Articles on Medical Diseases and Conditions

Tag: Chronic Iron Deficiency

  • Hemoglobin Synthesis Abnormalities

    Thalassemia

    Strictly speaking, there is no thalassemia hemoglobin. Thalassemia comprises a complex group of genetic abnormalities in globin chain synthesis. There are three major clinical categories: thalassemia major, associated with severe and often life-threatening clinical manifestations; thalassemia minor, with mild or minimal clinical manifestations; and a combination of the thalassemia gene with a gene for another abnormal hemoglobin.

    The genetic situation is much more complicated than arbitrary subdivision into the major clinical syndromes would imply.

    The globin portion of normal hemoglobin (Hb A1) is composed of two pairs of polypeptide (amino acid) chains, one pair (two chains) called alpha (a) and the other two chains called beta (b). All normal hemoglobins have two alpha chains, but certain hemoglobins have either one or both beta chains that have a polypeptide amino acid sequence different from usual beta chains. Thus, Hb A2 has two delta (d) chains, and Hb F has two gamma (g) chains in addition to the two alphas. All three of these hemoglobins (A1, A2, and F) are normally present in adult RBCs, but A2 and F normally are present only in trace amounts. One polypeptide chain from each pair of alpha and beta chains is inherited from each parent, so that one alpha chain and one beta chain are derived from the mother and the other alpha and beta chain from the father. The thalassemia gene may involve either the alpha or the beta chain. In the great majority of cases, the beta chain is affected; genetically speaking, it would be more correct to call such a condition a beta thalassemia. If the condition is heterozygous, only one of the two beta chains is affected; this usually leaves only one beta chain (instead of two) available for Hb A1 synthesis and yields a relative increase in Hb A2. This produces the clinical picture of thalassemia minor. In a homozygous beta thalassemia, both of the beta chains are affected; this apparently results in marked suppression of normal Hb A1 synthesis and leads to a compensatory increase in gamma chains; the combination of increased gamma chains with the nonaffected alpha chains produces marked elevation of Hb F. This gives the clinical syndrome of thalassemia major. It is also possible for the thalassemia genetic abnormality to affect the alpha chains. The genetics of alpha thalassemia are more complicated than those of beta thalassemia, because there are two alpha-globin gene loci on each of the two globin-controlling body chromosomes, whereas for beta-globin control there is one locus on each of the two chromosomes. In alpha thalassemia trait (“silent carrier”), one gene locus on one chromosome only is deleted or abnormal (one of the four loci). In alpha thalassemia minor, two of the four loci are affected. This may be produced either by deletion or abnormality in both loci on one of the two chromosomes (a genotype called alpha-thalassemia-1, more common in Asians), or by deletion of one of the two loci on each of the two chromosomes (a genotype called alpha-thalassemia-2, more common in Africans and those of Mediterranean descent). Hemoglobin A1 production is mildly curtailed, but no Hb A2 or F increase occurs because they also need alpha chains. Hemoglobin H disease results from deletion or inactivation of three of the four loci. All four globin chains of Hb H are beta chains. Hemoglobin H disease occurs mostly in Asians but occasionally is found in persons from the Mediterranean area. The most serious condition is that resulting from deletion or inactivation of all four alpha-globin gene loci; if this occurs, the hemoglobin produced is called Bart’s hemoglobin, and all four globin chains are the gamma type. In most cases functional hemoglobin production is curtailed enough to be lethal in utero or neonatal life. Another abnormal hemoglobin that should be mentioned is Hb-Lepore, which is called a fusion hemoglobin, and which consists of two normal alpha chains and two nonalpha fusion chains, each containing the amino terminal portion of a delta chain joined to the carboxy terminal portion of a beta chain.

    Thalassemia major (Cooley’s anemia). This globin variant is the homozygous form of beta thalassemia and consists of two alpha chains and two gamma chains. The condition generally does not become evident until substantial changeover to adult hemoglobin at about 2-3 months of age and clinically is manifest by severe hemolytic anemia and a considerable number of normoblasts in the peripheral blood. The nucleated RBCs are most often about one third or one half the number of WBCs but may even exceed them. There are frequent Howell-Jolly bodies and considerable numbers of polychromatophilic RBCs. The mature RBCs are usually very hypochromic, with considerable anisocytosis and poikilocytosis, and there are moderate numbers of target cells. The mean corpuscular volume (MCV) is microcytic. WBC counts are often mildly increased, and there may be mild granulocytic immaturity, sometimes even with myelocytes present. Platelets are normal. Skull x-ray films show abnormal patterns similar to those in sickle cell anemia but even more pronounced. Death most often occurs in childhood or adolescence.

    Diagnosis. Diagnosis is suggested by a severe anemia with very hypochromic RBCs, moderate numbers of target cells, many nucleated RBCs, and a family history of Mediterranean origin. The sickle preparation is negative. Definitive diagnosis depends on the fact that in thalassemia major, Hb F is elevated (10%-90% of the total hemoglobin, usually >50%). Hemoglobin F has approximately the same migration rate as Hb A1 on paper electrophoresis but may be separated by other electrophoretic techniques. In addition, Hb F is much more resistant to denaturation by alkali than is Hb A1. This fact is utilized in the alkali denaturation test. The hemoglobin solution is added to a certain concentration of sodium hydroxide (NaOH), and after filtration the amount of hemoglobin in the filtrate (the undenatured hemoglobin) is measured and compared with the original total quantity of hemoglobin. One report cautions that if the RBCs are not washed sufficiently before the hemoglobin solution is prepared, reagents of some manufacturers may produce a false apparent increase in Hb F.

    The gene producing the Mediterranean type of homozygous beta thalassemia does not synthesize any beta chains; sometimes referred to as b°. When homozygous beta thalassemia occurs in African Americans, the gene is apparently slightly different because small amounts of beta chains may be produced as well as the predominant gamma chains. This gene is referred to as b+, and the degree of anemia tends to be less severe than in the Mediterranean b° type.

    Thalassemia minor. This clinical subgroup of the thalassemias is most frequently the heterozygous form of beta thalassemia (beta thalassemia trait). Besides a relatively high incidence (1%-10%) in Americans of Mediterranean extraction, there is an estimated frequency of 1% (0.5%-2%) in African Americans. About 75% of patients have anemia, which is usually mild; fewer than 10% of patients with anemia have hemoglobin levels less than 10 gm/100 ml. Patients with beta thalassemia trait have microcytic MCV values in a great majority of cases (87%-100%) whether or not anemia is present. The mean corpuscular hemoglobin (MCH) value is also decreased below reference range in almost all cases. Peripheral blood smears typically contain hypochromic and somewhat microcytic RBCs, usually with some target cells and frequently with some RBCs containing basophilic stippling. Nucleated RBCs are not present. The reticulocyte count is frequently elevated (50% of patients in one study had reticulocytosis >3%).

    The main laboratory abnormality in beta thalassemia trait is an increased amount of Hb A2 (A2 is not elevated in alpha thalassemia trait). As noted previously, A2 is a variant of adult Hb A and is normally present in quantities up to 2.5% or 4%, depending on the method used. In beta thalassemia trait, A2 is elevated to some degree with a maximum of approximately 10%. (If >10% is reported using cellulose acetate electrophoresis, this suggests another Hb migrating in the A2 area, such as Hb C.) Hemoglobin F is usually normal but can be present in quantities up to 5%. Hemoglobin A2 cannot be identified on paper electrophoresis, and demonstration or quantitation necessitates cellulose acetate or polyacrylamide gel electrophoresis or resin column methods. DNA probe tests are available in some university centers for diagnosis of beta thalassemia.

    Thalassemia minor due to alpha thalassemia trait is probably more common in the United States than previously recognized. There is a relatively high incidence in persons of Southeast Asian origin and in African Americans. (Limited studies have detected 6%-30% affected persons from African Americans.) There is also a significant incidence in persons of Mediterranean extraction. The majority of affected persons do not exhibit any clinical symptoms of any anemia, and of the minority that do, symptoms of anemia are most often relatively mild. In one limited study of African Americans in Los Angeles, the majority of affected persons had decreased MCV but only about 10% were anemic. The average MCH value was about 2% less than the average value for normal persons, and the average MCH concentration (MCHC) was about the same as that of normal persons. Hemoglobin H disease can be detected by appropriate hemoglobin electrophoretic techniques. Hemoglobin H disease is mostly restricted to Asians and is manifest by a chronic hemolytic anemia of moderate degree (which may, however, be mild rather than moderate). There is also an acquired form of Hb H disease, reported in some patients with myelodysplastic or myeloproliferative syndromes.

    Currently there is no easy laboratory method to diagnose genetic alpha-globin abnormality. However, in the newborn’s cord blood, Bart’s hemoglobin is generally elevated (by electrophoresis) in rough proportion to the severity of the alpha thalassemia syndrome. Bart’s hemoglobin thus constitutes about 25% (range 20%-40%) in Hb H disease, about 5% (range 2%-10%) in alpha thalassemia trait, and about 1%-2% in the silent carrier state. After about 4 months (range 4-6 months) of age, Bart’s hemoglobin has mostly disappeared. Thereafter, globin chain synthesis studies or DNA probe techniques are the current methods used, but these techniques are available only in research laboratories. Hemoglobin H inclusions in RBCs can be seen using standard reticulocyte count methods, but their sensitivity is disputed (especially in alpha thalassemia trait), possibly due in part to differences in methodology.

    Thalassemia minor vs. chronic iron deficiency. Thalassemia minor must sometimes be differentiated from iron deficiency anemia because of the hypochromic-microcytic status of the RBCs. Certain guidelines have been suggested to increase suspicion for thalassemia minor or to presumptively rule it out in patients with microcytic anemia. The RBC distribution width (RDW; Chapter 2) is usually normal in uncomplicated thalassemia minor and elevated in chronic iron deficiency anemia. Uncomplicated thalassemia minor typically has an RBC count greater than 5 million/mm3 in spite of decreased MCV. Uncomplicated thalassemia minor very uncommonly has a hemoglobin level less than 9.0 gm/100 ml (90 g/L) and usually has a MCHC of 30% or greater (adult reference range, 33%-37% or 330-370 g/L), whereas 50% of patients with chronic iron deficiency anemia have a hemoglobin level less than 9.0 gm (90 g/L), and 50% or more have an MCHC less than 30% (300 g/L). There are also several formulas to segregate thalassemia trait from chronic iron deficiency, of which the best known is the discriminant function of England and Frazer. Unfortunately, there is enough overlap to severely limit the usefulness of these formulas for any individual patient. Serum iron is usually decreased in uncomplicated chronic iron deficiency anemia or anemia of chronic disease and is usually normal in uncomplicated beta thalassemia trait. An elevated total iron-binding capacity (TIBC) suggests iron deficiency, and a decreased TIBC suggests chronic disease. Serum ferritin is decreased in uncomplicated chronic iron deficiency and is normal in uncomplicated beta thalassemia trait. Bone marrow iron is absent in iron deficiency and normal or increased in thalassemia trait. The word “uncomplicated” is stressed because patients with beta thalassemia trait may have concurrent chronic iron deficiency and because anemia of chronic disease may be concurrent with either condition. The anemia of chronic disease may itself be microcytic and hypochromic in about 10% of cases.

    Definitive diagnosis of beta thalassemia trait involves measurement of Hb A2, which is increased in uncomplicated beta thalassemia trait but not in chronic iron deficiency or chronic disease. However, chronic iron deficiency decreases Hb A2 levels so that iron deficiency coexistent with beta thalassemia trait could lead to falsely normal Hb A2 results. In one study, 15% of children with beta thalassemia minor initially displayed normal A2 levels that became elevated after 2 months of therapy with oral iron. Conditions other than beta thalassemia that raise Hb A2 levels include folate or B12 deficiency, increase in Hb F level, and the presence of certain abnormal hemoglobins that migrate with A2, the particular variety of interfering hemoglobin being dependent on the A2 assay method used.

    Sickle thalassemia. This combination produces a condition analogous to SC disease, clinically similar in many respects to SS anemia but considerably milder. Sickle cell test results are positive. There frequently are considerable numbers of target cells. Approximately 60%-80% of the hemoglobin is Hb S. In the S-thalassemia b° (S-Thal-b°) type, most of the remaining hemoglobin is Hb F, so that the pattern resembles SS anemia. However, S-Thal-b° is clinically milder, and the peripheral smear may display RBCs that are more hypochromic than one would expect with SS disease. Also, Hb A2 is increased. The S-thalassemia b+ (S-Thal-b+) pattern might be confused with the SA pattern of sickle trait. However, in sickle trait Hb A predominates rather than Hb S, so that an electrophoretic SA pattern in which more than 50% of the total Hb is Hb S suggests S-thalassemia.

    Screening for thalassemia. Several reports indicate that a decreased MCV detected by automated hematology cell counters is a useful screening method for thalassemia as well as for chronic iron deficiency anemia. One report suggests an 85% chance of thalassemia when there is a combination of MCV of less than 75 femtoliters (fL) with a RBC count of more than 5 million/mm3 (5 Ч 106/L). A hemoglobin level less than 9.0 gm/100 ml (90 g/L) plus an MCHC less than 30% suggests chronic iron deficiency rather than thalassemia. As noted previously, cord blood has been used for detection of the alpha thalassemias. It is now becoming possible to screen for homozygous hemoglobinopathies and severe forms of thalassemia in utero employing DNA analysis of a chorionic villus biopsy at 8-10 weeks or amniotic fluid cells from amniocentesis at 16-18 weeks.

  • Diagnosis of Chronic Iron Deficiency

    The usual signals of iron deficiency are a decreased MCV (or anemia with a low-normal MCV) or elevated RDW. Hypochromia with or without microcytosis on peripheral blood smear is also suspicious. Conditions frequently associated with chronic iron deficiency (e.g., malabsorption, megaloblastic anemia, pregnancy, infants on prolonged milk feeding) should also prompt further investigation. The major conditions to be considered are chronic iron deficiency, thalassemia minor, and anemia of chronic disease. The most frequently used differential tests are the serum iron plus TIBC (considered as one test) and the serum ferritin. Although the serum ferritin test alone may be diagnostic, the test combination is frequently ordered together to save time (since the results of the serum ferritin test may not be conclusive), to help interpret the values obtained, and to provide additional information. Low serum iron levels plus low TIBC suggests chronic disease effect (Table 3-2 and Table 37-2). Low serum iron levels with high-normal or elevated TIBC suggest possible iron deficiency. If the serum iron level and %TS are both low, it is strong evidence against thalassemia minor. Laboratory tests for thalassemia minor are discussed elsewhere in more detail. If the serum ferritin level is low, this is diagnostic of chronic iron deficiency and excludes both thalassemia and anemia of chronic disease, unless they are coexisting with iron deficiency or the ferritin result was a lab error. If iron deficiency is superimposed on another condition, the iron deficiency can be treated first and the other condition diagnosed later. In some cases there may be a question whether chronic iron deficiency is being obscured by chronic disease elevation of the serum ferritin value. In some of these instances there may be indication for a bone marrow aspiration or a therapeutic trial of oral iron.

    Since RBC indices and peripheral blood smear may appear to be normal in some patients with chronic iron deficiency, it may be rational and justifiable to perform a biochemical screening test for iron deficiency (serum iron or serum ferritin) in patients with normocytic-normochromic anemia. In fact, no single biochemical test, not even serum ferritin determination, will rule out iron deficiency simply because the test result is normal. In 42 of our patients with chronic iron deficiency anemia, 9% had an MCV of 90-100 fL and 45% overall had a normal MCV. Fifteen percent had normal serum iron levels and 65% had normal TIBC. Ten percent had a serum iron level and TIBC pattern suggestive of anemia of chronic disease (presumably coexistent with the iron deficiency). Thirty percent had a serum ferritin level greater than 15 ng/100 ml (although all but one were less than 25 ng).

  • Laboratory Tests for Chronic Iron Deficiency

    Several laboratory tests are commonly used to screen for or establish a diagnosis of chronic iron deficiency. The sequence in which abnormal test results appear is given in the box below. In a normal adult on a normal diet made iron deficient by repeated phlebotomy (for experimental reasons), it takes about 3 months before significant anemia (Hb more than 2 gm/dl below normal) appears. The first laboratory indication of iron deficiency is lack of marrow iron on bone marrow aspiration. The next test to become abnormal is the serum iron level. When anemia becomes manifest, it is moderately hypochromic but only slightly microcytic; marked hypochromia and microcytosis are relatively late manifestations of iron deficiency. When the anemia is treated, results of these tests return to normal in reverse order. Even with adequate therapy it takes several months before bone marrow iron appears again.

    Peripheral blood smear. The peripheral blood smear in chronic iron deficiency anemia typically shows RBC hypochromia. There is also microcytosis, but lesser degrees of microcytosis are more difficult to recognize than hypochromia. Some of the peripheral blood changes may appear before actual anemia. However, examination of the peripheral blood smear cannot be depended on to detect iron deficiency, since changes suggestive of chronic iron deficiency either may not be present or may be missed. In one study the peripheral blood smear did not show typical RBC changes in as many as 50% of patients with chronic iron deficiency anemia. This happens more often in patients with mild anemia. Even if hypochromia is present, iron deficiency must be differentiated from other conditions that also may produce hypochromic RBC. In severe anemia there is anisocytosis and poikilocytosis in addition to microcytosis and rather marked hypochromia. The anisocytosis means that the RBCs may not all be microcytes. The microcytes of iron deficiency must be differentiated from spherocytes; such distinction is usually not difficult, since in chronic iron deficiency even the microcytes are hypochromic. Bone marrow aspiration reveals mild erythroid hyperplasia and no marrow iron (using iron stains).

    Hypochromic anemias

    Table 3-1 Hypochromic anemias

    Sequence of Test Abnormalities in the Evolution of Chronic Iron Deficiency

    EARLY PRECLINICAL CHANGES
    Negative iron balance
    Decreased bone marrow hemosiderin
    Decreased serum ferritin

    LATER PRECLINICAL CHANGES
    Increased RBC protoporphyrin levels
    Increased total iron-binding capacity
    Decreased serum iron

    RELATIVELY LATE CHANGES
    RBC microcytosis
    RBC hypochromia
    Anemia

    Red blood cell indices. The mean corpuscular volume (MCV) typically is decreased below reference range lower limits, and the RBC distribution width (RDW) is increased by the time iron deficiency anemia has appeared. However, the few studies available indicate that the MCV is normal in about 30%5% (range 24%-55%) of patients. The mean corpuscular hemoglobin (MCH) value is normal in about 20%. There is considerable disagreement on MCH concentration (MCHC) values, with a decrease reported in 21%-81% of patients. Although various factors could have biased these studies, it is probable that chronic iron deficiency will not be detected by RBC indices in a significant number of patients with chronic iron deficiency anemia. Some of these patients, but not all, may have other superimposed conditions that mask the morphologic effects of iron deficiency. Even if the MCV is decreased, iron deficiency must be differentiated from various other conditions that also produce microcytosis.

    Reticulocytes. The reticulocyte count is normal in uncomplicated chronic iron deficiency anemia. Superimposed acute blood loss or other factors, such as adequate iron in the hospital diet, may cause reticulocytosis. For a short time following recent (acute) hemorrhage, the Wintrobe MCV may be normal or even increased due to the reticulocytosis. The reticulocyte response to iron therapy (3%-7%) is somewhat less than that seen with treatment of megaloblastic anemia.

    Serum iron. Serum iron levels fall sometime between depletion of tissue iron stores and development of anemia. Therefore, the serum iron value should be a sensitive indicator of possible iron deficiency by the time a patient has anemia. Unfortunately, about 10%-15% (literature range 0%2%) of serum iron measurements in patients with iron deficiency anemia remain in the lower half of the reference range.

    CONDITIONS THAT AFFECT SERUM IRON LEVELS. The first is transferrin levels. Serum iron measurement predominantly reflects iron bound to serum proteins. Under usual conditions, most iron is bound to transferrin. Normally, transferrin is about one-third saturated. Therefore, serum iron values depend not only on the quantity of iron available but also on the amount of transferrin present. (If transferrin is increased, the serum iron measurement reflects not only the quantity of iron bound to the normal amount of protein but also the iron bound to the additional protein. The opposite happens when transferrin is decreased.) Second is the time of day. There is a 20%0% diurnal variation in serum iron levels (literature range 2%-69%); the time of day at which the peak value appears is most often in the morning, but it may occur in the early or late afternoon. In one study the peak was found at 8 A in 72% of 25 patients and at 4 P in 28%. Therefore, in some patients the time of day that the specimen is obtained can materially influence whether a result is interpreted as mildly decreased or still within the lower reference range. Third, it has also been found that serum iron displays considerable day-to-day variation among individuals, with changes averaging 20%0% but in some cases varying over 100%. Finally, in some cases there may be some degree of iron contamination of laboratory materials.

    SERUM IRON DECREASE IN VARIOUS CONDITIONS. Serum iron levels may be decreased in other conditions besides iron deficiency; the most frequent is probably the anemia associated with severe chronic disease such as the rheumatoid-collagen diseases, extensive malignancy, uremia, cirrhosis, and severe chronic infection (Table 3-1). There is usually a slight increase in serum iron levels in the first trimester of pregnancy, since increased estrogens tend to increase transferrin. However, by the third trimester the effect of estrogens is reversed, partially by hemodilution but also from utilization of maternal iron by the fetus. This leads to a decrease in serum iron in the third trimester. Severe stress (surgery, infection, myocardial or cerebral infarction) frequently produces a considerable decrease in serum iron (in one study by an average of 65% with a range of 38%-93%), which begins within 24 hours of the onset of the stress (sometimes as early as 4-6 hours). Its nadir occurs between 24 and 48 hours, and recovery begins toward baseline about 6-7 days after the original decrease.

    SERUM IRON INCREASE. Serum iron levels may be increased in hemolytic anemia, iron overload conditions, estrogen therapy (due to an increase in transferrin), acute hepatitis, and parenteral iron therapy. The effects of intramuscular iron-dextran (Imferon) administration persist for several weeks. The serum iron level is normal or increased in thalassemia minor without coexisting iron deficiency(Table 3-2 and Table 37-2).

    Serum iron not total iron-binding capacity patterns

    Table 3-2 Serum iron not total iron-binding capacity patterns

    SERUM IRON IN MEGALOBLASTIC ANEMIA. When megaloblastic anemia is treated, the serum iron level temporarily falls resulting from marked utilization of previously unused available iron. On the other hand, a significant minority of patients with megaloblastic anemia (20%-40%) have coexisting iron deficiency that eventually will be unmasked by correction of the folate or B12 deficiency. Since megaloblastic anemia can interfere with interpretation of tests for iron deficiency, it has been recommended that follow-up studies be done 1 months after the beginning of folate or B12 therapy to rule out iron deficiency.

    Serum total iron-binding capacity. Serum total iron-binding capacity (TIBC) is an approximate estimate of serum transferrin. Assay is usually performed by adding an excess of iron to serum to saturate serum transferrin, removing all iron not bound to protein, and then measuring the serum iron (which is assumed to be mostly bound to transferrin under these conditions). Since transferrin is not the only protein that can bind iron, the TIBC is not an exact measurement of transferrin and tends to be even less representative in cases of iron overload and certain other conditions.

    Serum TIBC is increased in uncomplicated chronic iron deficiency, most studies indicating abnormality at the same time as a decrease in serum iron levels or even before. Unfortunately, the TIBC is not elevated above reference limits in 30%-40% (29%-68%) of patients with chronic iron deficiency anemia. In the best-known study published, 69% of iron deficiency anemia patients with low serum iron levels had an elevated TIBC, 11% had a TIBC within reference limits, and an additional 21% had decreased TIBC values. Transferrin is a “negative” acute-phase reaction protein and decreases both with various acute diseases and with severe chronic diseases (the same chronic diseases that decrease serum iron levels). Decrease in transferrin depresses TIBC to low or low-normal levels. Hypoproteinemia and iron overload conditions are also associated with a decreased TIBC. Unfortunately, conditions that decrease TIBC can mask the TIBC elevation of coexisting chronic iron deficiency. Some conditions increase transferrin levels and therefore increase TIBC; these include pregnancy, estrogen therapy, alcoholism, and acute hepatitis (Table 3-2 and Table 37-2).

    Transferrin saturation. The textbook pattern of iron tests in chronic iron deficiency shows a decrease in serum iron levels and an increase in TIBC. This will increase the unsaturated binding capacity of transferrin and decrease the percent of transferrin that is bound to iron (percent transferrin saturation, or %TS). A %TS of 15% or less is the classic finding in chronic iron deficiency anemia. The %TS is said to be a more sensitive screening test for chronic iron deficiency than either serum iron levels or the TIBC, since a decreased serum iron level that still remains in the lower end of the reference range plus a TIBC still in the upper end of the TIBC reference range may produce a %TS below 15%. A decrease in %TS is also found in many patients with anemia of chronic disease, so that decreased %TS is not specific for iron deficiency. Also, about 15% (10%4%) of patients with iron deficiency have a %TS greater than 15%, especially in the early stages or when iron deficiency is superimposed on other conditions. The %TS is increased in hemolytic or megaloblastic anemia, sideroblastic anemia, and iron overload states and is normal or increased in thalassemia minor (Table 3-2; a more complete list of conditions that affect TIBC and %TS is included in Table 37-2).

    Serum ferritin. Ferritin is the major body iron-storage compound. Routine tissues or bone marrow iron stains, however, detect hemosiderin but not ferritin. Ferritin in serum can be measured by radioassay or enzyme immunoassay. A serum ferritin level decrease accompanies a decrease in tissue ferritin level, which, in turn, closely mirrors decrease of body iron stores in iron deficiency. The decrease in tissue ferritin occurs before changes in serum iron tests, changes in RBC morphology, or anemia. Except for bone marrow iron stains, serum ferritin is currently the most sensitive test available for detection of iron deficiency. The major factors that modify its efficacy as an indicator involve the technical aspects of present-day ferritin immunoassay kits, some of which have less than desirable reproducibility and accuracy at the low end of the reference range. A major reason for this is the fact that the lower edge of the reference range (20-150 ng/ml or µg/L) is not far from zero. Another problem is the extreme difficulty most laboratories have in establishing their own ferritin reference range, since there is no good way to exclude subclinical iron deficiency from the clinically “normal” population without performing bone marrow aspiration. A third problem, partially arising from inadequately validated reference ranges, is disagreement in the literature as to what cutoff level should be used to confirm or exclude iron deficiency. The majority of investigators use 12 ng/ml as the cutoff level (literature range 10-20 ng/ml). A fourth problem (discussed later) is increase in ferritin levels by various conditions that may coexist with iron deficiency.

    Ferritin levels at birth are very high and are the same for boys and girls. Ferritin values decrease rapidly by age 3 months and reach their lowest point at about age 9 months. At some time during the teenage years the reference ranges for boys and girls being to diverge somewhat, with the lower limit of the reference range for girls being approximately 10 ng/100 ml lower than that for boys. The upper limit in men tends to increase slowly until old age, whereas the upper limit in women tends to remain relatively stationary until menopause and then slowly increases. The lower limits of reference ranges for both sexes are affected only to a small degree by age. There is approximately a 10%-15% average daily variation in ferritin values in the same individual; about one half the variation is due to fluctuation in serum iron values.

    INTERPRETATION OF SERUM FERRITIN RESULTS. A serum ferritin level less than 12 ng/ml is considered almost diagnostic of iron deficiency. Presumably false positive results in the literature based on bone marrow iron stains (displaying decreased serum ferritin levels with bone marrow iron present) range from 0%-4% of cases. False negative results have been reported in 2.6% of bone marrow-proven uncomplicated iron-deficient cases. However, if iron deficiency coexists with a condition that raises the serum ferritin, the ferritin value in a substantial number of patients may be higher than the cutoff value for iron deficiency. Serum ferritin level is decreased to a variable degree during pregnancy; the amount of decrease may be reduced as much as 50% if iron supplements are given.

    Many conditions can elevate ferritin levels. Serum ferritin is one of a group of proteins that become elevated in response to acute inflammation, infection, or trauma; elevation begins between 24 and 48 hours, peaks in about 3 days, and lasts 5 days to 5 weeks. In addition, a more sustained increase in ferritin levels may be produced by various chronic diseases (see Table 3-1), including those that decrease serum iron and serum TIBC values. Fortunately, some patients with coexisting chronic disease and iron deficiency still have decreased serum ferritin levels. Ferritin values may also be increased in some patients who have had blood transfusions, in megaloblastic anemia, and in hemolytic anemias. Ferritin is greatly increased in iron overload states such as hemochromatosis and acute iron poisoning. One study reports that about one third of patients with chronic hepatitis virus had elevated serum ferritin and some also had elevated serum iron and TIBC, simulating hemochromatosis.

    The serum ferritin level has been used in chronic renal failure to monitor iron status. Because chronic disease raises serum ferritin levels, the ferritin lower limit used for this purpose (approximately 100 ng/ml) is much higher than the lower limit of reference range used for the general population.

    Free erythrocyte protoporphyrin (zinc protoporphyrin). This test is discussed in detail in Chapter 35. The last step in heme synthesis occurs when the heme precursor protoporphyrin IX forms a complex with an iron atom with the help of the enzyme ferrochelatase (see Fig. 34-1). If iron is not available, or if ferrochelatase is inhibited (as occurs in lead poisoning), a zinc ion becomes complexed with protoporphyrin IX (zinc protoporphyrin; ZPP) instead of iron. When ZPP is assayed using manual biochemical techniques, the zinc ion is removed during acid extraction of RBC hemoglobin, and the metal-free substance measured is then called free erythrocyte protoporphyrin. Zinc protoporphyrin can be measured directly and quickly using one or two drops of whole blood by means of a small commercially available instrument called a hematofluorometer.

    ZINC PROTOPORPHYRIN IN IRON DEFICIENCY. Zinc protoporphyrin levels are elevated in iron deficiency and in lead poisoning. In iron deficiency, ZPP levels become elevated after several weeks of deficient iron stores and return to normal only after 2 to 3 months of iron therapy. In two studies, elevated ZPP levels detected 83%-94% of patients who were iron deficient on the basis of low serum ferritin levels.

    PROBLEMS WITH ZINC PROTOPORPHYRIN ASSAYS. Some hematofluorometers report ZPP per unit of whole blood; this reporting system may be affected by changes in hematocrit values. This problem is avoided with instruments that report results as a ZPP/heme ratio. Another potential difficulty is falsely decreased results due to a shift in the protoporphyrin fluorescent maximal absorption peak if the Hb is not fully oxygenated. This can be avoided in several ways. More troublesome is ZPP elevation by acute or chronic infections, noninfectious inflammation, various malignancies, chronic liver disease, and moderate or severe hemolytic anemias. Therefore, ZPP levels are elevated in many of the same conditions that falsely elevate serum ferritin levels. Although ZPP is a good screening method for iron deficiency and lead poisoning, most laboratories do not own a hematofluorometer.

    Bone marrow iron stain. The gold standard for chronic iron deficiency has been bone marrow aspiration or biopsy with Prussian blue chemical reaction for iron (hemosiderin). Although there is some disagreement, a clot section is generally considered more reliable for iron staining than an aspiration smear. Bone biopsy specimens must be decalcified, and some decalcifying reagents (but not others) may destroy some iron. The major problem with bone marrow aspiration has been reluctance of patients to undergo the procedure. Occasionally, bone marrow aspiration may be necessary to diagnose patients with hypochromic anemia without clear-cut evidence from other tests for or against iron deficiency. However, a therapeutic trial of iron might provide the same information.