on 01-Apr-2021 (Thu)

For those with microcytic or normocytic anemia, a reticulocyte count should be used to determine whether there is decreased RBC production, which is consistent with iron deficiency; increased RBC destruction (hemolysis); or blood loss.

Review of the peripheral blood smear is likely to provide valuable information regarding the characteristic morphologies seen in iron deficiency anemia ( picture 1) versus other causes of anemia. The history, CBC, RBC indices, and findings on the peripheral blood smear usually allow the clinician to make a presumptive diagnosis of iron deficiency anemia.

There are two complementary ways to confirm (or exclude) the diagnosis of iron deficiency: iron studies (see 'Iron studies (list of available tests)' below) and assessment of the response to a trial of iron therapy (see 'Response to a therapeutic trial of iron' below). In the vast majority of individuals, iron studies should be obtained. The results help to distinguish iron deficiency from other conditions, document the severity of the deficiency (if present), and provide a baseline prior to initiating iron administration.

In many developed countries, iron deficiency is more likely to be due to blood loss in menstruating or pregnant females and adults of either sex, and it is more likely to have a dietary component in children and menstruating women, reflecting an imbalance between physiologic demands and intake, and in vegetarians. In the less developed world, parasitic infestations may be a significant contributing issue [78].

The gold standard for documenting iron deficiency is an iron stain (Prussian blue stain) of a bone marrow aspirate smear to assess iron stores in bone marrow macrophages and erythroid precursors (sideroblasts) on marrow spicules. Lack of stainable iron in erythroid precursors as well as bone marrow macrophages is consistent with iron deficiency, whereas in anemia of chronic disease, increased stainable iron is seen in marrow macrophages but stainable iron is absent or reduced in erythroid precursors (picture 8)

Changes in the CBC occur in proportion to the severity of iron deficiency and tend to lag behind changes in iron studies; reduced storage iron precedes anemia. In turn, a slight decline in hemoglobin (usually 1 to 2 g/dL) precedes microcytosis (table 4). Thus, in early iron deficiency and in many individuals in high-resource settings, the CBC may be relatively normal.

As iron deficiency progresses and the individual becomes anemic, the following findings may be seen on the CBC:

● Low red blood cell (RBC) count (typical RBC count for a patient with a hemoglobin of 9 g/dL would be approximately 3 million cells per microL)

● Low hemoglobin and hematocrit

● Low absolute reticulocyte count

● Low mean corpuscular volume (MCV) and low mean corpuscular hemoglobin (MCH)

The platelet count may be increased in iron deficiency anemia. This is thought to result from stimulation of platelet precursors by erythropoietin.

The low MCV and MCH are reflected on the peripheral blood smear by microcytic, hypochromic RBCs (picture 1). As anemia progresses, increasingly abnormal forms (poikilocytosis) may be seen.

In some studies, a reticulocyte hemoglobin content of <26 pg/cell has correlated well with the finding of iron deficiency [81,82].

Iron deficiency anemia is characterized by reduced or absent iron stores and increased levels of transferrin proteins that facilitate iron uptake and transport to RBC precursors in the bone marrow (table 4). Of these iron studies, ferritin remains the most useful test if low. Otherwise, patients require iron and total iron binding capacity (TIBC), from which one calculates transferrin saturation (TSAT) [15,16,73]. In order to avoid the inconvenience and cost of an additional office visit, our practice is to order all three tests (serum ferritin, iron, and TIBC) in nearly all patients.

Iron can be measured in serum (preferred) or plasma. The test measures circulating iron, most of which is bound to the transport protein transferrin. Serum iron is low in iron deficiency as well as in anemia of chronic disease/anemia of inflammation (ACD/AI). This is because levels of serum iron depend on the efficiency of iron recycling by bone marrow and reticuloendothelial macrophages, which is reduced in both conditions.

Serum iron can also fluctuate with dietary intake and normal diurnal variation. By itself, low serum iron is not diagnostic of any condition but must be evaluated in light of other tests such as transferrin saturation and ferritin. As serum iron may be transiently affected by absorption of dietary or pharmacologic iron, it is recommended that the sample be drawn after an overnight fast.

Transferrin is a circulating transport protein for iron. It is increased in iron deficiency but can be decreased in ACD. Transferrin can also be reported as TIBC. The transferrin concentration (in mg/dL) can be converted to the TIBC (in mcg/dL) by multiplying by 1.389 [73].

Transferrin saturation (TSAT) is the ratio of serum iron to TIBC: (serum iron ÷ TIBC x 100). In iron deficiency, iron is reduced and TIBC is increased, resulting in a lower transferrin saturation. Normal values are in the range of 25 to 45 percent [83,84]. Values below 10 percent are common in individuals with iron deficiency, and a cutoff of below 19 percent is generally used to screen for iron deficiency, although other thresholds may be used in some settings such as pregnancy

Because the TSAT is a ratio, in principle, an increase in the serum iron (eg, due to hemolysis or recent ingestion of an iron tablet) can raise the value, even in an individual who has iron deficiency and an increased TIBC.

Ferritin is a circulating iron storage protein that is increased in proportion to body iron stores. However, ferritin is also an acute phase reactant (see "Acute phase reactants") that can increase independently of iron status in disorders associated with inflammation, infection, liver disease, heart failure, and malignancy [85].

The ferritin concentration that predicts the absence of marrow iron is debated. While many sources use a cutoff level of 12 to 15 ng/mL (99 percent specific but only 57 percent sensitive), our practice is to use a cutoff of 30 ng/mL, which is supported by bone marrow correlations and international guidelines [ 86,87]. The sensitivity and specificity for a cutoff at 30 ng/mL is estimated to be 92 percent and 98 percent, respectively [88]

Soluble transferrin receptor (sTfR), also called circulating transferrin receptor or serum transferrin receptor, is a circulating protein derived from cleavage of the membrane transferrin receptor on bone marrow erythroid precursor cells. Its concentration in serum is directly proportional to erythropoietic rate and inversely proportional to tissue iron availability, similar to serum transferrin [89]. Thus, iron-deficient patients generally have increased levels of sTfR, with reference ranges determined by the individual laboratory performing the testing. Different laboratories may report sTfR as mg/L or as nmol/L. sTfR is not used in routine practice but can be helpful in complex cases (see 'Patients with inconclusive initial testing or comorbidities' below).

The major advantage of sTfR is that it reflects overall erythropoiesis, which is increased in iron deficiency. However, sTfR can be elevated in patients with hemolysis or with administration of erythropoiesis-stimulating agents (ESAs).

sTfR-ferritin index is calculated as the ratio of the sTfR (in mg/L) to the logarithm of the serum ferritin (in mcg/L): (sTfR ÷ log[ferritin]). The sTfR reflects erythropoiesis, while the ferritin reflects the tissue iron stores; thus, a high sTfR-ferritin index (eg, above 2 to 3) is very likely to be a sign of iron deficiency due to increased erythropoietic drive and low iron stores.

Patients with ACD/AI are likely to have an sTfR-ferritin index <1, whereas those with isolated iron deficiency or iron deficiency plus ACD/AI are likely to have an sTfR-ferritin index >2.

In iron deficiency, intestinal zinc absorption increases, and zinc is incorporated into protoporphyrin in developing RBCs. Thus, elevated erythrocyte (RBC) zinc protoporphyrin (eg, >80 mcg/dL) is consistent with iron deficiency. However, zinc protoporphyrin is not specific for iron deficiency as it may be elevated in inflammatory states, hemodialysis, and lead poisoning

The reticulocyte hemoglobin content (CHr, also called Ret-He) is available on some autoanalyzers and has the potential of providing very rapid information about iron status (at same time as CBC), especially in the presence of iron-restricted erythropoiesis [97]. Unlike the serum ferritin, the CHr is not influenced by inflammation. This parameter is used more extensively in individuals with chronic kidney disease; data are lacking regarding its role in managing anemia and iron therapy in other adults.

Recent intake of iron-rich foods or of oral iron supplements (even in multiple vitamins) may affect serum iron and therefore TSAT. For this reason, testing obtained in a fasting state are the most reliable. Ferritin concentration is not affected by food.

● Oral iron can transiently increase the serum iron concentration, with a dose-dependent peak at approximately four hours after the oral dose [98,99]. This could falsely increase the TSAT (which represents a ratio of serum iron to transferrin) and thus make an individual appear to have more normal iron stores than they actually have. Therefore, TSAT should not be drawn immediately after a morning dose of oral iron.

● Intravenous iron also increases serum iron and TSAT, and as a result, may alter the results of iron studies testing, similar to oral iron [100]. After a dose of intravenous iron, we typically wait and retest iron studies at the time of repeat CBC (eg, after approximately four weeks).

Data evaluating the effects of recent blood transfusion on iron studies are limited. One study showed transient elevation of the serum iron and resultant TSAT within the first 24 hours of a blood transfusion [104]. However, other studies have shown only small, subclinical changes in iron parameters.

Individuals without comorbidities — Testing can be done using an iron studies panel, which includes serum iron, transferrin or total iron binding capacity (TIBC), calculated transferrin saturation (TSAT), and ferritin (algorithm 1).

A ferritin level below 30 ng/mL is considered diagnostic of iron deficiency regardless of the patient's underlying condition or hemoglobin concentration. In individuals with anemia, a ferritin <30 ng/mL is sufficient to diagnose iron deficiency. However, a ferritin in the normal range does not exclude iron deficiency in an individual for whom there is a strong suspicion for iron deficiency. A TSAT <19 percent can also be used, as discussed below.

In contrast to a low ferritin, a low serum iron cannot be used to diagnose or exclude iron deficiency. Serum iron may be low in anemia of chronic disease/anemia of inflammation (ACD/AI) or increased by recent ingestion of an iron tablet.

In this review, serum ferritin radioimmunoassay had the greatest predictive value for iron deficiency (figure 4):

● A ferritin level ≤15 ng/mL had a 99 percent specificity for iron deficiency. Ferritin ≤15 ng/mL was highly specific in individuals with inflammatory states.

● A ferritin ≤15 ng/mL had a sensitivity of only 59 percent, meaning that a large proportion of individuals with iron deficiency would be missed.

Using a higher ferritin cutoff may improve sensitivity while maintaining a reasonable specificity. Various observational studies comparing ferritin levels with bone marrow stainable iron have found that a ferritin cutoff of <30 ng/mL provides a sensitivity in the range of 90 to 92 percent and a specificity in the range of 75 to 98 percent [86,110-112].

In some patients the clinical situation is more complex, and additional evaluations and/or management considerations may predominate. Individuals with poorly controlled heart failure or diabetes may require a more thorough assessment of the reasons for poor control; individuals with other unexplained findings (eg, weight loss, adenopathy) may require further diagnostic testing for the cause of their symptoms. In such cases, it may be reasonable to defer a more extensive evaluation for iron deficiency and/or a therapeutic trial of iron until after these other issues are resolved.

The increase in ferritin level conferred by a chronic inflammatory state was demonstrated in a study that retrospectively reviewed records for several thousand patients who had measurements of ferritin as well as C-reactive protein (CRP) and albumin [119]. Median ferritin levels for increasing CRP were as follows:

● CRP <10 mg/L (least inflammation) – ferritin 85 mcg/L

● CRP 10 to 80 mg/L – ferritin 193 mcg/L

● CRP >80 mg/L (greatest inflammation) – ferritin 342 mcg/L

Lower serum albumin levels were also associated with higher serum ferritin levels.

A presumptive diagnosis of iron deficiency anemia may be made using a therapeutic trial of iron in a patient with anemia who has an obvious cause of iron deficiency such as individuals in resource-limited settings where it is not possible to obtain iron studies routinely, or in young women with heavy menstrual periods or pregnancy. In such cases, patients with iron deficiency anemia are expected to have a rapid and complete response to iron administration that includes resolution of symptoms, reticulocytosis, and normalization of hemoglobin level (typically by three weeks).

Additionally, administration of iron to an individual with thalassemia will worsen the existing iron overload commonly seen in this condition. Thus, it may be prudent to obtain iron studies to confirm the diagnosis even in cases where iron deficiency is considered extremely likely. For patients who wish to avoid a return appointment, we find it cost-effective to order iron studies and prescribe iron therapy at the same encounter, with plans to obtain additional testing only if the initial testing was inconclusive.

Diagnosis — We consider the diagnosis of iron deficiency to be confirmed by any one of the following findings in the appropriate clinical setting:

● Serum ferritin <30 ng/mL

● Transferrin saturation <19 percent, mostly used in patients for whom the ferritin is thought to be unreliable due to an inflammatory state

● Anemia that resolves upon iron administration

● Absence of stainable iron in the bone marrow (providing that adequate staining controls are performed)

We diagnose functional iron deficiency in patients with chronic kidney disease or a malignancy who are candidates for treatment with an erythropoiesis-stimulating agent (ESA) if the serum ferritin is in the range of 100 to 500 ng/mL and the transferrin saturation is less than 20 percent

Even before the diagnosis of iron deficiency is confirmed, individuals with suspected iron deficiency should be asked to provide information that might identify the source of the deficiency, which is more likely to be dietary in individuals in resource-poor settings and more likely to be due to blood loss in menstruating or pregnant females and adults of either sex.

This initial evaluation may involve the following:

● Dietary history for infants (eg, use of cow's milk rather than iron-supplemented formula or breastfeeding)

● Menstrual/pregnancy/lactation history for females (table 6)

● History of gastrointestinal blood loss, melena, hematemesis, and hematuria

● History of other gastrointestinal symptoms that might suggest celiac disease, autoimmune gastritis, or H. pylori infection

● History of multiple blood donations

● Marathon running [115]

● Use of non-steroidal anti-inflammatory drugs (NSAIDS) or anticoagulants

● Personal or family history of bleeding diathesis, including platelet disorders, von Willebrand disease, hereditary hemorrhagic telangiectasia

● Personal or family history of celiac disease, colon cancer, or other gastrointestinal disorders

● Review of the results of prior gastrointestinal evaluations (eg, routine colon cancer screening)

● Testing the stool for occult blood in adults 50 years of age or older

The use of an anticoagulant or the presence of severe thrombocytopenia are important to note as they may contribute to bleeding. However, anticoagulation or thrombocytopenia do not diminish the importance of searching for the site(s) of bleeding such as a gastrointestinal or colonic lesion, as these hemostatic changes are often more likely to unmask a bleeding source (and potentially result in earlier diagnosis) than to cause bleeding from a normal mucosa [122].

Celiac disease is important to identify if present; this can present at any age and symptoms may be absent (or may only be appreciated in retrospect). Celiac disease is also a common cause of a lack of response to iron therapy in patients with known iron deficiency because it interferes with iron absorption.

As mentioned above, the prevalence of autoimmune gastritis may be even higher than that of celiac disease, and testing may be appropriate for anti-parietal cell antibodies as well [27].

Deficiencies of vitamin B12 and/or folate can cause megaloblastic anemia (macrocytic anemia with other features due to impaired cell division). Vitamin B12 deficiency can also cause neuropsychiatric findings.

Vitamin B12 (also called cobalamin) is present in many animal products, including meats, dairy products, and eggs [1]. The highest concentrations are in clams and liver, which explains the efficacy of consuming large amounts of liver for the treatment of pernicious anemia before the role of vitamin B12 was discovered [2]. Many breakfast cereals are fortified with vitamin B12. In contrast, vitamin B12 is not present in foods derived from plants, with the exception of those that contain animal products or added vitamin B12. Seaweed is not a source of vitamin B12, although certain edible algae used in Japanese soups may contain some vitamin B12 [3].

Folate (also called vitamin B9 or pteroylglutamic acid) is present in many plant and animal products, especially dark green leafy vegetables and liver [6,7]. Naturally occurring folates include a number of polyglutamated compounds. These are mostly in the reduced form, such as 5-methyltetrahydrofolate (5-methyl-THF) or f-formyltetrahydrofolate (also called folinic acid, which is the active drug in leucovorin) [8]. Folic acid is the term used to refer to the chemically synthesized form of the vitamin, which is used in dietary supplements and fortified foods; folic acid is an oxidized monoglutamate that is more chemically stable than food-derived folates.

The recommended daily intake of folate is expressed in dietary folate equivalents because folic acid is approximately two-fold more bioavailable than naturally occurring folates from foods [9].

Vitamin B12 is a chemically complex molecule. A number of mechanisms ensure its stability and absorption, which is illustrated in the figure (figure 1) [12]:

● Vitamin B12 in foods is protein-bound, and this is dissociated in the acid milieu of the stomach with the help of pepsin.

● Additional vitamin B12-binding proteins known as R-binders or haptocorrins (HC) are secreted in the saliva and bind to the vitamin B12 in the stomach.

● Gastric parietal cells produce intrinsic factor. Pancreatic proteases secreted into the higher pH duodenum cleave off the R-binders, allowing the vitamin B12 to bind intrinsic factor.

● The vitamin B12-intrinsic factor complex is taken up by mucosal receptors in the ileum. The exact nature of the receptor is under investigation; it is thought to be composed of the proteins cubilin and amnionless and was named "cubam" to reflect these components [13-17].

● A small amount of ingested vitamin B12 (<1 percent) can be absorbed by passive diffusion [18]. This is the basis for the use of high-dose oral vitamin B12 in pernicious anemia. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Prevention of vitamin B12 deficiency'.)

● Vitamin B12 is exported into the bloodstream by an ATP-binding cassette protein [19]. The vitamin B12 is bound to a family of transcobalamins sometimes referred to as cobalophilins.

● Vitamin B12 bound to transcobalamin is taken up by other cells throughout the body by receptor-mediated endocytosis.

● Intracellular vitamin B12 is metabolized into adenosylcobalamin or methylcobalamin. The functions of these proteins are described below. (See 'Physiologic roles' below.)

Total body stores of vitamin B12 are in the range of 2 to 5 mg, with approximately half of this stored in the liver. If vitamin B12 intake or absorption ceases, deficiency typically does not develop for at least one to two years, sometimes longer.

Folate polyglutamates are cleaved to monoglutamates and absorbed in the lumen of the jejunum. Absorption is predominately carrier-mediated, but passive absorption also occurs [8,20]. Carrier systems include the reduced folate carrier folate receptors and the proton-coupled folate transporter. Absorption is optimal at slightly acidic pH (pH 5.5 to 6.0).

Folate must be reduced to be effective in its biological role in one-carbon transfer. It is reduced to dihydrofolate and then to tetrahydrofolate (THF) by a series of enzymatic steps. THF is subsequently converted to 5,10-methylene THF, which is converted to L-5-methyl-THF by the enzyme methylenetetrahydrofolate reductase.

The predominant form of folate in plasma is L-5-methyl-THF. This is rapidly cleared by hepatocytes and other cells. Surgical biliary drainage results in a reduction in serum folate within six hours, whereas dietary restriction does not produce a comparable decrease for three weeks [21]. This observation indicates that there is a large enterohepatic circulation of folate.

Folate enters cells by binding to a folate receptor. Megalin is a receptor related to the low-density lipoprotein receptor that can take up various proteins, including folates [13]. Once inside the cell, folate is again polyglutamated to a biologically active form that cannot diffuse back out of the cell [21]. Polyglutamated tetrahydrofolate acts as a one-carbon donor for biologic processes, including synthesis of the purines and pyrimidines used in DNA synthesis. (See 'DNA synthesis, RNA synthesis, DNA methylation' below.)

Total body folate stores are estimated to be approximately 500 to 20,000 mcg (0.5 to 20 mg). If folate intake ceases, deficiency may develop within weeks to months, or more rapidly if demands for folate are increased.