Tag Archives: Iron Deficiency

Injectable Iron

Iron deficiency, the commonest cause of anaemia, is treated by iron supplementation. Oral iron, introduced by the French physician Pierre Blaud in the 19th century is the mainstay of therapy of iron deficiency. Oral iron is inexpensive and safe but is poorly absorbed and causes abdominal adverse effects in 35-59% of the patients. Injectable iron preparations were initially developed for the treatment of iron deficiency in patients intolerant to iron. Their use became more prevalent with it became clear that iron deficiency was a common cause of failure of erythropoiesis stimulating agent therapy and that oral iron was insufficient for this indication.


Is it possible to inject an iron salt, say ferric chloride, for iron deficiency, like it is to inject calcium chloride for hypocalcaemia? Iron, unlike calcium or potassium,  produces free radicals (oxyradicals) that can damage macromolecules and result in cellular injury. Ferric hydroxide,  the first injectable iron preparation used, caused an immediate release of iron in circulation resulting in severe reactions.  When an iron oxide, hydroxide or an iron salt is used, only a small dose can be safely administered. One estimate calculated the maximum iron permissible as 8mg/day (this is equivalent of the unbound iron binding capacity of transferrin). It would take about 6 months to bring up haemoglobin at this dose.


About  25mg of iron is delivered each day to the erythroid precursors for haemoglobin synthesis. Iron is a poorly absorbed micronutrient and stores are needed to  provide for sudden increase in demand as may be seen in patients with acute blood loss. Given the propensity of iron to cause free radical induced cellular injury, transport and storage systems capable of protecting the body from iron have evolved. Iron is transported bound to transferrin and stored as ferritin and haemosidin. In both the situations iron is bound to apoproteins (apotrasferrin and apoferritin) and this binding does not allow free radical generation.

Erythrocyte destruction takes place in the macrophages and macrophages have systems for the handling, storage and recycling of iron. Injectable iron preparations have a carbohydrate shell that prevents exposure of the plasma to free iron.  Injectable iron preparations are taken up by macrophages of the reticuloendothelial system by endocytosis. The iron is released and enters the macrophages iron pool. This iron has a fate similar to iron reaching the macrophage from other sources viz. intestinal absorption and erythrocyte destruction. Binding iron to carbohydrate shell has made it possible to administer as high as dose as 1000mg over as short a time as 15 minutes. This is a 125 fold increase over the amount of iron that can be administered without the carbohydrate shell.


Injectable iron preparations share a common structure. They have a core of iron oxide/hydroxide that is associated with a carbohydrate shell. The carbohydrate shell  keeps free iron from entering the plasma and in a way performs the same function as transferrin. The antigenicity of the shell and the strength of binding between the iron core and the carbohydrate shell determines the side effects and the maximum dose per injection.


Preparations of Injectable Iron

The preperations of injectable iron include

  1. Preparations with a strong association between iron core and the carbohydrate shell
    1. High-Molecular weight iron dextran
    2. Low -molecular weight iron dextran
    3. Ferric carboxymaltose
    4. Ferumoxytol
  2. Perperations with a weak association between iron core and carbohydrate shell
    1. Iron Sucrose
  3. Preperations that have a labile low molecular weight components
    1. Ferric Gluconate
    2. Iron-Sorbitol-Citric Acid Complex, Dextrin-Stabilized


Table 1 – parentral iron preparations
Drug Shell MW T1/2 Labile
Dose and administration
HMW ID High moleculer weight dextran 265 60 1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 5 minutes) Maximum dose 20mg/kg. Administered as intravenous bolus 100mg/day slowly. A total dose infusion may be given over a prolonged period.
LMW ID  Low molecular weight dextan  165  20  1-2% Should be administered 1 hr after an intravenous test dose (0.5ml over at least 30 seconds). It may be administered as daily iv bolus of 100mg over 2 mins or a a total dose infusion over 3-4 hours may be administered 20mg/kg
Iron Sucrose  Sucrose 30-60  6  4-5% up to 300mg in a single dose, higher doses have been administered over as a prolonged infusion. May be administered undiluted as an iv bolus 200mg over at least 10 mins or as an infusion in 0.9% saline over 15-60 minutes
Ferric Gluconate  Gluconate  289-440  1  5-6% 125mg, iv bolus at 12.5mg/min or as a 1 hr infusion in 0.9% saline
Ferric Carboxy-maltose Carboxy-maltose  150  16  1-2% The maximum dose is 20mg/kg (max 1000mg). Doses of 200-500mg should be administered at 100mg/min, doses between 500mg and 1000mg should be administered over 15 minutes. It may be administered as an iv infusion diluted in 0.9% saline
Ferric Isomaltoside Isomaltoside 150  20  <1% May be administered as an intravenous bolus dose of 500mg un to three times a week at the rate of 50mg/min or 20mg/kg (maximum 1000mg) in 0.9% saline over 1 hour
Ferumoxytol Carboxy-methyl Dextran 750 15 <1% 510mg as a iv bolus at the rate of 30mg/sec. A second dose may be administered after 3-8 days

The parental iron preparations differ in the carbohydrate that surrounds the iron core. The therapeutic implications of these differences are:

  1. Antigenicity: Dextran is antigenic. Patinets may have performed antibodies to dextran or may develop antibodies during therapy. All iron preparations  containing dextran can give rise to anaphylaxis. The risk is greatest with high molecular weight iron dextran. Anaphylaxis may also be seen with low molecular weight iron dextran though the risk is lower and has also been reported with ferumoxytol that contains carboxymethyl dextran.
  2. Maximum dose per injection: All intravenous iron preparations tend to release labile iron. There is an inverse relationship between the amount of labile iron released and the strength of association between the iron core and carbohydrate shell. Higher the molecular weight of the iron preparation, stronger is the association. Labile iron is taken up by transferrin. Non-transferrin bound iron (NTBI) appears when the binding capacity of transferrin is overwhelmed. NTBI is responsible for tissue damage and restricts the dose of a injectable preparation that can be given in a single infusion. Iron preparations may be classified on the basis of strength of the associations between iron core and the carbohydrate shell. Iron dextran, ferric carboxymaltose and ferumoxytol, that have a high molecular weights have a tight binding between iron core and the carbohydrate shell, release up to 2% labile iron. These can be given in a large single dose and are suitable for total dose infusion. Iron sucrose has a weaker association between iron core and carbohydrate shell and has 4-5% labile iron. This limits the amount of drug that can be administered at one time to 300mg. Ferric gluconate is a large molecule but has two types of polymers. Lower weight polymers (about 18,000) have a weaker association between iron and carbohydrate limiting the dose to 125mg per dose.

Dose and Administration

The total dose of parenteral iron is calculated as follows

Total iron dose (mg) = weight (kg) X Hemoglobin deficit  X 0.24 + 500

Haemoglobin deficit (g/L) = Target Haemoglobin (g/L) – Actual Haemoglobin (g/L)


Adverse Effects

  1. Infusion Related Events: the risk of infusion related events (per million) is 0.6 for iron sucrose, 0.9 for ferric gluconate, 3.3 for low molecular weight dextran iron and 11.3 for high molecular weight iron dextran.  Iron sucrose has been safely administered to patients who having sensitivity to iron dextran.  Despite the fact that some studies suggest that low molecular weight iron dextran in as safe as iron sucrose, a test dose must be administered before any iron dextran preparation.
  2. Delayed Reactions to Intravenous Iron: A syndrome charecterized by fever, arthralgia and lymphadenopathy may be seen as a delayed consequence of intravenous iron therapy. Premedication with steroids may reduce the risk of this manifestation.

Further Reading

  1. Rodolfo Delfini Cançado, Manuel Muñoz. Intarvenous iron therapy: How far have we come? Rev Bras Hematol Hemoter. 2011; 33(6): 461–469.
  2. Hayat A. Safety Issues With Intravenous Iron Products in the Management of Anemia in Chronic Kidney Disease. Clin Med Res. Dec 2008; 6(3-4): 93–102.
  3. Peter Geisser and Susanna Burckhardt The Pharmacokinetics and Pharmacodynamics of Iron Preparations. Pharmaceutics. Mar 2011; 3(1): 12–33.



Clinical Manifestation of Iron Deficiency

The manifestations of Iron deficiency evolve insidiously and are multi-systemic. They include


Iron deficiency causes hypochromic microcytic anaemia. The anaemia has an insidious onset and the patient may become severely anaemic before symptoms of anaemia become evident.

Non-Specific Symptoms

Iron deficiency can cause fatigue, irritability, dizziness, breathlessness, and headache. One may attribute these symptoms to anaemia but  these symptoms are seen in patients with iron deficiency even in the absence of anaemia. Iron therapy has been shown to correct many of these manifestations.

Skin and Hair

Iron deficiency results in brittle nails that have longitudinal ridges. The nails become thin, brittle and become flat (platonychia) or concave (koilonychia). Hair may become thin and brittle.

Gastrointestinal System

Next to blood the effects of iron deficiency are most pronounced on the gastrointestinal system

  1. Tongue: Iron deficiency causes atrophy of the papillae of the tongue. The filiform papillae of the anterior part of the tongue are the first to be affected. The fungiform papillae of the posterior tongue atrophy later resulting in a smooth red tongue.  Papillary atrophy results in soreness of the tongue. The changes reverse after about 1-2 weeks of iron therapy.
  2. Mouth: Iron deficiency results in angular stomatitis. This anomalies is not specific to iron deficiency may be seen in pyridoxine and riboflavin deficiency.
  3. Dysphagia: Iron deficiency results in the formation of webs at the junction of hypopharynx and oesophagus. These manifest as dysphagia that has an insidious onset. The patients complains of discomfort on swallowing, initially for solids, at the level of the cricoid cartilage. Mild dysphagia may be corrected by iron replenishment but dysphagia persists in most patients despite iron replenishment and dilatation may be needed. Oesophageal webs predispose to carcinoma. (See Plummer-Vinson Syndrome/Peterson-Kelly Syndrome)
  4. Pica: Pica is craving for non-nutritive substances like chalk, paint and ice. Pagophagia, the craving for ice is a common form of pica.

Neuromuscular Symptoms

Iron deficiency impairs performance of muscles. Children with iron deficiency show irritability, are disruptive, have impaired attention span and may show scholastic impairment. Iron deficiency has also been shown to be associated with developmental delay, ischaemic stroke and raised intracranial pressure.

Effect on Immunity and Infections

Iron deficiency decreases the number of T lymphocytes and impairs phagocytosis. This however does not appear to result in an increased risk of infections.

Menstrual Anomalies

While menstrual anomalies can be a cause of iron deficiency, they can often be a consequence of iron deficiency. Only response to treatment can tell which of the two is the case in a given patient.

Skeletal Expansion

Young patients who have prolonged iron deficiency may develop bone changes line haemolytic anaemia or thalassaemia because of bone marrow expansion.

Intestinal Iron Absorption

Intestinal absorption of iron

Intestinal Iron Absorption
Figure 1 Absorption of Iron

Iron absorption occurs in two steps

  1. Absorption of iron into the enterocyte at the luminal surface of the enterocytes
  2. Transport of iron to the lamina propria at the basilateral surface of the enterocyte

Absorption of iron into the enterocyte

Dietary iron is in two forms, heme and non-heme. Animal foods have 40% iron as heme iron and 60% as non-heme iron. Plant foods contain only non-heme iron. Most of the iron available for absorption comes from non-meat sources even in meat eaters. The widely held perception that vegetarian diets contain less iron than meat eaters is not true. Vegetarian diets contain as much or more iron than meat diets (MJA Open 2012; 1 Suppl 2: 11-16). Vegetarian foods have less heme iron. Different pathways absorb heme and non-heme iron.

Absorption of heme iron

Digestion of proteins releases heme which is oxidized to haemin and absorbed by the enterocytes. A receptor for the absorption of heme has been postulated but not identified. Once within the cell the haemin is acted upon by heme oxygenase 1 to release the iron. The heme iron is added to the enterocyte iron pool and follows the same path out of the cell as non-heme iron. Food does not affect the absorption of heme iron. Some heme may be absorbed directly and is bound to hemoprixin.

Non-Heme Iron

Dietary non-Heme iron is present in the ferric state. Ferric iron is insoluble and needs to be converted to the more soluble ferrous state for absorption. The reduction of ferric iron is aided by the acidic environment of the stomach, dietary components like ascorbic acid and duodenal cytochrome b (Dcytb). Dcytb does not appear to be essential for iron absorption. Ferrous iron is absorbed by the divalent metal ion transporter (DMT1). In addition to  Fe2+,DMT 1 also transports Mn2+, Co2+, Ca2+, Zn2+, Cd2+ and Pb2+. DMT1 is expressed at the brush-border of the enterocyte near the tips of small intestinal villi. DMT1 needs protons to co-transport with iron. Protons come from the gastric juice and are most abundant in the duodenum. Most of the iron is absorbed in the duodenum. Antacid, H2 antagonists and proton pump inhibitors hamper iron absorption by reducing hydrogen ion availability. All these drugs have been shown to impair the efficacy of medicinal iron.

The absorption of non-heme iron, unlike heme iron, is affected by food.

  1. Foods enhancing iron absorption: Ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates with iron enhance absorption of non-heme iron.
  2. Inhibiting iron absorption: `Inhibitors of absorption of non-heme iron include
    1. Phytates present in grains and vegetables
    2. Dietary fibre
    3. Polypohenols present in tea, coffee and wine,
    4. Phosphates and phosphoproteins present in egg yolk, bovine milk
    5. Calcium and zinc.

Transport of iron to the lamina propria

Iron is transported to the lamina propria by an iron transporter ferroportin. Ferroportin transports iron in the ferrous form. This needs to be oxidized to the ferric form for binding to transferrin. Hephaestin oxidizes ferrous iron to ferric iron.

Ferroportin is the key to controlling body iron. In iron deficient state ferroportin expression at the basolateral surface of the enterocyte is increased and more iron is transferred to the blood. When the body is iron repleted ferroportin expression is low and the iron remains in the enterocyte as ferritin.  Iron stored as ferritin is lost with the enterocytes when it is shed at the end of it’s lifespan of 5-6days.

Ferroportin levels are controlled by enhancing it’s degradation when iron stores are adequate. The liver, on sensing adequate iron stores, secretes a peptide hepcidin that binds to ferroportin. Binding of ferroportin to hepcidin causes internalisation and degradation of ferroportin preventing iron transport. Mutations ferroportin, hepcidin and molecules that promote the secretion of hepcidin  (haemachromatosis protein (HFE),  haemojuvelin (HJV), transferrin receptor 2 (TFR2)) result in increased iron absorption and haemachromatosis.  Mutations in TMPRSS6 a negative regulator of hepcidin results in iron refractory iron deficiency anaemia.

Hepcidin and Ferroportin

Iron is an essential micronutrient that is toxic because of its ability to form oxygen free radicals that can damage macromolecules. It is this property of iron that makes it essential for total body iron content be controlled and tissue be protected while iron is being transported from the sources of iron (macrophages and enterocytes) to consumers of iron (erythroid cells and syncytitrophoblast). Iron has no significant excretory pathways controlling absorption is the only way to check against iron overload. Ferroportin the only known transporter of iron and hepcidin a peptide that binds and degrades ferroportin are central to control body iron content. Mutations in both have been implicated in haemochromatosis.


Ferroportin, the product of the SLC40A1 (Solute carrier family 40 (iron-regulated transporter) member 1) gene (OMIM: 604653) situated at chromosome 2q32.2,  is the only known iron transporter. It is a 571 amino acid peptide with a yet to be defined. It is believed to contain between 9-12 transmembrane domains. Ferroportin is expressed on the macrophages, Küpffer cells, hepatocytes, intestinal enterocytes and placental cells. All these cells are involved in iron transport.

Regulation of expression
  1. Transcriptional level: IRE in the untranslated 5′ region. The effect or ion loading is tissue specific. Iron loading increases the expression of ferroportin in liver cells and macrophages . Iron deficiency increases the expression of ferroportin the duodenum. These findings are consistent with the functions of the hepatocytes and macrophages on one hand (releasing iron at a rate dictated by iron deficiency) and the duodenum on the other (transporting iron to the lamina propria in the presence of iron deficiency).
  2. Post-translational Modification: Hepcidin binds to and phosphorylation ferroportin leading to internalisation and  degradation of ferroportin.
Mutations in Ferroportin Gene

Mutations in the Ferroportin Gene result in haemochromatosis type 4 or ferroportin disease.  Mutations result in synthesis of ferroportin molecules that can not bind hepcidin


Hepcidin (Hepcidin Antimicrobial Peptide, HAMP) is an 25 amino acid peptide secreted predominantly by the liver. It is encoded by the HAMP (OMIM 60464)gene located in chromosome 19q13.12. It is synthesised as an 84 amino acid precursor. It has 8 cysteine residues and four disulphide bond. It circulates in the plasma associated with α2-macroglobulin and albumin.

Function of Hepcidin

Hepcidin  was firs identified as an antimicrobial peptide but is now known to the main regulator of iron metabolism. Hepcidin binds ferroportin to bring about its internalisation and degradation. Hepcidin impairs release or iron from the cells.  Hepcidin levels are decreased by depletion of iron stores, phlebotomy, haemolysis and elevated erythropoietin levels. Hepcidin regulates the absorption and distribution of iron

  1. Absorption of Iron: Hepcidin levels increase iron overload resulting in decreased ferroportin expression on the basilateral aspect of the duodenal cell.  The cell is not able to transport absorbed iron to the plasma and addition of more iron to the body iron pool is controlled.
  2. Distribution of Iron: Increased hepcidin in states of iron overload decreases the ferroportin expression of macrophages and hepatocytes. Hepcidin prevents entry of recycled iron from the macrophages and stored iron from the hepatocytes into the plasma.

Mice with disruption id the USF2 gene have a complete suppression of hepcidin levels and develop an illness like human haemochromatosis. Inducing over expression of hepcidin by placing the gene under transgenic control of the liver-specific transthyretin promoter resulted in a mice that were severely anaemic with hypochromia and microcytosis that survived only a few hours (Proc Natl Acad Sci USA2002;99(7):4596-4601).

Regulation of Hepcidin
  1. Positive Regulators: Bone morphogenic Proteins 6 (BPM6), hemojuvelin (HJV), mothers against decapentaplegic homolog 4 (SMAD4), Transferrin receptor 2 (TFR2) and haemochromatois protein (HFE)
  2. Negative Regulators: TMPRSS6
hypochromic Micro Anaemia

Microcytic Hypochromic 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.


Iron Studies in Microcytic Anaemia

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.

Laboratory diagnosis of Iron Deficiency

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.

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]


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
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.

Evaluating Anaemia

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
  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.

Plummer-Vinson Syndrome/Paterson Kelly Syndrome

In 1912 Plummer described dysphagia and spasm of the upper oesophagus without anatomical stenosis in patients with longstanding iron deficiency. In 1919, Vinson, Paterson and Kelly independently described patients with similar findings. The triad of iron deficiency anaemia, dysphagia and oesophageal webs has been called Plummer-Vinson syndrome or Paterson-Kelly syndrome (depending in the side of Atlantic you reside) after these physicians. It is a rare disease possibly related to iron deficiency and is also known as sideropenic dysphagia.

Etiology and Pathogenesis

Etiology of Plummer –Vinson syndrome is not known, iron deficiency is the most likely cause but malnutrition and genetic predisposition may contribute. Plummer-Vinson syndrome has been associated with coeliac disease, thyroiditis and rheumatoid arthritis raising the possibility of autoimmunity playing a role in Plummer-Vinson syndrome.

The oesophagus of patients with Plummer –Vinson syndrome shows webs at the junction of the hypopharynx and oesophagus, usually extends backwards from the anterior wall. The webs may take a cuff shape and the opening may be reduced to a pinhole. Oesophageal stricture may be seen. Histologically the webs are made of normal oesophageal epithelium, sometimes with chronic inflammation.

Clinical Manifestations

Plummer –Vinson syndrome is a mainly affects women in the fourth to the seventh decade of life but has been described in other age groups. The patients present with gradual onset of dysphagia, initially for solids. Examination reveals evidence of iron deficiency including pallor, koilonachia, glossitis and cheilosis. Splenomegaly and thyroid enlargement may occur.


The webs are best demonstrated videofluoroscopically by a barum swallow. Endoscopically they appear as 2-3mm smooth grey thin extension of normal mucosa usually with an eccentric lumen. Endoscopy can rupture the webs and the scope should be introduced under direct vision. Laboratory investigations show hypochromic microcytic anaemia, low transferrin saturation and low serum ferritin characteristic of iron deficiency.


The iron deficiency should be treated. Dysphagia may be treated by dilatation or endoscopic disruption using a biopsy forceps. The prognosis is good. Patinets with Plummer –Vinson syndrome are predisposed to carcinoma of pharynx and oesophagus and a close follow up of patients is needed.