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.