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TESTS USED TO ASSESS IRON STATUS
Iron status can be assessed through several laboratory tests. Because each test assesses a different aspect of iron metabolism, results of one test may not always agree with results of other tests. Hematological tests based on characteristics of red
blood cells (i.e., Hb concentration, hematocrit, mean cell volume, and red blood cell distribution width) are generally more available and less expensive than are biochemical tests. Biochemical tests (i.e., erythrocyte protoporphyrin concentration, serum ferritin concentration, and transferrin saturation), however, detect earlier changes in iron status.
Although all of these tests can be used to assess iron status, no single test is accepted for diagnosing iron deficiency (70). Detecting iron deficiency in a clinical or field setting is more complex than is generally believed.
Lack of standardization among the tests and a paucity of laboratory proficiency testing limit comparison of results between laboratories (71). Laboratory proficiency testing is currently available for measuring Hb concentration, hematocrit, red blood cell count, serum ferritin concentration, and serum iron concentration; provisional proficiency testing was added in 1997 for total iron-binding capacity in the College of American Pathologists survey and was added to the American Association of Bioanalysts survey in 1998. As of April 1998, three states (New York, Pennsylvania, and Wisconsin) had proficiency testing programs for erthrocyte protoporphryin concentration.
Regardless of whether test standardization and proficiency testing become routine, better understanding among health-care providers about the strengths and limitations of each test is necessary to improve screening for and diagnosis of iron-deficiency anemia, especially because the results from all of these tests can be affected by factors other than iron status.
Only the most common indicators of iron deficiency are described in this section.
Other indicators of iron deficiency (e.g., unbound iron-binding capacity and the concentrations of transferrin receptor, serum transferrin, and holo-ferritin) are less often used or are under development.
Hb Concentration and Hematocrit
Because of their low cost and the ease and rapidity in performing them, the tests
most commonly used to screen for iron deficiency are Hb concentration and hematocrit (Hct). These measures reflect the amount of functional iron in the body. The concentration of the iron-containing protein Hb in circulating red blood cells is the more direct and sensitive measure. Hct indicates the proportion of whole blood occupied by the red blood cells; it falls only after the Hb concentration falls. Because changes in Hb concentration and Hct occur only at the late stages of iron deficiency, both tests are late indicators of iron deficiency; nevertheless, these tests are essential for determining iron-deficiency anemia.
Because iron deficiency is such a common cause of childhood anemia, the terms
anemia, iron deficiency, and iron-deficiency anemia are often used interchangeably (3). The only cases of anemia that can be classified as iron-deficiency anemia, however, are those with additional evidence of iron deficiency. The concept of a close association between anemia and iron deficiency is closest to correct when the prevalence of iron deficiency is high. In the United States, the prevalence and severity of anemia have declined in recent years; hence, the proportion of anemia due to causes other than iron deficiency has increased substantially. As a consequence, the effectiveness of anemia screening for iron deficiency has decreased in the United States.
Iron deficiency may be defined as absent bone marrow iron stores (as described on
bone marrow iron smears), an increase in Hb concentration of >1.0 g/dL after iron treatment, or abnormal values on certain other biochemical tests (17). The recent recognition that iron deficiency seems to have general and potentially serious negative effects (3234) has made identifying persons having iron deficiency as important as identifying persons having iron-deficiency anemia.
The case definition of anemia recommended in this report is <5th percentile of the distribution of Hb concentration or Hct in a healthy reference population and is based on age, sex, and (among pregnant women) stage of pregnancy (45,72). This case definition for anemia was shown to correctly identify 37% of women of childbearing age and 25% of children aged 15 years who were iron deficient (defined as two of three positive test results [i.e., low mean cell volume, high erythrocyte protoporphyrin, or low transferrin saturation]) (sensitivity) and to correctly classify 93% of women of childbearing age and 92% of children aged 15 years as not having iron deficiency (specificity) (73). Lowering the Hb concentration or Hct cut-off would result in identifying fewer people who have anemia due to causes other than iron deficiency (false positives) but also in overlooking more people with iron deficiency (true positives) (74).
The distributions of Hb concentration and Hct and thus the cutoff values for anemia differ between children, men, nonpregnant women, and pregnant women and by age or weeks of gestation (Table 6). The distributions also differ by altitude, smoking status, and race.
Among pregnant women, Hb concentration and Hct decline during the first and
second trimesters because of an expanding blood volume (18,3942). Among pregnant women who do not take iron supplements, Hb concentration and Hct remain low in the third trimester, and among pregnant women who have adequate iron intake, Hb concentration and Hct gradually rise during the third trimester toward the prepregnancy levels (39,40). Because adequate data are lacking in the United States, the cutoff values for anemia are based on clinical studies of European women who had taken iron supplementation during pregnancy (3942,72). For pregnant women, a test result >3 standard deviations (SD) higher than the mean of the reference population (i.e., a Hb concentration of >15.0 g/dL or a Hct of >45.0%), particularly in the second trimester, likely indicates poor blood volume expansion (72). High Hb concentration or Hct has been associated with hypertension and poor pregnancy outcomes (e.g., fetal growth retardation, fetal death, preterm delivery, and low birthweight) (7578). In one study, women who had a Hct of  43% at 2630 weeks gestation had more than a twofold increased risk for preterm delivery and a fourfold increased risk for delivering a child having fetal growth retardation than did women who had a Hct of 33%36% (76). Hence, a high Hb concentration or Hct in the second or third trimester of pregnancy should not be considered an indicator of desirable iron status.
Long-term residency at high altitude (3,000 ft) (79) and cigarette smoking (80) cause a generalized upward shift in Hb concentration and Hct (Table 7). The effectiveness of screening for anemia is lowered if the cutoff values are not adjusted for these factors (72,79,80). Adjustment allows the positive predictive value of anemia screening to be comparable between those who reside near sea-level and those who live at
high altitude and between smokers and nonsmokers (72).
In the United States, the distribution of Hb concentration values is similar among whites and Asian Americans (81), and the distribution of Hct values is similar among whites and American Indians (82). The distributions are lower among blacks than
whites, however, even after adjustment for income (83,84). These different distributions are not caused by a difference in iron status indicators (e.g., iron intake, serum ferritin concentration, or transferrin saturation); thus, applying the same criteria for anemia to all races results in a higher rate of false-positive cases of iron deficiency for blacks (84). For example, in the United States during 19761980, 28% of nonpregnant black women but only 5% of nonpregnant white women had a Hb concentration of <12 g/dL and, according to the anemia criteria, would be classified as iron deficient, even
though other tests for iron status suggested these women were not iron deficient (84). For this reason, the Institute of Medicine recommends lowering Hb concentration and Hct cutoff values for black children aged <5 years by 0.4 g/dL and 1%, respectively, and for black adults by 0.8 g/dL and 2%, respectively (5). Because the reason for this disparity in distributions by race has not been determined, the recommendations in this report do not provide race-specific cutoff values for anemia. Regardless, health-care providers should be aware of the possible difference in the positive predictive value of anemia screening for iron deficiency among blacks and whites and consider using other iron status tests (e.g., serum ferritin concentration and transferrin saturation) for their black patients.
Accurate, low-cost, clinic-based instruments have been developed for measuring Hb concentration and Hct by using capillary or venous blood (85,86). Small diurnal variations are seen in Hb concentration and Hct measurements, but these variations are neither biologically nor statistically significant (87,88). A potential source of error of using capillary blood to estimate Hb concentration and Hct in screening is improper sampling technique. For example, excessive squeezing (i.e., "milking") of the finger contaminates the blood with tissue fluid, leading to false low readings (89). Confirmation of a low reading is recommended by obtaining a second capillary blood sample from the finger or by venipuncture.
Although measures of Hb concentration and Hct cannot be used to determine the cause of anemia, a diagnosis of iron-deficiency anemia can be made if Hb concentration or Hct increases after a course of therapeutic iron supplementation (23,51). Alternatively, other laboratory tests (e.g., mean cell volume, red blood cell distribution width, and serum ferritin concentration) can be used to differentiate iron-deficiency
anemia from anemia due to other causes.
In the United States in recent years, the usefulness of anemia screening as an indicator of iron deficiency has become more limited, particularly for children. Studies using transferrin saturation (a more sensitive test for iron deficiency) have documented that iron deficiency in most subpopulations of children has declined such that screening by Hb concentration no longer efficiently predicts iron deficiency (3,45,51,90) . Data from NHANES II, which was conducted during 19761980, indicated
that <50% of children aged 15 years and women in their childbearing years who had anemia (as defined by Hb concentration <5th percentile) were iron deficient (i.e., had at least two of the following: low mean cell volume, high erythrocyte protoporphyrin concentration, or low transferrin saturation) (70,73,83). Causes of anemia other than iron deficiency include other nutritional deficiencies (e.g., folate or vitamin B12 deficiency), hereditary defects in red blood cell production (e.g., thalassemia major and sickle cell disease), recent or current infection, and chronic inflammation (91). The current pattern of iron-deficiency anemia in the United States (28,45) indicates that selective anemia screening of children at known risk for iron deficiency or additional measurement of indicators of iron deficiency (e.g., erythrocyte protoporphyrin concentration and serum ferritin concentration) to increase the positive predictive value of screening are now suitable approaches to assessing iron deficiency among most U.S. children (3,73). The costs and feasibility of screening using additional indicators of iron deficiency may preclude the routine use of these indicators.
Mean Cell Volume
Mean cell volume (MCV), the average volume of red blood cells, is measured in femtoliters (10-15 liters). This value can be calculated as the ratio of Hct to red blood cell count or measured directly using an electronic counter. MCV is highest at birth, decreases during the first 6 months of life, then gradually increases during childhood to adult levels (23,51). A low MCV corresponds with the 5th percentile for age for the
reference population in NHANES III (28).
Some anemias, including iron-deficiency anemia, result in microcytic red blood cells; a low MCV thus indicates microcytic anemia (Table 8). If cases of lead poisoning and the anemias of infection, chronic inflammatory disease, and thalassemia minor
can be excluded, a low MCV serves as a specific index for iron-deficiency anemia (28,87,94,95).
Red Blood Cell Distribution Width
Red blood cell distribution width (RDW) is calculated by dividing the SD of red blood cell volume by MCV and multiplying by 100 to express the result as a percentage:
RDW (%) = [SD of red blood cell volume (fL)/MCV (fL)] × 100
A high RDW is generally set at >14.0%, which corresponds to the 95th percentile of RDW for the reference population in NHANES III (20). The RDW value obtained depends on the instrument used (51,95).
An RDW measurement often follows an MCV test to help determine the cause of a low MCV. For example, iron-deficiency anemia usually causes greater variation in red blood cell size than does thalassemia minor (96). Thus, a low MCV and an RDW of >14.0% indicates iron-deficiency anemia, whereas a low MCV and an RDW  14.0% indicates thalassemia minor (51).
Erythrocyte Protoporphyrin Concentration
Erythrocyte protoporphyrin is the immediate precursor of Hb. The concentration of erythrocyte protoporphyrin in blood increases when insufficient iron is available for Hb production. A concentration of >30 µg/dL of whole blood or >70 µg/dL of red blood cells among adults and a concentration of >80 µg/dL of red blood cells among children aged 12 years indicates iron deficiency (28,45,91). The normal range of erythrocyte
protoporphyrin concentration is higher for children aged 12 years than for adults, but no consensus exists on the normal range for infants (28,90). The sensitivity of free erythrocyte protoporphyrin to iron deficiency (as determined by response to iron therapy) in children and adolescents aged 6 months17 years is 42%, and the estimated specificity is 61% (74).
Infection, inflammation, and lead poisoning as well as iron deficiency can elevate erythrocyte protoporphyrin concentration (23,92). This measure of iron status has several advantages and disadvantages relative to other laboratory measures. For example,
the day-to-day variation within persons for erythrocyte protoporphyrin concentration is less than that for serum iron concentration and transferrin saturation (87). A high erythrocyte protoporphyrin concentration is an earlier indicator of iron-deficient erythropoiesis than is anemia, but it is not as early an indicator of low iron stores as is low serum ferritin concentration (30). Inexpensive, clinic-based methods have been developed for measuring erythrocyte protoporphyrin concentration, but these methods can be less reliable than laboratory methods (92).
Serum Ferritin Concentration
Nearly all ferritin in the body is intracellular; a small amount circulates in the plasma. Under normal conditions, a direct relationship exists between serum ferritin concentration and the amount of iron stored in the body (97), such that 1 µg/L of
serum ferritin concentration is equivalent to approximately 10 mg of stored iron (98). In the United States, the average serum ferritin concentration is 135 µg/L for men (28), 43 µg/L for women (28), and approximately 30 µg/L for children aged 624 months (23).
Serum ferritin concentration is an early indicator of the status of iron stores and is the most specific indicator available of depleted iron stores, especially when used in conjunction with other tests to assess iron status. For example, among women who
test positive for anemia on the basis of Hb concentration or Hct, a serum ferritin concentration of 15 µg/L confirms iron deficiency and a serum ferritin concentration of >15 µg/L suggests that iron deficiency is not the cause of the anemia (93). Among women of childbearing age, the sensitivity of low serum ferritin concentration (15 µg/L) for iron deficiency as defined by no stainable bone marrow iron is 75%, and the specificity is 98%; when low serum ferritin concentration is set at <12 µg/L, the sensitivity for iron deficiency is 61% and the specificity is 100% (93). Although low serum ferritin concentration is an early indicator of low iron stores, it has been questioned whether a normal concentration measured during the first or second trimester of pregnancy can predict adequate iron status later in pregnancy (6).
The cost of assessing serum ferritin concentration and the unavailability of clinic-based measurement methods hamper the use of this measurement in screening for iron deficiency. In the past, methodological problems have hindered the comparability
of measurements taken in different laboratories (87), but this problem may be reduced by proficiency testing and standardized methods. Factors other than the level of stored iron can result in large within-individual variation in serum ferritin concentration (99). For example, because serum ferritin is an acute-phase reactant, chronic infection, inflammation, or diseases that cause tissue and organ damage (e.g., hepatitis,
cirrhosis, neoplasia, or arthritis) can raise its concentration independent of iron status (97). This elevation can mask depleted iron stores.
Transferrin Saturation
Transferrin saturation indicates the extent to which transferrin has vacant iron-binding sites (e.g., a low transferrin saturation indicates a high proportion of vacant iron-binding sites). Saturation is highest in neonates, decreases by age 4 months, and
increases throughout childhood and adolescence until adulthood (23,28). Transferrin saturation is based on two laboratory measures, serum iron concentration and total iron-binding capacity (TIBC). Transferrin saturation is calculated by dividing serum iron concentration by TIBC and multiplying by 100 to express the result as a percentage:
Transferrin saturation (%) = [serum iron concentration (µg/dL)/TIBC (µg/dL)] × 100
Serum iron concentration is a measure of the total amount of iron in the serum and is often provided with results from other routine tests evaluated by automated, laboratory chemistry panels. Many factors can affect the results of this test. For example, the concentration of serum iron increases after each meal (71), infections and inflammations can decrease the concentration (69), and diurnal variation causes the concentration to rise in the morning and fall at night (100). The day-to-day variation of serum iron concentration within individuals is greater than that for Hb concentration and Hct (88,101).
TIBC is a measure of the iron-binding capacity within the serum and reflects the availability of iron-binding sites on transferrin (94). Thus, TIBC increases when serum iron concentration (and stored iron) is low and decreases when serum iron concentration (and stored iron) is high. Factors other than iron status can affect results from this test. For example, inflammation, chronic infection, malignancies, liver disease, nephrotic syndrome, and malnutrition can lower TIBC readings, and oral contraceptive use and pregnancy can raise the readings (87,102). Nevertheless, the day-to-day variation is less than that for serum iron concentration (87,101). TIBC is less sensitive to iron deficiency than is serum ferritin concentration, because changes in TIBC occur after iron stores are depleted (17,31,94).
A transferrin saturation of <16% among adults is often used to confirm iron deficiency (93). Among nonpregnant women of childbearing age, the sensitivity of low transferrin saturation (<16%) for iron deficiency as defined by no stainable bone marrow iron is 20%, and the specificity is 93% (93).
The factors that affect serum iron concentration and TIBC, such as iron status, diurnal variation (87,103), and day-to-day variation within persons (101), can affect the measured transferrin saturation as well. The diurnal varation is larger for transferrin saturation than it is for Hb concentration or Hct (87,103). Transferrin saturation is an indicator of iron-deficient erythropoiesis rather than iron depletion; hence, it is less sensitive to changes in iron stores than is serum ferritin concentration (30,31). The cost of assessing transferrin saturation and the unavailability of simple, clinic-based methods for measuring transferrin saturation hinder the use of this test in screening for iron deficiency.
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