficiency from other causes of microcytic hypochromic anaermia.  The red cell indices that have been proposed to be useful include

  1. Red cell distribution width (RDW): RDW is a measure of anisocytosis. Iron deficiency anaemia shows more anisocytosis than β-thalassaemia and is associated with a higher RDW. The promise held out by RDW to differentiate iron deficiency and thalassaemia has not fulfilled.
  2. Reticulocyte haemoglobn content: Changes in erythropoiesis are reflected earliest in the reticulocyte. Reticulocytes form a small fraction of erythrocytes. Change in reticulocyte indices do not change erythrocyte indices. Some automated counters are able to measure reticulocyte indices. Reticulocyte haemoglobin content falls with iron deficiency anaemia but the finding is not specific. It has been found to asses iron deficiency in patient of chronic renal failure being treated with erythropoietin accurately. It has not been found to be useful in diagnosing iron deficiency in patients with thalassaemia.

Bone Marrow Iron Staining

Bone marrow is stained for iron content by the Prussian blue reaction and graded in a semiquantative method. Bone marrow iron is the gold standard for diagnosis of iron deficiency. The test is invasive and suffers from an inter-observer variation. Bone marrow iron staining is resorted to only when diagnosis can not be reached by other methods.

Serum iron, total iron binding capacity and transferrin saturation

Iron deficiency is diagnosed by a transferrin saturation of less than 16%. The serum iron is low and the total iron binding capacity is usually increased. Patients with low total iron binding capacity have anaemia of chronic disease if the transferrin saturation is ≥16%  or iron deficiency along with anaemia of chronic disease if the transferrin saturation is <16%.

Serum ferritin

Ferritin is one of the iron storage proteins. Serum ferritin levels co-relates with body iron content. A ferritin level less than 12ng/mL is diagnostic of iron deficiency. Inflammation increases ferritin. Chronic inflammatory diseases like rheumatoid arthritis and ulcerative colitis have anaemia of chronic disease and may also have iron deficiency. A patient with iron deficiency in the setting of an inflammatory disease may not have a low ferritin. There is no consensus for diagnosing iron deficiency in patients with anaemia of chronic disease. Inflammation rarely increases the serum ferritin values more than 60-100ng/mL. Iron deficiency can be excluded in patients with ferritin above this cutoff.

Zinc Portoporphyrin

Iron is added to propoporphyrin in the final step of heme synthesis. Zinc takes the place of iron in patients with iron deficiency. A rise in the concentration of zinc protoporphyrin is the earliest manifestations of iron deficiency. Zinc protoporphyrin levels rise in about 2 weeks from the onset of iron deficiency and need more than a month to normalize after restoration of normal iron levels.

Soluble transferrin Receptor

Iron deficiency results in an increase in soluble transferrin receptor (sTfR). Inflammation impacts serum ferritin but not sTfR making it a potentially useful investigation for differentiating anaemia of chronic disease and iron deficiency. The assay has been difficult to standardize. A ratio of sTfR/log ferritin is more useful.

The sTfR levels reflect the density of transferrin receptors cells and number of cells. sTfR increases when there is erythroid hyperplasia due to any cause like haemolytic anaemia and may not reflect iron deficiency in these disorders.

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Iron Storage Compounds

12 Feb

Ferritin

Ferritin is made of apoferritin and iron. Apoferritin is a protein shell made of 24 subunits that is 13nm in diameter. The shell has six channels for entry of ions. The core of ferritin holds ferric-hydroxyl phosphate. Ferritin can hold 4500 atoms of iron but usually close to 2000 atoms are present.

Apoferritin is made of two units, the H and L units. About twenty types of ferritins (isoferritins) having different ratios subunits are been found in humans. The H (heavy or heart) subunit has a molecular weight of 21000 Da. The gene for the subunit is present at 11q12.3. It has a ferroxidase centre that is responsible for oxidation of Fe2+ ion to Fe3+ ion before incorporation into ferritin. The L (Liver or light) subunit has a molecular weight of 19000 Da. The gene for the L subunit is at 19q13.33. Ferritins rich in H unit acquire iron rapidly where as those rich in the L subunits are more stable.

Synthesis of ferritin begins with the apoferritin shell. Iron in the ferrous form then passes to the core through the channels, is oxidized by the ferroxidase to the ferric form and incorporated in the ferritin. Fully assembled ferritin has molecular weight of 400-500kDa. Iron promotes ferritin synthesis.

Mitochondrial ferritin is transcribed from a different gene situated on chromosome 5q23. Its function is not fully understood. As mitochondrial ferritin is highly conserved, it must have an important role.

Hemosiderin

Hemosiderin, unlike ferritin, is not s defined chemical entity. It is formed by the partial degradation of ferritin. It contains more iron than ferritin and is less soluble than ferritin. This iron however is not as freely available as iron from ferritin. Hemosiderin only releases iron after prolonged or severe iron depletion.

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Case No 1 – Severe Microcytic Anaemia

1 Nov

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.

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Red Cell Indices

13 Oct

Estimation of haemoglobin concentration, erythrocyte count, haematocrit, total and differential leucocyte count and platelet count along with examination of the peripheral smear are a part of evaluation of anaemia. Examination of the peripheral smear detects abnormalities in size (microcytosis or microcytosis, anisocytosis), shape and haemoglobnization. Reporting a peripheral smear need experience and is subjective. Red cell indices evolved to bring objectivity and increase reproducibility reporting of red cell characteristics. Red cell indices have allowed a reproducible algorithmic approach to the diagnosis of anaemia.

Normal red cell counts were described in 1852 . This measurement made on four subjects was the normal standard for the next 70 years. Wintrobe in the 1930s determined the normal red cell count described the method for calculating red cell indices (Wintrobe MM Am J Med Sci 1929;177:513–23) and classified anaemia into four groups, macrocytic, normocytic, microcytic and microcytic hypochromic (Wintrobe MM Arch Intern Med. 1934. 54:256-280). Though the methods of determining red cell indices has changed, the concept of classification of red cells by size and the classes of anaemia described by Wintrobe still hold. The three indices are

  1. Mean corpuscular volume (MCV)
  2. Mean corpuscular haemoglobin (MCH)
  3. Mean corpuscular haemoglobin concentration (MCHC)

Mean Corpuscular Volume (MCV)

MCV is the average volume of erythrocytes and is defined as follows:

MCV = [Haematocrit (in percentage) X 10]/RBC count (in millions/mm3)

The normal value is 92±9fl

At the time MCV was described it was calculated as described above. Erythrocyte counts performed manually were cumbersome, technician dependent and inaccurate limiting the value of MCV. Automated haematology counters may be based on impedance or light scatter. Sensors of counters, impedance as well as those based on light scatter, generate signals according to cell size and number. The size of each particle is calculated by the pulse it generates and the MCV reported as an average of these measurements. The standard deviation of observation of MCV is reported as red cell distribution width. When automated counters are used MCV is a measured parameter rather than a calculated parameter. These measurements are accurate and reproducible.

Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC)

Mean corpuscular haemoglobin is defined as follows.

MCV = [Haemoglobin (in g/dL) X 10]/RBC count (in millions/mm3)

The normal value for MCH is 29.5±2.5pg.

Mean corpuscular haemoglobin concentration is calculated as follows

MCHC = [Haemoglobin (in g/dL) X100]/[Haematocrit (in percentage)]

Normal 33±1.5g/dL

The MCV and MCHC are of limited value in diagnosis of anaemias. A low MCH has the same significance as a low MCV. MCHC is low in microcytic anaemias. Macrocytic anaemias may have a high MCH but the MCHC is normal in these case. Some anaemias like hereditary spherocytosis, sickle cell anaemia and haemoglobin C disease are characterized by a high MCHC indicating cellular dehydration

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Red Cell Distribution Width

1 Oct

Features of peripheral smear like erythrocyte size, haemoglobinization and variability in size have long been useful in evaluation of patients with anaemia. The definitions of these parameters before the advent of haematological counters were subjective. The haematological counters have allowed an accurate measurement of erythrocyte size and numbers allowing reliable determination of mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cellular haemoglobin concentration (MCHC). Mean cellular volume is used to classify anaemias (see Evaluating Anaemias). MCV is of little value in differentiating two of the commonest causes of anemia, iron deficiency and β-thalassaemia trait as both are microcytic hypochromic.

Anisocytosis is and abnormal variation in erythrocyte size. It is a feature of nutritional deficiencies, myelofibrosis, bone marrow infiltrations, microangiopathic haemolytic anaemia and in the presence of erythrocyte aggregates. Thalassaemias do not show anisocytosis. The erythrocyte is a disc approximately the diameter of the nucleus of a small lymphocyte and the central one-third is pale. The assessment of anisocytosis is subjective. Red cell distribution width(RDW) is quantitation of aniscytosis.

Standard deviation of a parameter is a measure of its scatter from the mean. MCV is an average of erythrocyte volumes measured by the counter. RDW is the standard deviation of these observations. It is expressed as a percentage of MCV. The normal values are 11.5-14.5%. High RDW means more anisocytosis.

Despite the apparent promise RDW has a limited role in diagnosis. A normal RDW in a microcytic anaemia can suggest the presence of on thalssaemia but can not be relied on as a sole criteria for separating iron deficiency from thalassaemia. The presence of a high RDW should alert one to the presence of one of the causes of anaemia listed above but again RDW can not be relied on for making a final diagnosis of any of these conditions. Blood transfusion in an anaemic patient increases the RDW.

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Evaluating Anaemia

28 Sep

Anaemia is a manifestation of many diseases. Figure 1 gives the outline for evaluation of a patients with anaemia. The details of evaluation are given below.

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 type of anaemia, the cause of anaemia and complications caused by anaemia. Red cell transfusions in anaemic patients are for relieving symptoms due to a low haemoglobin. Patients without symptoms should not be transfused. Patients with symptoms because of low haemoglobin should be teated urgently with blood transfusion. 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).
  2. 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.
  3. Iron deficiency is the commonest cause of anaemia. 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.

  1. Manifestations of of anaemia: The symptoms of anaemia depend on the degree of anaemia, rate of fall of haemoglobin and 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. Thus a patient with a gradually developing anaemia and without coronary heart disease becomes symptomatic at a lower haemoglobin than one with a rapidly developing anaemia, one having a coronary or one with both.  Nutritional anaemias have a slow onset and thus present with a lower haemoglobin than anaemia or blood loss or acute haemolysis.
    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.
  2. 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.
  3. Manifestations due to compensatory changes: Tachycardia, wide pulse pressure and decreased exercise tolerance result from cardiovascular compensation to anaemia. Chronic haemolytic anaemias result in marrow hyperplasia that results in a 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.
Clinical Finding <strong >Significance
Painful Crisis Sudden onset of pain is a feature of sickle cell disease. Four regions are commonly affected: bone, chest, abdominal and joint
Jaundice
  1. Achloruric Januduce: Achloruric jaundice is a result of increased haemoglobin catabolism (see haemoglobin catabolism) It is seen in patients with haemolytic anaemia, where the erythrocyte turnover is increased, and megaloblastic anaemia, where the turnover of haemoglobinized erythroid precursors in the bone marrow is increased. 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 no foot ware is 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 under three circumstances

  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 or rarely 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 with progression. 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. Features that may be present include
  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.

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
  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. 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.

Evaluation of Microcytic Anaemia

As haemoglobin forms about 97% of the erythrocyte proteins. Disorders impairing haemoglobin synthesis cause microcytic anaemia. Haemoglobin synthesis needs an intact 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 3.5-8% have heterozygous β-thalassaemia. Higher HbA2 values should be considered to be due to an abnormal haemoglobin that separates with HbA2. 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. Unless HbH disease is prevalent in the ethenic group the patients belongs to, further investigation for patient with suspected thlassaemia is not necessary. Patients with transferrin saturation more than 50% need to have  bone marrow aspiration for diagnosis of sideroblastic 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 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.

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Megaloblasts

25 Jul 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 orthochromatophilic normoblast and the bottom right 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.

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Pathophysiology of Anaemia

3 Jul Oxyhaemoglobin Dissociation Curve

Low haemoglobin decreases the oxygen carrying capacity of the blood. This is offset by the following mechanisms.

  1. Increased cardiac output: Tissue hypoxia causes peripheral vasodialatation and decreased haemoglobin decreases blood viscosity. Together these reduce peripheral resistance allowing a higher cardiac output without increasing blood pressure.  Increased cardiac output increases tissue oxygen delivery but decreases the patient’s  exercise tolerance. Increase in cardiac output is seen at haemoglobin values less than 7g/dL.
  2. Redistribution of cardiac output: Blood flow from non-critical areas like the skin is diverted to critical organs like heart and brain.
    The Figure shows three oxyhaemoglobin dissociation curves. The blue curve is the physiological curve. This may be shift to the right (red) or left (green). When the curve shifts to the right the oxygen affinity of haemoglobin falls. Haemoglobin is more desaturated ar a given P02. The opposite happens when curve shifts to the left. The three arrows show the change in oxygen saturation between arterial and venous blood at venous and arterial PO2
  3. Increased Oxygen Extraction: Anaemic patients have increased tissue oxygen extraction from the blood. This is not seen in organs like the heart and brain that already have high oxygen extraction.
  4. Changes in oxygen affinity of haemoglobin: Patients with chronic anaemia have increased of 2,3 diphosphoglycerate (2,3-DPG). 2,3-DPG shifts oxyhaemoglobin dissociation curve to the right and increases oxygen delivery. The figure above shows three curves. The blue curve is the curve of normal haemoglobin the red shows shift to left and the green shift to right. Shift to left increases the oxygen delivery.
  5. Isovoluaemic haemodilution: Patinets with anaemia have increased plasma volume. This allows maintaining a normal cardiac output.

 

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