Articles on Medical Diseases and Conditions

Tag: Factor Deficiency Anemia

  • Pyridoxine

    Pyridoxine (vitamin B6) is necessary for synthesis of D-aminolevulinic acid, a precursor of heme. Pyridoxine-deficient patients develop anemia with microcytic-hypochromic RBCs that can simulate chronic iron deficiency. Both hereditary and acquired (secondary) forms exist. The hereditary form is rare and the acquired form is uncommon, with the most frequently mentioned secondary type being due to tuberculin therapy with isoniazid (INH).

    Sideroblastic Anemias*

    HEREDITARY (SIDEROACHRESTIC)
    Acquired
    Idiopathic
    Alcoholism (most common etiology)
    Lead poisoning
    Drug induced (isoniazid, cycloserine, chloramphenicol)
    Some patients with various diseases
    Thalassemia
    Other hemolytic anemias
    Megaloblastic anemia
    Rheumatoid-collagen
    Myeloproliferative and myelodysplastic disorders
    Lymphomas/carcinomas/myeloma
    Infection
    Uremia
    Hypothyroidism or hyperthyroidism

    *Marrow sideroblasts more than 20% of nucleated RBC.

    Sideroblastic anemias. Pyridoxine deficiency anemia is included in the sideroblastic anemias. These conditions by definition have conversion of at least 20% of all bone marrow nucleated RBC to ringed sideroblasts. Ring sideroblasts are normoblasts with abnormal numbers of iron-stainable cytoplasmic granules that appear to form a ring around the nucleus when stained with iron stains. The sideroblastic anemia group includes the rare hereditary form that frequently responds to pyridoxine and a secondary or acquired category that includes various conditions that may be associated with sideroblastic marrows (see the box above). Of these, the most likely to have sideroblastic marrows are alcoholism, thalassemia, and some of the myelodysplastic syndromes. Hematologically, the sideroblastic anemias are characterized by hypochromic RBCs, sometimes predominant and sometimes coexisting with a minority or majority population of nonhypochromic RBC (“dimorphic” RBC population). If the hypochromic microcytic RBC are predominant, the MCV may be decreased. There typically is an elevated serum iron level with increased saturation of iron-binding capacity.

  • Folic Acid Deficiency

    Folic acid (folate) is necessary for adequate synthesis of certain purines and pyrimidines, which, in turn, are precursors of cell DNA. Folate is also necessary for methionine synthesis, histadine catabolism, and metabolism of serine and glycine. Vitamin B12 converts inactive 5-methyltetrahydrofolate to tetrahydrofolate, which is able to transfer one-carbon groups.

    Folic acid deficiency causes a megaloblastic anemia that may be indistinguishable from pernicious anemia in every laboratory test except the Schilling test without IF. It may also be indistinguishable clinically, except that neurologic symptoms do not occur from folic acid deficiency. Folic acid therapy improves most hematologic abnormalities of PA, even though the PA defect is a deficiency of vitamin B12, not folic acid, but folic acid therapy alone can worsen PA neurologic damage. Therefore, it is necessary to differentiate B12 from folic acid problems.

    Causes of folic acid deficiency. The most frequent cause of folic acid deficiency is dietary deficiency. This is especially common in chronic alcoholics. However, some investigators report that alcohol can inhibit folate absorption and interfere with folate metabolism. Another important cause is malabsorption, especially that category due to primary small bowel disease. Ten percent to 25% of pregnant women are reported to have some degree of folic acid deficiency, although by far the most common cause of deficiency anemia in pregnancy is iron deficiency. Folic acid deficiency in pregnancy may be due to dietary defect plus fetal demands; sometimes no good explanation is available. Uncommonly (<5%) a more severe folate deficiency state can occur in the last half of the third trimester. Some reports suggest that oral contraceptive pills can be associated with folic acid and vitamin B6 deficiency, but this is disputed by others. Drug-induced folate deficiency includes several drug categories. Certain cytotoxic medications such as methotrexate exert an antitumor effect by interfering with folate metabolism. Anticonvulsant drugs, especially phenytoin (about 30% of cases, range 14%-50%) and primidone (Mysoline), frequently show macrocytosis without anemia, but in a few patients induce a macrocytic megaloblastic anemia that responds best to folic acid. Phenytoin is associated with some degree of folate deficiency in about 40% of patients (27%-76%). It should be noted that megaloblastic anemia due to diet, pregnancy, or anticonvulsant drugs shows normal Schilling test results. Sulfasalazine (Azulfidine), used in therapy of ulcerative colitis, is also sometimes associated with macrocytosis due to folic acid deficiency. Colchicine, para-aminosalicylic acid (PAS), and neomycin interfere with folate absorption in some patients.

    Serum folate assay. Folic acid deficiency can be proved by serum folic acid measurement. If the test is done by the original microbiologic assay system, any antibiotic therapy must cease for a full week before the serum is drawn. Immunoassay (EIA or RIA) is less complicated than bacterial methods and is not affected by antibiotics; therefore, RIA has made folate measurement more practical. Unfortunately, because serum folate measurement is not ordered frequently, smaller laboratories will probably not do the test for economic reasons. Serum folate levels fall below normal limits 3-4 weeks after dietary or absorption-induced deficiency begins. Tissue folate levels (measured by RBC folate assay) become abnormal about 3 months later than serum folate and also return to normal after therapy somewhat later than serum folate. Anemia may not develop until 5 months after onset of deficiency in folate. In some patients with folate deficiency from deficient diet, a few meals with adequate folic acid may elevate serum folate values into the folate reference range, but RBC folate levels may still be low. My personal experience, as well as that of some others, indicates that RBC folate levels are more frequently low than serum folate levels in patients with suspected folate deficiency. Another problem with serum folate is a decreased level sometimes found in patients with severe liver or kidney disease. However, the RBC folate method also has its difficulties. Some manufacturers have kits that permit B12 and folate assay to be performed simultaneously on the same serum specimen (thus saving time and money), whereas RBC folate assay requires a different specimen and different processing than does serum B12 assay. Another problem is that B12 deficiency interferes with incorporation of folate into RBC. Therefore, B12 deficiency without folate deficiency can produce low RBC folate levels even though the serum folate level is normal; the combination of low serum B12 and low RBC folate levels might be misinterpreted as the combined B12 and folate deficiency seen in malabsorption.

  • Pernicious Anemia (PA)

    Pernicious anemia (PA) typically is seen in Northern Europeans, but recently is being recognized more in African Americans and Hispanics. In Northern Europeans, age of onset is usually after age 40 years, with significantly higher incidence beginning at age 50-55 years. In African Americans, onset tends to occur several years earlier (especially in African-American women) so that the patient is somewhat less likely to be elderly.

    PA is an interesting disease in which a combination of specific anatomical lesions and factor deficiency leads to a characteristic clinical picture. Briefly, patients with PA have atrophic gastritis of the body of the stomach, a complete lack of gastric hydrochoric acid, and a deficiency of IF. IF is a substance produced by the parietal cells of the body of the stomach that is necessary for the normal absorption of vitamin B12 in the ileum. Although PA may be caused by total (occasionally partial) gastrectomy, more often PA is idiopathic. Laboratory findings vary according to the duration and severity of the disease, but classically consist of a macrocytic anemia typically featuring oval macrocytes and megaloblastic changes in the bone marrow. An elevated MCV sometimes precedes onset of anemia. The bone marrow changes are often followed by decrease in peripheral blood cells, then anemia, thrombocytopenia, and finally leukopenia (due to neutropenia) and pancytopenia. However, the frequency of these changes depends on the severity of the B12 deficiency. In a Mayo Clinic series of PA patients, only 29% had anemia; so that only 64% had macrocytosis (MCV over 100 fL), 12% had thrombocytopenia, and 9% had leukopenia. There is frequently hypersegmentation (five nuclear segments or more) of some mature neutrophil nuclei. Definitive diagnosis of PA is made by the Schilling test without and with IF. PA may be excluded on the basis of a normal Schilling test result without IF (if certain technical problems are avoided, to be discussed next).

    Schilling test. The Schilling test entails oral administration of vitamin B12 that has been tagged with radioactive cobalt. The usual dose is 0.5 µg, although a 1981 publication of the International Committee for Standardization in Hematology (ICSH) recommends 1.0 µg. Next, 1,000 µg of nonisotopic vitamin B12 is given subcutaneously or intramuscularly to saturate tissue-binding sites to allow a portion of any labeled B12 absorbed from the intestine to be excreted or flushed out into the urine. This “flushing dose” of nonradioactive B12 is usually administered 2 hours after the radioactive B12(investigators have reported administration times of 2 to 6 hours, with extremes ranging from simultaneous injection with the radioactive dose to as much as 12 hours later). In a normal person, more than 8% (literature range, 5%-16%) of the oral radioactive B12 dose appears in the urine. In classic PA, the Schilling test result is positive; that is, there is less than 8% urinary excretion of the radioisotope if no IF has been given with the test dose. The test is then completely repeated after at least 24 hours, administering adequate amounts of oral IF with the radioactive B12 dose. Urinary B12 excretion should then become normal, since the added intrinsic factor permits normal vitamin B12 absorption.

    Urine collection. Although the ICSH recommends a single 24-hour urine collection and the majority of laboratories follow this protocol, some reports indicate that certain patients without B12 malabsorption may take up to 72 hours to excrete normal amounts of the radioactive B12. One report indicated that 14% of patients with normal B12 excretion took 48 hours and 7% took 72 hours. Some of those with delayed excretion had an elevated blood urea nitrogen (BUN) level, but some did not. Poor renal function is known to delay Schilling test B12 excretion and frequently produces low 24-hour results. Reports indicate that cumulative results over a 48-hour time period, and sometimes even a 72-hour period, may be required to reach a normal recovery level. Normal values for a 48- or 72-hour excretion period are the same as those for a 24-hour collection. Therefore, at least a 48-hour collection should be considered if the BUN level is elevated. If extended urine collection periods are used, it is recommended that one additional nonradioactive B12 parenteral dose be administered each 24 hours, since this has been reported to increase the B12 excretion. Because renal function can be significantly decreased with the BUN level remaining within reference range, and because of uncertainty reported for individual patient response, some laboratories routinely collect two consecutive 24-hour urine specimens. If the first 24-hour specimen contains a normal amount of radioactive B12, the second collection can be discontinued. If the total 48-hour specimen radioactivity is low but relatively close to the lower limit of normal, a third 24-hour specimen might be desirable. One relatively frequent 24-hour Schilling test problem is inadvertent loss of a portion of the urine collection. If this occurs, in my experience it is useful to order completion of the 48-hour collection period. In the meantime, if the urine actually collected before the accident is counted, it may contain a normal amount of radioactivity, in which case the additional collection can be terminated. If not, the radioactivity in the initial specimen can be added to that in the remainder of the collection.

    One precaution to be observed when performing a Schilling test is having the patient fast overnight since food may contain B12 and also to prevent food interference with gastric emptying or protein in food from binding any radioactive B12. Two hours after the radioactive dose is administered, the patient is permitted a light meal that does not contain any B12 (the ICSH recommends toast and jelly as an example). A 12- to 24-hour urine specimen should be obtained just before starting the test to demonstrate that no previously administered radioactivity (e.g., from a nuclear medicine scan) is present.

    Repeat Schilling tests. If the Schilling test result without IF is abnormal (low result), the test is repeated with IF. In some cases it may have to be repeated without IF to correct a test problem or to verify results. There is some debate over what time interval should elapse between the end of the first test and beginning of the second (with or without IF). Various investigators have recommended intervals ranging from 1 to 7 days. The ICSH states that it is not necessary to wait more than 24 hours. However, there are some reports that nonradioactive B12 from the flushing dose might be excreted in bile sufficient to compete with radiolabeled B12 for absorption in the ileum. Also, if only a 24-hour collection is used for the first Schilling test, a few persons with considerably delayed excretion might contribute a significant, although small, amount of radioactivity to the next test. Therefore, a 48-hour wait might be a reasonable compromise. Schilling tests can be performed while a patient is having B12 therapy as long as the patient is not receiving IF. The nonradioactive “flushing dose” of B12 administered during the Schilling test is actually a therapeutic dose.

    FALSE POSITIVE RESULTS. Falsely low values may be produced by bladder retention of urine (incomplete voiding) and by spilled or lost urine already voided; in either case the problem is incomplete urine collection (normal output is >600 ml/24 hours and usually is >800 ml). Measurement of urine creatinine excretion is also helpful as a check on complete collection. Also, delayed B12 excretion could produce falsely decreased values in a 24-hour collection. As noted previously, some laboratories routinely collect two consecutive 24-hour urine specimens, since the initial 24-hour collection may be incomplete, and vitamin B12 excretion during the second 24 hours may be sufficient to bring total 48-hour excretion to reference range both with incomplete first-day collection or with delayed excretion. In my series of 34 consecutive patients with Schilling tests, 10 patients (29%) had low B12 excretion without IF at 24 hours; 4 of these had normal results at 48 hours. Therefore, 4 of 10 patients would have had falsely low results if a second 24-hour specimen had not been obtained. Of the thirty-four patients, 82% had >2% B12 excretion in the second 24-hour specimen (average 8.7%, range 2.0%-18.9%).

    FALSE NEGATIVE RESULTS. Falsely normal values are much less of a problem than false positive results but may be caused by fecal contamination of the urine or by presence of radioactivity from a previous nuclear medicine procedure. One report indicates that folic acid deficiency (manifested by low blood folate level) significantly increases urine B12 excretion with or without IF, which potentially might produce a borderline or low normal Schilling test result with and without IF in PA with concurrent folate deficiency when the result would ordinarily be low. Therapy with B12 and folate for 1 month resulted in a repeat Schilling test diagnostic for PA.

    In some patients the Schilling test shows decreased labeled B12 excretion both with and without IF. Some of these patients may have PA but with ileal mucosal epithelial cells that are malfunctioning because of megaloblastic change. (“megaloblastic bowel syndrome”). Therapy with parenteral B12 will restore the ileum to normal function, although this sometimes takes several weeks or months (in one report, 15% of patients with megaloblastic bowel syndrome from PA still did not have Schilling test results diagnostic of PA by 4 months after beginning therapy). Some patients have intestinal bacterial overgrowth or the “blind loop” syndrome. Two weeks of appropriate antibiotic therapy should permit correct repeat Schilling test results. Other patients with abnormal Schilling results with and without IF may have primary small intestine mucosal malabsorption disease (sprue) or widespread mucosal destruction from regional ileitis or other causes, severe chronic pancreatitis (40%-50% of patients), the fish tapeworm (rarely), or drug-induced malabsorption (e.g., from colchicine, dilantin, or neomycin).

    In some cases it may not be possible to obtain a Schilling test. Some partial alternatives include assay for intrinsic factor antibody and a “therapeutic trial.”

    The therapeutic trial. In deficiency diseases, treatment with the specific agent that is lacking will result in a characteristic response in certain laboratory tests. This response may be used as a confirmation of the original diagnosis. Failure to obtain the expected response casts doubt on the original diagnosis; suggests that treatment has been inadequate in dosage, absorption, utilization, or type of agent used; or suggests that some other condition is present that is interfering with treatment or possibly is superimposed on the more obvious deficiency problem. The two usual deficiency diseases are chronic iron deficiency and vitamin B12 or folic acid deficiency. When a test agent such as iron is given in a therapeutic dose, a reticulocyte response should be manifested in 3-7 days, with values at least twice normal (or significantly elevated over baseline values if the baseline is already elevated). Usually, the reticulocyte count is normal or only slightly elevated in uncomplicated hematologic deficiency diseases. If the baseline reticulocyte values are already significantly elevated over normal range in a suspected deficiency disease, this suggests either previous treatment (or, in some cases, a response to hospital diet), wrong diagnosis, or some other superimposed factor (e.g., recent acute blood loss superimposed on chronic iron deficiency anemia). If the baseline values are already more than twice normal, it may not be possible to document a response. Once the correct replacement substance is given in adequate dosage, hemoglobin values usually rise toward normal at a rate of approximately 1 gm/100 ml/week.

    Two major cautions must be made regarding the therapeutic trial: (1) it never takes the place of a careful, systematic search for the etiology of a suspected deficiency state, and (2) the patient may respond to one agent and at the same time have another factor deficiency or more serious underlying disease.

    A therapeutic trial usually is initiated with therapeutic doses of the test agent. This standard procedure does not differentiate vitamin B12 deficiency from folic acid deficiency, since therapeutic doses of either will evoke a reticulocyte response in cases of deficiency due to the other. If a therapeutic trial is desired in these circumstances, a small physiologic dose should be used, such as 1 µg of vitamin B12/day for 10 days, or 100 µg of folic acid/day for 10 days. At least 10 days should elapse between completion of one trial agent and beginning of another. Also, the patient should be on a diet deficient in folic acid or B12, and baseline reticulocyte studies should be performed for 1 week with the patient on this diet before initiation of the actual trial. Generally, in vitamin B12 deficiency, the Schilling test (without IF) gives the same information and can be repeated with the addition of IF to pinpoint the cause. In folic acid deficiency, a therapeutic trial may be helpful in establishing the diagnosis. The main drawback to using the therapeutic trial in diagnosis is the time involved.

    Reticulocyte response. Treatment with oral vitamin B12 plus IF evokes a reticulocyte response of 5%-15%. The same response occurs after a Schilling test as a result of the nonisotopic B12 given parenterally. One B12 therapeutic or Schilling test dose is sufficient to produce a reticulocyte response in patients with folic acid deficiency, and therapeutic doses of folate can produce a reticulocyte response in B12 deficiency. In addition, folate therapy can increase serum levels of B12.

    Assay for intrinsic factor and for antibody to intrinsic factor. In vitro techniques have been described for assay of these substances. For assay of IF, aspiration of gastric juice is necessary. The specimens from routine gastric analysis are satisfactory. Antibody to IF is said to be present in the serum of 50% to 70% (range, 33%-75%) of patients with PA. Actually, two different antibodies have been found. Type I is a blocking antibody that prevents binding of B12 to IF. This is the antibody usually present in PA (60%-75%) of patients). The type II antibody is a binding-type antibody that binds to intrinsic factor or to the intrinsic factor-vitamin B12 complex. This antibody is less common in serum from patients with PA (30% to 50% of patients). The presence of the type I antibody is considered by some to be almost diagnostic of pernicious anemia. However, presently available kits have been reported to give false positive results when serum B12 levels are very high (most commonly, within 24 hours after a B12 dose injection. One institution obtained false results as long as 1 week). False positive results have also been reported in a small number of patients with diabetes mellitus, adrenal insufficiency, thyroid diseases, and various gastric abnormalities. The test is available at present only in some medical centers and a few large reference laboratories. More work should be done with commercial kits to ascertain their level of performance and what false results can be expected. IF antibody assay, if positive, may be useful to help confirm a diagnosis of PA in equivocal cases. Antiparietal cell antibody can be detected in 76% to 91% of patients with PA. However, it is much more nonspecific than IF antibody, being found in 30%-60% of patients with idiopathic atrophic gastritis, in 12%-28% of diabetics, 25%5% with thyrotoxicosis, 25% with Hashimoto’s thyroiditis, and 5%-10% of clinically normal persons.

  • 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.

  • 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.

  • Iron Deficiency

    Iron deficiency may be produced in three ways: (1) iron intake not sufficient to replace normal iron losses, (2) iron not available for erythropoiesis despite adequate body iron, and (3) increased loss of body iron (blood loss) not adequately replaced by normal intake.

    Acute blood loss can usually be handled without difficulty if the bleeding episode is not too prolonged and if tissue iron stores are adequate. The anemia that develops from acute bleeding is normocytic and normochromic and is not the type characteristic of chronic iron deficiency (acute changes in hematocrit level are discussed elsewhere. Chronic bleeding, however, is often sufficient to exhaust body iron stores from continued attempts by the bone marrow to restore the blood Hb level. If this occurs, a hypochromic-microcytic anemia eventually develops. Chronic bleeding may be in the form of slow, tiny, daily losses; intermittent losses of small to moderate size not evident clinically; or repeated, more widely spaced, larger bleeding episodes. Chronic iron deficiency may develop with normal diet but is hastened if the diet is itself borderline or deficient in iron.

    Chronic iron deficiency is common among infants, usually from deficient dietary intake. The infant grows rapidly and must make Hb to keep up with expanding blood volume. Most of the infant’s iron comes from fetal Hb present at birth. Since premature infants have a smaller birth weight on the average, they also tend to have a smaller Hb mass than term infants. The premature infant is thus more likely than the term infant to develop clinical iron deficiency at age 6 months to 2 years, when the demands of rapid growth are most likely to produce iron depletion. Breast milk contains a marginally adequate iron content, but cow’s milk is definitely low in available iron. Whether iron deficiency occurs will therefore depend on infant birth weight, the type of milk received, and the length of milk feeding before supplementation or replacement by other foods. It has been estimated that 15%-20% of infants between 9 and 12 months of age have some degree of iron deficiency and possibly up to 50% in low socioeconomic groups.

    In adults, iron deficiency from inadequate diet alone more frequently is subclinical rather than severe enough to produce anemia. However, poor iron intake may potentiate the effects of iron deficiency from other etiologies, such as menstruation. In one study, about 5% of clinically healthy white female adolescents were found to have chronic iron deficiency anemia, with about 9% of others having nonanemic iron deficiency. Iron deficiency is frequent in malabsorption diseases such as sprue, which, strictly speaking, represents inability to use available dietary iron rather than a true dietary deficiency. Another cause is pregnancy, in which iron deficiency is caused by maternal iron utilization by the fetus superimposed on previous iron deficiency due to excessive menstrual bleeding or multiple pregnancies. About 50% of chronic alcoholics and 20%-40% of patients with megaloblastic anemia are said to have some degree of iron deficiency. An interesting related condition is long-distance runner or jogger anemia, said to occur in about 40% of women active in these sports and to be caused by a combination of iron deficiency from poor diet, gastrointestinal bleeding, hematuria, and hemolysis.

    By far the most common cause of chronic iron deficiency in adolescents or adults severe enough to cause anemia is excessive blood loss. In men, this is usually from the gastrointestinal (GI) tract. In women it may be either GI or vaginal bleeding. It has been estimated that 20%-50% of women in the menstrual age group have some degree of iron deficiency; menstrual loss is frequently aggravated by poor diet. Therefore, careful inquiry about the frequency, duration, and quantity of menstrual bleeding is essential in treating women. An estimate of quantity may be made from the number of menstrual pads used. GI bleeding is most frequently due to peptic ulcer in men and women below age 40. After age 40 GI carcinoma is more common and should always be ruled out. Hemorrhoids are sometimes the cause of chronic iron deficiency anemia, but since hemorrhoids are common it should never be assumed that the anemia is due only to hemorrhoids.

    If excessive vaginal bleeding is suspected, a careful vaginal/uterine examination with a Papanicolaou smear should be done. If necessary, a gynecologist should be consulted. For possible GI bleeding an occult blood test on a stool sample should be ordered on at least three separate days. However, one or more negative stool guaiac results do not rule out GI cancer or peptic ulcer, since these lesions may bleed intermittently. If a patient is over age 40 and stool guaiac results are negative, many gastroenterologists recommend a barium enema and possibly sigmoidoscopy or colonoscopy. If the barium enema or endoscopy result is negative, and no other cause for the iron deficiency anemia can be demonstrated, they recommend repeating the barium enema in 3-4 months in case a lesion was missed. Lower GI tract studies are particularly important for detection of carcinoma because colon carcinoma has an excellent cure rate if discovered in its early stages. Gastric carcinoma, on the other hand, has a very poor cure rate by the time it becomes demonstrable. In addition to peptic ulcer, gastric hiatal hernia is sometimes associated with iron deficiency anemia.

  • Iron Metabolism

    Hemoglobin (Hb) contains about 70% of the body iron, and storage iron accounts for most of the remainder. One gram of Hb contains 3.4 mg of iron, and 1 ml of packed red blood cells (RBCs) contains about 1 mg of iron. Iron intake averages about 10 mg/day, of which about 10% is absorbed. Iron loss averages about 1 mg/day in men and nonmenstruating women and about 2 mg/day in menstruating women. There is an additional iron requirement for pregnant and lactating women.

    Most body iron originates from dietary iron in the ferric state, which is converted to the ferrous state after ingestion and absorbed predominantly in the duodenum and jejunum. Iron circulates in the blood coupled to a beta globulin, transferrin. Bone marrow RBC precursors use a portion of the available iron; about 60% of the remainder is stored within reticulum cells in bone marrow, liver, and spleen as ferritin and about 40% as hemosiderin. Iron from ongoing RBC death and hemoglobin breakdown is the primary source of iron storage material.

    Approximately 50% more oral iron is absorbed if taken 45-60 minutes before food ingestion than if taken with food. Meat inhibits absorption less than dairy products or foods with high fiber content. Coffee and tea also inhibit absorption, whereas vitamin C (given with iron) enhances absorption. Iron in human milk is absorbed much more readily than that in cow’s milk.

  • Anemia

    Although anemia may be defined as a decrease in Hb concentration, it may result from a pathologic decrease in the RBC count. Since mature RBCs are fully saturated with Hb, such a decrease means that total blood Hb value will also be affected.

    Before commencing transfusion therapy or an extensive workup for the etiology of anemia, one should consider the possibility of pseudoanemia Pseudoanemia may be associated with either a Hb value below reference range limits or a drop in Hb level of 2 gm/100 ml (20 g/L) or more from a previous value. Accuracy of the values, especially those below the reference range, should be verified by redrawing the specimen. It is also possible that a patient could have true anemia simultaneously with one of the conditions in the box on this page.

    One frequently overlooked cause of Hb decrease is iatrogenic blood loss due to laboratory test specimens. Several studies document that a surprising amount of blood can be withdrawn, especially in critically ill patients or those with diagnostic problems. Whereas the majority of patients contributed an average of 10-20 ml of blood per day, those in critical care units may average 40-50 ml/day and sometimes even as much as 150-500 ml per patient per day for several days’ time. In some cases this may total more than 1,000 ml during the hospital stay. (The data sometimes include blood withdrawn to clear arterial lines and sometimes not; this blood also may represent a considerable total quantity.)

    Some Conditions That Produce or Contribute to False Anemia
    Overhydration or rehydration of a dehydrated patient
    Specimen obtained with intravenous (IV) fluid running
    Fluid retention
    Pregnancy
    Hypoalbuminemia
    Posture changes (from upright to recumbent)
    Laboratory variation in hemoglobin assay (approximately ± 0.5 gm/dl) or laboratory error

    Classification of Anemia

    Classification of anemia is helpful because it provides a handy reference for differential diagnosis. There are several possible classifications; each is helpful in some respects.

    Anemia may be classified by pathogenesis. According to pathogenesis, three mechanisms may be responsible.

    1.
    Deficiency of vital hematopoietic raw material (factor deficiency anemia). The most common causes of factor deficiency anemia are iron deficiency and deficiency of vitamin B12, folic acid, or both.
    2.
    Failure of the blood-forming organs to produce or to deliver mature RBCs to the peripheral blood (production-defect anemia). This may be due to (1) replacement of marrow by fibrosis or by neoplasm (primary or metastatic); (2) hypoplasia of the bone marrow, most commonly produced by certain chemicals; or (3) toxic suppression of marrow production or delivery without actual marrow hypoplasia, found to a variable extent in some patients with certain systemic diseases. The most common of these diseases are severe infection, chronic renal disease, widespread malignancy (without extensive marrow replacement), rheumatoid-collagen diseases, and hypothyroidism. (These conditions may sometimes be associated with an element of hemolytic anemia.)
    3.
    RBC loss from the peripheral blood (depletion anemia). This is commonly due to (1) hemorrhage, acute or chronic (causing escape of RBCs from the vascular system), (2) hemolytic anemia (RBCs destroyed or RBC survival time shortened within the vascular system), or (3) hypersplenism (splenic sequestration).

    A second classification is based on RBC morphology. Depending on the appearance of the RBC on a peripheral blood smear, Wintrobe indices, or both, anemias may be characterized as microcytic, normocytic, or macrocytic. They may be further subdivided according to the average amount of RBC hemoglobin, resulting in hypochromia or normochromia. (Macrocytic RBCs may appear hyperchromic on peripheral smear, but this is an artifact due to enlarged and thicker cells that, being thicker, do not transmit light through the central portion as they would normally.) The box on this page lists the more common etiologies.

    Investigation of a Patient With Anemia

    Anemia is a symptom of some underlying disease and is not a diagnosis. There always is a cause, and most of the causes can be discovered by a relatively few simple procedures. Knowing the common causes of anemia, getting a good history, doing a thorough physical examination, and ordering a logical sequence of laboratory tests based on what the clinical picture and other findings suggest provide the greatest assistance in identifying which underlying disease is responsible. When anemia is discovered (usually by the appearance of a low Hb or Hct value), the first step is to determine whether anemia really exists. The abnormal result should be confirmed by drawing a second specimen. Then, if the patient is not receiving excess intravenous (IV) fluid that might produce hemodilution, the next step is to obtain a WBC count, differential, RBC indices, reticulocyte count, and a description of RBC morphology from the peripheral smear. It is wise for the physician to personally examine the peripheral smear, because many technicians do not routinely pay much attention to the RBCs. A careful history and physical examination must be performed. To some extent, the findings on peripheral smear and RBC indices (including the RDW, if available) help suggest areas to emphasize:

    1.
    If the RBCs are microcytic, the possibility of chronic blood loss must always be carefully excluded.
    2.
    If the RBCs are macrocytic, the possibility of megaloblastic anemia or reticulocytosis due to acute bleeding must always be investigated.
    3.
    If the RBCs are not microcytic, if megaloblastic anemia is ruled out in patients with macrocytosis, and if the reticulocyte count is significantly elevated, two main possibilities should be considered: acute blood loss and hemolytic anemia. The reticulocyte count is usually 5% or higher in these cases. However, the possibility of a deficiency anemia responding to therapy should not be forgotten.
    4.
    In a basically normocytic-normochromic anemia without significant reticulocytosis and in which either leukopenia or thrombocytopenia (or both) is present, hypersplenism, bone marrow depression, or a few systemic diseases (e.g., systemic lupus erythematosis) are the main possibilities. In patients over age 40 with normocytic-normochromic anemia and without WBC or platelet decrease, myeloma should be considered, especially if rouleaux or other abnormalities are present that are commonly associated with myeloma. The possibility of chronic iron deficiency should not be forgotten, even though the typical RBC morphology of iron deficiency is microcytic-hypochromic. Occasionally, patients with B12 or folate deficiency have an MCV in the upper normal range.
    5.
    Appearance of certain RBC abnormalities in the peripheral blood suggests certain diseases. A considerable number of target cells suggests one of the hemoglobinopathies or chronic liver disease. Marked basophilic stippling points toward lead poisoning or reticulocytosis. Sickle cells mean sickle cell anemia. Nucleated RBCs indicate either bone marrow replacement or unusually marked bone marrow erythropoiesis, most commonly seen in hemolytic anemias. Significant rouleaux formation suggests monoclonal gammopathy or hyperglobulinemia. Spherocytes usually indicate an antigen-antibody type of hemolytic anemia but may mean congenital spherocytosis or a few other types of hemolytic anemia. Schistocytes (burr cells) in substantial numbers are usually associated with microangiopathic hemolytic anemias or with uremia, alcoholism, and hypothyroidism. Macrocytes are frequently produced by reticulocytosis but are also associated with megaloblastic anemias, cirrhosis, chronic alcoholism, hypothyroidism, and aplastic anemia.

    Some Common Causes of Anemia According to RBC Morphology*
    MICROCYTIC
    Hypochromic

    Chronic iron deficiency (most frequent cause)
    Thalassemia
    Occasionally in chronic systemic diseases
    Normochromic
    Some cases of chronic systemic diseases
    (May be simulated by spherocytosis or polycythemia in some patients)

    NORMOCYTIC
    Hypochromic

    Some cases of anemia due to systemic diseases
    Many cases of lead poisoning

    Normochromic

    Many cases of anemia due to systemic disease (most common cause)
    Many cases of anemia associated with pituitary, thyroid, or adrenal disease
    Acute blood loss
    Hemolytic anemia
    Bone marrow replacement or hypoplasia
    Hypersplenism
    Distance-runner anemia (most persons)

    MACROCYTIC
    Hypochromic

    Some cases of macrocytic anemia with superimposed iron deficiency

    Normochromic

    Vitamin B12 or folic acid deficiency
    Malabsorption (vitamin B12 or folic acid)
    Chronic alcoholism
    Reticulocytosis
    Some cases of chronic liver disease and some cases of hypothyroidism
    Myelodysplasia syndromes/aplastic anemia
    Drug-induced

    *All patients with any disease do not fit into any one category.

    Once the basic underlying process is identified, the cause can usually be identified by using selected laboratory tests with the help of history, physical findings, and other diagnostic procedures. In general it is best to perform diagnostic laboratory studies before giving blood transfusions, although in many cases the diagnosis can be made despite transfusion. Blood specimens for the appropriate tests can usually be obtained before transfusion is actually begun, since blood for type and crossmatching must be drawn first. Serum can be saved or frozen for additional studies, if needed.