Articles on Medical Diseases and Conditions

Tag: Red blood cell indices

  • Vitamin B12 Deficiency

    Vitamin B12 (cyanocobalamin) is necessary for adequate DNA synthesis through its role in folic acid metabolism. Vitamin B12 transforms metabolically inactive 5-methyl-tetrahydrofolate to tetrahydrofolate, which can then be converted to an active coenzyme. Vitamin B12 also acts as a single-carbon transfer agent in certain other metabolic pathways, such as metabolism of propionic acid. Most vitamin B12 comes from food of animal origin, primarily meat, eggs, or dairy products. In the stomach, the B12 is detached from binding proteins with the help of gastric acid. Vitamin B12 is absorbed through the ileum with the aid of “intrinsic factor” (IF) produced by parietal cells in the fundus of the stomach.

    Vitamin B12 deficiency may be produced in several ways. The most common cause is inability to absorb B12 due to deficiency of intrinsic factor (pernicious anemia). Another cause is failure to release B12 from binding proteins caused by severe deficiency of gastric acid. A third cause is other malabsorption syndromes involving the ileum mucosa or B12 absorption by the ileum. Besides direct damage to ileal mucosa, various causes of vitamin B12 malabsorption include bacterial overgrowth in the intestine (blind loop syndrome), infestation by the fish tapeworm, severe pancreatic disease, and interference by certain medications. Finally, there is dietary B12 deficiency, which is rare and found mainly in strict vegetarians.

    Vitamin B12 assay. Vitamin B12 deficiency can usually be proved by serum B12 assay. Therefore, a therapeutic trial of oral B12 is seldom needed. In patients with severe anemia of unknown etiology it is often useful to freeze a serum specimen before blood transfusion or other therapy begins, since vitamin B12 and folic acid measurements (or other tests) may be desired later. Chloral hydrate is reported to increase B12 levels in some patients using certain assay kits but not others. Pregnancy, large doses of vitamin C, and folic acid deficiency may be associated with reduced B12 assay levels. Achlorhydria is reported to produce decreased release or utilization of vitamin B12 from food, although intestinal absorption of crystalline vitamin B12 used in the Schilling Test dose or in oral therapeutic B12 is not affected. Vitamin B12 deficit may be accompanied by folic acid deficiency in some conditions and by chronic iron deficiency.

    Vitamin B12 is transported in serum bound to several serum proteins, among which the most important are transcobalamin and “R-protein.” Severe liver disease or myeloproliferative disorders with high white blood cell (WBC) counts produce elevated vitamin B12-binding protein levels and falsely raise total serum B12 values. In 1978, reports were published demonstrating that certain metabolically inactive analogues of B12 were present in serum bound to R-protein. It was found that commercial vitamin B12 assay kits contained varying amounts of R-protein in the material used to segregate patient B12 for measurement and that the B12 analogues could bind to the reagent R-protein and be included with active B12 in the measurement, thereby falsely increasing the apparent B12 value. A low vitamin B12 value could be elevated into the reference range. Most manufacturers have redesigned their B12 assay kits to eliminate the effects of R-protein. Nevertheless, case reports of patients with symptoms of B12 deficiency but serum B12 assay results within reference range continue to appear. In these cases some additional test, such as intrinsic factor antibodies or a therapeutic trial with B12, may be desirable.

    A decreased B12 level does not guarantee that actual B12 deficiency exists. Several investigators have reported that less than 50% of their patients with decreased serum B12 levels had proven B12 deficiency.

    Megaloblastic changes. Deficiency of either vitamin B12 or folic acid eventually leads to development of megaloblastic anemia. Vitamin B12 deficiency may take 1 to 2 years for the MCV to become elevated and megaloblastic changes to appear. The RBC precursors in the marrow become slightly enlarged, and the nuclear chromatin develops a peculiar sievelike appearance, referred to as megaloblastic change. This affects all stages of the precursors. The bone marrow typically shows considerable erythroid hyperplasia as well as megaloblastic change. Not only RBCs are affected by folate or B12 deficiency but also WBCs and platelets. Actual anemia develops 6 to 18 months later (2 to 3 years after the original disease onset). In far-advanced megaloblastic anemia there are peripheral blood leukopenia and thrombocytopenia in addition to anemia; in early cases there may be anemia only. The bone marrow shows abnormally large metamyelocytes and band neutrophils. Macrocytes are usually present in the peripheral blood, along with considerable anisocytosis and poikilocytosis. Hypersegmented polymorphonuclear neutrophils are characteristically found in the peripheral smear, though their number may be few or the degree of hypersegmentation (five lobes or more) may be difficult to distinguish from normal variation. The reticulocyte count is normal.

    Methylmalonic acid assay. In patients with borderline serum B12 values or clinical suspicion of B12 deficiency but serum B12 within reference range, serum or urine methylmalonic acid assay may be helpful. B12 is a necessary cofactor in the reaction converting methylmalonate to succinate. If insufficient B12 is available, the reaction is partially blocked and methylmalonic acid (MMA) accumulates in serum and is increased in urine. Both serum and urine MMA are said to be increased in 95% of patients with B12 deficiency, even when serum B12 assay is within population reference range. One study found that only 60% of patients with increased urine MMA has decreased serum B12 values. MMA assay usually has to be sent to a reference laboratory.

    Bone marrow examination. For a long time, bone marrow aspiration was the classic way to confirm the diagnosis of megaloblastic anemia. Now that B12 and folate assays are available, most physicians no longer obtain bone marrow aspiration. The major difficulty occurs if megaloblastic anemia is strongly suspected and the B12 and folate assay results are within normal limits. To be useful, a bone marrow aspiration should be performed as early as possible and definitely before blood transfusion. Blood transfusion, hospital diet, or vitamin B12 administered as part of a Schilling test may considerably reduce or eliminate diagnosable megaloblastic change from the bone marrow in as little as 24 hours’ time, even though the underlying body deficiency state is not cured by the small amount of B12 and folate in blood transfusion.

    Megaloblastic changes in the bone marrow are not diagnostic of folic acid or vitamin B12 deficiency. Some cytologic features of megaloblastic change (“megaloblastoid” or “incomplete megaloblastic” change) may appear whenever intense marrow erythroid hyperplasia takes place, such as occurs in severe hemolytic anemia. Similar changes may also be found in chronic myelofibrosis, in sideroblastic anemias, in certain myelodysplasia syndromes, in erythroleukemia (formally called Di Guglielmo’s syndrome), in some patients with neoplasia, cirrhosis, and uremia, and in association with certain drugs, such as phenytoin (Dilantin) and primidone, methotrexate and folic acid antagonists, and alcohol (substances affecting folate metabolism); colchicine and neomycin (affecting absorption); and antineoplastic drugs such as 5-fluorouracil and 6-mercaptopurine (that interfere with DNA synthesis).

    Red blood cell indices. The MCV in megaloblastic anemia is typically elevated. However, 15%-30% of patients (0%-96%) with folic acid or B12 deficiency, more often folic acid deficiency, had an MCV in the upper half of the reference range. In some patients normal MCV was probably due to early or mild disease. In others, these findings were explained on the basis of a coexisting condition that produced microcytosis (most commonly chronic iron deficiency, thalassemia minor, and anemia of infection, and less commonly malnutrition. In one study, 21% of patients with pernicious anemia had a significant degree of iron deficiency. If chronic iron deficiency coexists, therapy for megaloblastic anemia alone may only partially (or not at all) correct the anemia but will unmask the chronic iron deficiency picture. If the iron deficiency alone is treated, the megaloblastic defect is unmasked (unless it is a deficiency that is inadvertently treated by hospital diet or vitamins).

    The MCV is less likely to be elevated in B12 deficiency without anemia. In one study, only 4% of nonanemic patients with decreased serum B12 during one time period had elevated MCV. On the other hand, the MCV may become elevated before there is hemoglobin decrease to the level of anemia; therefore, an elevated MCV, with or without anemia, should not be ignored.

    Serum lactic dehydrogenase. Serum lactic dehydrogenase (LDH) levels are elevated in most patients with hemolytic anemia and in 85% or more of patients with megaloblastic anemia. The electrophoretically fast-migrating LDH fraction 1 is found in RBCs, myocardial muscle cells, and renal cortex cells. Megaloblastic change induces increased destruction of RBCs within bone marrow, which is reflected by the increase in serum LDH levels. If the LDH is included in patient admission chemistry screening panels, an LDH elevation might provide a clue to the type of anemia present. In hemolytic and megaloblastic anemia, LDH isoenzyme fractionation typically displays elevated LDH fraction 1 with fraction 1 greater than fraction 2 (reversal of the LDH-1/LDH-2 ratio). However, the usefulness of serum LDH or LDH isoenzymes is limited by their nonspecificity (since LDH fraction 1 is not specific for RBCs, and other LDH isoenzymes are present in other tissues such as liver) and by the fact that the degree of LDH change is roughly proportional to the severity of anemia, so that patients with mild cases may have total LDH or fraction 1 values still within reference range.

    Neutrophil hypersegmentation. Hypersegmentation is most often defined as a segmented neutrophil with five or more lobes (although some restrict the definition to six or more lobes). An elevated neutrophil lobe count (defined as five or more lobes in more than 5% of all neutrophils) is reported to be more sensitive and reliable than elevated MCV or peripheral smear macrocytosis in detecting megaloblastic anemia and also is not affected by coexisting iron deficiency.

  • 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.